Name: c. elegans chemotaxis assay
Uploaded: 2013-04-27T14:00:00.000+00:00
Duration: 6 min 28 s
Description: Watch this Scientific Journal Video about C. elegans Chemotaxis Assay at JoVE.com

我们感谢生命科学与艺术和科学学院在皇后大学为这项工作提供资金。同时，我们感谢钦生实验室提供必要的试剂，设备和技术支持。我们也感谢2011年QGEM，尤其是托尼，他为他的贡献的讨论。

1。准备/洗涤蠕虫<ol><li>同步蠕虫年轻成人11。 </li><li>吸取2毫升到5厘米的趋化板的S基础上演OP50 E.刚刚清除草坪蠕虫大肠杆菌 。倾斜板，根据需要，以确保到缓冲区中的板面从洗涤蠕虫。 </li><li>吸取1毫升离心管蠕虫-S基础的解决方案。 </li><li> ，持续10秒使用PicoFuge，在6,600 rpm转速下离心。 </li><li>吸的S基础，离开颗粒蠕虫不受干扰的。 </li><li> ML小号基础的解决方案添加到离心管中，颠倒几次洗蠕虫。 </li><li>重复步骤1.4〜1.6另外三次。 </li><li>离心的第五时间，这段时间吸取上清液，至终体积为约10秒。 100微升。 </li><li>取2微升到NGM板，以确保各2微升样品中，有50和250之间的蠕虫的蠕虫。调整根据需要通过重悬体积更小或更大的S基底中的蠕虫的蠕虫在S基底的浓度。 </li><li>使用后立即洗了长达1小时的蠕虫蠕虫检测。 </li></ol> 2。准备测试板<ol><li>标志着一个5厘米的板的底面，分为4象限划分的菜。趋化琼脂或NGM的也可使用。需要最少3个试验正在测试每遗传应变。 </li><li>马克的圆的半径为0.5厘米的原点周围（ 图1）。 </li><li>使用一个“T”为“测试”或“C”为“控制”，确保该网站是等距离的起源和相互点标记在每个象限。点必须远离原点至少2厘米。马克左上角和右下角的象限作为测试象限的右上方和左下象限作为对照（ 图1）。 </li></ol> 3。运行试验</P&gt; <ol><li>相结合的试验化合物和0.5M磷酰基叠氮化物（麻醉剂使用逮捕的蠕虫在到达一个象限）的等体积混合供试品溶液。混合控制解决方案相结合，所用的溶剂稀释试验化合物用0.5 M的叠氮化物。 </li><li>吸取2μl的蠕虫溶液（1.8）编制沉淀到原点（两线相交）。 </li><li>紧接着吸管2微升的两个“T”网站上的测试解决方案。同样地，移液管的控制解决方案的相同数量的两个“C”的网站上。 </li><li>一旦蠕虫和嗅觉下降已在琼脂吸收，更换盖子，翻转板。 </li><li> 60分钟后，将蠕虫病毒在4℃培养箱。只有从孵化器中取出板时，它可以算作。离开蠕虫在室温下，可能让他们再次动员。 </li><li>记录每一个象限中的蠕虫数目，完全越过内保监会文件中。 </li><li>重复使用控制解决方案在测试和控制象限步骤3.2至3.6。这将作为控制板。三板应该和运行服务作为一个适当的控制。 </li></ol> 4。解读比分/确定趋化指数<ol><li>使用公式1计算趋化指数。这将产生一个趋化指数-1.0和+1.0之间。 </li></ol> （1） 趋化指数=（＃蠕虫在两个测试象限 - ＃蠕虫两个控制象限）/（总进球蠕虫） 1.0的比分实现的目标，并表示最大的吸引力，蠕虫抵达含有化学目标象限代表100％。指数-1.0最大排斥的证据。类似的检测方法已经被聘用3名（ 见表1）。 <ol start="2"><li>报告平均ChemotaxiS指数（CI）和标准误差（SEM）。执行一个Student T检验比较的数据，从试验组和对照板。 </li></ol>

<ol>
	<li>Bargmann, C. I., Hartwieg, E., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-selective+genes+and+neurons+mediate+olfaction+in+C.+elegans.">Odorant-selective genes and neurons mediate olfaction in C. elegans.</a> Cell. 74, 515-527 (1993).</li><li>Bargmann, C. I., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemosensory+neurons+with+overlapping+functions+direct+chemotaxis+to+multiple+chemicals+in+C.+elegans.">Chemosensory neurons with overlapping functions direct chemotaxis to multiple chemicals in C. elegans.</a> Neuron. 7, 729-742 (1991).</li><li>Lee, J., Jee, C., McIntire, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+preference+in+C.+elegans.">Ethanol preference in C. elegans.</a> Genes Brain Behav. 8, 578-585 (2009).</li><li>Swierczek, N. A., Giles, A. C., Rankin, C. H., Kerr, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+behavioral+analysis+in+C.+elegans.">High-throughput behavioral analysis in C. elegans.</a> Nat. Methods. 8, 592-598 (2011).</li><li>Ward, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+by+the+nematode+Caenorhabditis+elegans:+identification+of+attractants+and+analysis+of+the+response+by+use+of+mutants.">Chemotaxis by the nematode Caenorhabditis elegans: identification of attractants and analysis of the response by use of mutants.</a> Proc. Natl. Acad. Sci. U.S.A. 70, 817-821 (1973).</li><li>Colbert, H. A., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-specific+adaptation+pathways+generate+olfactory+plasticity+in+C.+elegans.">Odorant-specific adaptation pathways generate olfactory plasticity in C. elegans.</a> Neuron. 14, 803-812 (1995).</li><li>Kauffman, A., Parsons, L., Stein, G., Wills, A., Kaletsky, R., Murphy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+Positive+Butanone+Learning,+Short-term,+and+Long-term+Associative+Memory+Assays.">C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays.</a> J. Vis. Exp. (49), e2490 (2011).</li><li>Troemel, E. R., Kimmel, B. E., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+chemotaxis+responses:+sensory+neurons+define+olfactory+preferences+in+C.+elegans.">Reprogramming chemotaxis responses: sensory neurons define olfactory preferences in C. elegans.</a> Cell. 91, 161-169 (1997).</li><li>Pierce-Shimomura, J. T., Morse, T. M., Lockery, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fundamental+role+of+pirouettes+in+Caenorhabditis+elegans+chemotaxis.">The fundamental role of pirouettes in Caenorhabditis elegans chemotaxis.</a> J Neurosci. 19, 9557-9569 (1999).</li><li>Frokjaer-Jensen, C., Ailion, M., Lockery, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ammonium-acetate+is+sensed+by+gustatory+and+olfactory+neurons+in+Caenorhabditis+elegans.">Ammonium-acetate is sensed by gustatory and olfactory neurons in Caenorhabditis elegans.</a> PLoS One. 3, e2467 (2008).</li><li>Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A., Cer&#243;n, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Caenorhabditis+elegans+Methods:+Synchronization+and+Observation.">Basic Caenorhabditis elegans Methods: Synchronization and Observation.</a> J. Vis. Exp. (64), e4019 (2012).</li><li>Sengupta, P., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+fate+specification+and+differentiation+in+the+nervous+system+of+Caenorhabditis+elegans.">Cell fate specification and differentiation in the nervous system of Caenorhabditis elegans.</a> Dev. Genet. 18, 73-80 (1996).</li><li>Hart, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior.">Behavior.</a> WormBook. , (1895).</li></ol>

我们什么都没有透露。

趋化，虽然控制的一组复杂的神经元和细胞机制，能够容易且客观的定量使用趋化实验。为了获得最佳的检测结果，必须采取某些关键步骤。首先，举办蠕虫产生一致的实验结果是必不可少的。蠕虫在不同的人生阶段有不同的行为13，所以混合阶段蠕虫可能会歪斜实验结果。其次，确保所有E.大肠杆菌洗掉的蠕虫是至关重要的，因为残留的细菌可能会干扰检测。同样重要的是，要注?...

沃德第一次在1973年5开发的趋化实验，并从那时起，它有深远的应用。神经生物学是一个领域，受惠于使用各种趋化实验。 C.嗅觉适应，学习和记忆的一种简单形式，已被证明在线虫趋化实验6。它们也被用来表明C。线虫可以开发乙醇耐受性的结果不仅展现了蠕虫的行为可塑性，但也表明，蠕虫可以是非常有用的，在研究酒精依赖人类3。检测甚至已经发展到展示的能力C.线虫短期和长期记忆存储协会是由趋化因子之间的蠕虫和食品（OP50）7。此外，由于广泛的信息，目前有关C.线虫基因组，chemotaxiC.行为已经改变了无数次的线虫进行基因突变1,8。这使得许多令人兴奋的工程的可能性，如发展C.线虫作为一个生物修复工具。因此，在1973年以来的初步发展的趋化实验，已经频繁地改变，用来阐明多种学科的奥秘。 某些实验旨在发现蠕虫朝着一个目标所采取的具体路线。这种原型试验是由沃德5。三虫熔化的琼脂上放置15分钟。他们的运动跟踪它们留下的印记，因为他们从一个梯度板在板的中心引诱的周边。盘上的所有蠕虫病毒被逮捕用氯仿在每次试验结束。这种方法的一个后裔在板中间放置一个单一的蠕虫的引诱和控制从原点2吨的距离相等，方向相反。 皮尔斯下村等 。开发了一个试验，以观察在趋化9所涉及的移动的确切性质。个人蠕虫放置在9厘米的培养皿中可以含有引诱剂或径向形状的梯度均匀的浓度，最终在源的引诱剂。确认该蠕虫的计算机软件程序，是用来记录所观察到的行为。一种摄像机连接到显微镜一起工作的阶段来调整培养皿中，自动检测运行，以确保保持在视场中的蠕虫。由此看来，更详细的信息被发现的原因下所显示的回旋线虫 。 其他检测，更类似于此处所述，测试一个人口众多的蠕虫病毒测试化合物的反应。二象限趋化实验已经用于探索各种神经细胞，受体和信号转导分子的角色时播放C.线虫暴露于各种化合物1。 20-50之间水洗蠕虫被放置在板的中心附近与引诱剂，麻醉剂，叠氮化钠（NaN 3的）一起在极性端和一个控制。 60分钟后，趋化指数值从-1.0到1.0，产生多少蠕虫贴的引诱剂或控制之间的差异的基础上。类似的趋化指数是用于测定在这篇文章中报道，虽然未能严格评估非能动的蠕虫早期检测。此法进一步趋化测试神经消融的影响。 进行上述测定法的另一种变化，其中含有四个象限3的板的中心被放置在100-200蠕虫。相邻象限包含测试或控制物质。在以前的实验中，蠕虫叠氮化钠的作用被固定之前被拿下。这里描述的类似的方法作为一种评价的反应C.线虫的各种化合物。但是，下面的方法有额外的好处，仅评估已通过阈值距离分离手机动的蠕虫蠕虫。 其他实验已纳入忽略不动的蠕虫类似指引。 frøkjær-Jensen 等 。开发出一种通用的检测，可以用来测试易失性和水溶性化合物10。培养皿分为四个象限。顶部和底部的象限内不含有溶剂。左象限包含水，以及所载的权利，引诱剂。测试时，挥发性的气味中，分析物被放置在盖子上的菜，在适当的象限，而水溶性化合物被直接放置在琼脂糖河目前存在一种方法，用于评估下的趋化反应线虫正在不断地被改进，以优化其便于使用，效率和准确性。所以，虽然这里描述的检测有能力评估的最大数量的蠕虫（最大吞吐量：250的蠕虫/小时每板，略大于由Lee 等人表现出的吞吐量）;这种方法是真正的实力简洁的高潮许多早期检测（ 表1）的属性。

<table><tbody><tr><td>S Basal (- cholesterol) [5.8 g NaCl; 1 M K phosphate buffer, pH 6.0; dH&lt;sub&gt;2&lt;/sub&gt;O to 1 liter.] Autoclave</td><td></td><td></td><td></td></tr><tr><td>PicoFuge</td><td>Stratagene</td><td>400552</td><td></td></tr><tr><td>Microscopes</td><td>Leica</td><td></td><td></td></tr><tr><td>Dissecting</td><td>Leica</td><td></td><td></td></tr><tr><td>P1000 Pipette</td><td>Gilson</td><td></td><td></td></tr><tr><td>P10 Pipette</td><td>Gilson</td><td></td><td></td></tr><tr><td>0.5 M Sodium Azide</td><td></td><td></td><td></td></tr><tr><td>Chemotaxis Agar [1.6% BBL-agar (Benton-Dickinson) or 2% Difco-agar. Autoclave. Add 5 mM potassium phosphate, pH 6.0; 1 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, 1 mM MgSO&lt;sub&gt;4&lt;/sub&gt;]</td><td></td><td></td><td></td></tr><tr><td>Ethanol/Distilled Water</td><td></td><td></td><td></td></tr><tr><td>Test Compounds (eg. 0.5% diacetyl)</td><td></td><td></td><td></td></tr><tr><td>Agar</td><td>Bio-Rad</td><td>166-0600</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>Amresco</td><td>CA97062-046</td><td></td></tr><tr><td>MOPS</td><td>VWR</td><td>CA12001-120</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;OH</td><td>BDH</td><td>CABDH8641-2</td><td></td></tr><tr><td>0.25% Tween 20</td><td>Bio-Rad</td><td>170-6531</td><td></td></tr></tbody></table>

c. elegans chemotaxis assay

许多生物都使用趋化寻求食物来源，避免有毒物质，并寻找配偶。 线虫具有令人印象深刻的趋化行为。 后面测试蠕虫的加臭剂的反应的前提是将它们放置在一个区域，并观察响应的加臭剂诱发的运动。即使有许多可用的检测，优化蜗杆相对于起始位置的控 ​​制和测试区，同时最大限度地减少与对方，同时保持了显着的样本大小的相互作用蠕虫仍然正在进行中的1-10项的工作。这里描述的方法的目的是为解决这些问题，通过修改巴格曼等人开发的测定。甲培养皿分为四个象限，两个相对的象限标记为“测试”和两个被指定为“控制”。麻醉是摆在所有的测试和控制的网站。蠕虫放置在板的中心与精华É围绕原点标记，以确保非能动的蠕虫将被忽略。利用一个四象限系统，而不是一个或两个1消除偏置蠕虫的运动，因为它们是从试验组和对照样品的距离相等，而不管他们开始的哪一侧的起源。这就避免蠕虫被迫穿越其他蠕虫的集群，以应对气味，可延缓蠕虫或迫使他们采取更迂回的路线，其拟定的路径产生一个不正确的解释的问题。这种方法也显示了实用的优点，具有较大样本的大小和允许研究员无人值守运行分析和得分蠕虫一旦在规定的时间已过期。

比较野生型（N2）C. ODR-10（KY10）突变的线虫 。 我们使用的丁二酮，公知的C。线虫趋化双乙酰1,12缺乏受体的突变，比较野生型蠕虫。对于野生型（N2）蠕虫的趋化指数为（0.100±0.066）乙醇和0.839±0.031至0.5％的二乙酰。正如预期的那样，二乙酰引出一个显着chemoattractive的野生型蠕虫（P &lt;0.003）的响应。与此相反，在ODR-10（KY10）突变体缺?...

Watch this Scientific Journal Video about C. elegans Chemotaxis Assay at JoVE.com

C. elegans Chemotaxis Assay

定量评价的趋化反应的方法<em&gt;线虫</em&gt;进行说明。趋化指数（CI）作为一种能够精确地评估蠕虫的反应到一定的目标，并作为一个平台之间的比较利益的菌株和化合物。

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 ...

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

C. elegans Chemotaxis Assay

45.6K 次观看.  Queen's University. 定量评价的趋化反应的方法线虫进行说明。趋化指数（CI）作为一种能够精确地评估蠕虫的反应到一定的目标，并作为一个平台之间的比较利益的菌株和化合物。

视频: C. elegans Chemotaxis Assay

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment through neuron-specific detection of food and chemical odors1, 2, and can associate nutritive states with chemical odors3, temperature4, and the pathogenicity of a food source5.
Here, we describe assays of C. elegans associative learning and short- and long-term associative memory. We modified an aversive olfactory learning paradigm6 to instead produce a positive response; the assay involves starving ~400 worms, then feeding the worms in the presence of the AWC neuron-sensed volatile chemoattractant butanone at a concentration that elicits a low chemotactic index (similar to Toroyama et al.7). A standard population chemotaxis assay1 tests the worms' attraction to the odorant immediately or minutes to hours after conditioning.
After conditioning, wild-type animals' chemotaxis to butanone increases ~0.6 Chemotaxis Index units, its "Learning Index". Associative learning is dependent on the presence of both food and butanone during training. Pairing food and butanone for a single conditioning period ("massed training") produces short-term associative memory that lasts ~2 hours. Multiple conditioning periods with rest periods between ("spaced training") yields long-term associative memory (&lt;40 hours), and is dependent on the cAMP Response Element Binding protein (CREB),6 a transcription factor required for long-term memory across species.8
Our protocol also includes image analysis methods for quick and accurate determination of chemotaxis indices. High-contrast images of animals on chemotaxis assay plates are captured and analyzed by worm counting software in MatLab. The software corrects for uneven background using a morphological tophat transformation.9 Otsu's method is then used to determine a threshold to separate worms from the background.10 Very small particles are removed automatically and larger non-worm regions (plate edges or agar punches) are removed by manual selection. The software then estimates the size of single worm by ignoring regions that are above a specified maximum size and taking the median size of the remaining regions. The number of worms is then estimated by dividing the total area identified as occupied by worms by the estimated size of a single worm.
We have found that learning and short- and long-term memory can be distinguished, and that these processes share similar key molecules with higher organisms.6,8 Our assays can quickly test novel candidate genes or molecules that affect learning and short- or long-term memory in C. elegans that are relevant across species.

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

Here we describe methods to test C. elegans associative learning and short- and long-term associative memory. These population assays employ the worms abilities to chemotax toward volatile odorants, and form positive associations upon pairing food with the chemoattractant butanone. Increasing the number of conditioning periods induces long-term memory.

线虫的积极丁酮学习，短期，长期联想记忆检测

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment ...

科研

神经科学

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

基于微流体Electrotaxis的，为按需定量分析<em&gt;线虫</em&gt;&#39;LOCOMOTION的

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

生物工程

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection of the neural circuits underlying these behaviors, the development of robust and reproducible behavioral assays is necessary. Previous C. elegans behavioral studies have used variations of a "drop test", a "chemotaxis assay", and a "retention assay" to investigate the response of C. elegans to soluble compounds. The method described in this article seeks to combine the complementary strengths of the three aforementioned assays. Briefly, a small circle in the middle of each assay plate is divided into four quadrants with the control and experimental solutions alternately placed. After the addition of the worms, the assay plates are loaded into a behavior chamber where microscope cameras record the worms' encounters with the treated regions. Automated video analysis is then performed and a preference index (PI) value for each video is generated. The video acquisition and automated analysis features of this method minimizes the experimenter's involvement and any associated errors. Furthermore, minute amounts of the experimental compound are used per assay and the behavior chamber's multi-camera setup increases experimental throughput. This method is particularly useful for conducting behavioral screens of genetic mutants and novel chemical compounds. However, this method is not appropriate for studying stimulus gradient navigation due to the close proximity of the control and experimental solution regions. It should also not be used when only a small population of worms is available. While suitable for assaying responses only to soluble compounds in its current form, this method can be easily modified to accommodate multimodal sensory interaction and optogenetic studies. This method can also be adapted to assay the chemosensory responses of other nematode species.

We present a behavior recording setup and protocol that enables automated analysis of the nematode, Caenorhabditis elegans' preference for soluble compounds in a population-based assay. This article describes the construction of a behavior chamber, the behavioral assay protocol, and video analysis software usage.

基于线虫人群的化学感应优选测定的自动分析

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection ...

行为学

<meta charset="utf8"></head><body>秀丽隐杆线虫中挥发性信息素反应的提取和定量

Volatile Sex Pheromone Extraction and Chemoattraction Assay in Caenorhabditis elegans

Chemical communication is vital in organismal health, reproduction, and overall well-being. Understanding the molecular pathways, neural processes, and computations governing these signals remains an active area of research. The nematode Caenorhabditis elegans provides a powerful model for studying these processes as it produces a volatile sex pheromone. This pheromone is synthesized by virgin females or sperm-depleted hermaphrodites and serves as an attractant for males.
This protocol describes a detailed method for isolating the volatile sex pheromone from several C. elegans strains (WT strain N2, daf-22, and fog-2) and C. remanei. We also provide a protocol for quantifying the male chemotaxis response to the volatile sex pheromone. Our analysis utilizes measurements such as chemotaxis index (C.I.), arrival time (A.T.), and a trajectory plot to compare male responses under various conditions accurately. This method allows for robust comparisons between males of different genetic backgrounds or developmental stages. Furthermore, the experimental setup outlined here is adaptable to investigating other chemoattraction chemicals.

<meta charset="utf8"></head><body>作者聚焦：检查挥发性信息素对雄性 秀丽隐杆线 虫行为的影响

This protocol establishes methods for extracting and quantifying responses to the volatile sex pheromone in C. elegans, providing tools to study chemical communication and navigation trajectory.

秀丽隐杆线虫中的挥发性信息素提取和趋化试验

Chemical communication is vital in organismal health, reproduction, and overall well-being. Understanding the molecular pathways, neural processes, and ...

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

ECIS的/出租车细胞趋化测量

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

生物学

Aversive Associative Learning and Memory Formation by Pairing Two Chemicals in Caenorhabditis elegans

The nematode Caenorhabditis elegans is an attractive model organism to study learning and memory at molecular and cellular levels because of the simplicity of its nervous system, whose chemical and electrical wiring diagrams were completely reconstructed from serial electron micrographs of thin sections. Here, we describe detailed protocols for the conditioning of C. elegans by massed and spaced training for the formation of short-term memory (STM) and long-term memory (LTM), respectively. By pairing 1-propanol and hydrochloric acid as conditioned and unconditioned stimuli, respectively, C. elegans was successfully trained to form aversive associative STM and LTM. While na&#239;ve animals were attracted to 1-propanol, the trained animals were no longer or very weakly attracted to 1-propanol. Like in other organisms such as Aplysia and Drosophila, "learning and memory genes" play essential roles in memory formation. Particularly, NMDA-type glutamate receptors, expressed in only six pairs of interneurons in C. elegans, are required for the formation of both STM and LTM, possibly as a coincidence factor. Therefore, the memory trace may reside among the interneurons.

Aversive Associative Learning and Memory Formation by Pairing Two Chemicals in Caenorhabditis elegans

We have previously developed protocols for Caenorhabditis elegans to form short- and long-term associative memories by massed and spaced training, respectively. Here, detailed protocols are described for the conditioning of C. elegans by pairing 1-propanol and hydrochloric acid as conditioned and unconditioned stimuli, respectively, to form aversive associative memory.

通过配对秀丽隐杆线虫中的两种化学物质来形成厌恶联想学习和记忆形成

The nematode Caenorhabditis elegans is an attractive model organism to study learning and memory at molecular and cellular levels because of the ...

Chemotaxis is a process in which cells or organisms move in response to a chemical stimulus. In nature, chemotaxis is important for organisms to sense and move toward food sources and move away from stimuli that may be toxic or harmful. Chemotaxis is also important at the cellular level. For example, chemotaxis is required for the movement of sperm toward an egg prior to fertilization. In the lab, chemotaxis is frequently examined in the nematode, C. elegans, which is known to migrate towards food sources in soil, but away from toxins such as heavy metals, substances with a low pH, and detergents. This video demonstrates how to perform a chemotaxis assay, which includes preparing the chemotaxis plates and the worms, running the assay, and analyzing the data. Then, we discuss examples of how chemotaxis assays can be used in C. elegans as a tool to understand learning and memory, olfactory adaptation, and neurological disease such as Alzheimer&quot;s disease. Chemotaxis experiments in C. elegans have near-limitless possibilities for learning more about the cellular and genetic mechanisms of many biological processes, and may lead to a greater understanding of human biology, development, and disease.

Chemotaxis Assay in Caenorhabditis elegans

线虫的化学趋向性介绍

Chemotaxis is a process in which cells or organisms move in response to a chemical stimulus. In nature, chemotaxis is important for organisms to sense and ...

教育

JoVE Science Education

基础生物学

模式生物I:酵母，果蝇和秀丽线虫

C. elegans Survival Assay: A Short-Term Toxicity Test

Source: Possik, E. and Pause, A. <a href="https://jove.unicartagenaproxy.elogim.com/video/2496/measuring-caenorhabditis-elegans-life-span-96-well-microtiter">Measuring Oxidative Stress Resistance of&#160;Caenorhabditis elegans&#160;in 96-well Microtiter Plates.</a>&#160;J. Vis. Exp. (2015).
This video introduces a short-term survival assay that tests a treatment for acute toxicity or tests a genetic strain for resistance to a toxic condition.&#160; The example protocol shows an example survival assay in which worms are tested for their ability to&#160; resist oxidative stress induced by paraquat (PQ).

秀丽隐杆线虫存活试验:短期毒性试验

Source: Possik, E. and Pause, A. Measuring Oxidative Stress Resistance of&#160;Caenorhabditis elegans&#160;in 96-well Microtiter Plates.&#160;J. Vis. Exp. ...

线虫趋含量的

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此视频中的章节

线虫的积极丁酮学习，短期，长期联想记忆检测

基于微流体Electrotaxis的，为按需定量分析

基于线虫人群的化学感应优选测定的自动分析

秀丽隐杆线虫中的挥发性信息素提取和趋化试验

秀丽隐杆线虫中的挥发性信息素提取和趋化试验

秀丽隐杆线虫中的挥发性信息素提取和趋化试验

ECIS的/出租车细胞趋化测量

通过配对秀丽隐杆线虫中的两种化学物质来形成厌恶联想学习和记忆形成

线虫的化学趋向性介绍

秀丽隐杆线虫存活试验:短期毒性试验