Name: large-scale gravitaxis assay of caenorhabditis dauer larvae
Uploaded: 2022-05-31T15:00:00
Description: Watch this Scientific Journal Video about Large-Scale Gravitaxis Assay of Caenorhabditis Dauer Larvae at JoVE.com

这项研究得到了美国国立卫生研究院对JHR的研究资助（#R01 5R01HD081266和#R01GM141493）。一些菌株由CGC提供，CGC由NIH研究基础设施计划办公室（P40 OD010440）资助。我们要感谢Pradeep Joshi（UCSB）的编辑投入。统计咨询由UCSB DATALAB提供。

本研究中使用的菌株是 秀丽隐杆线虫 （N2）和 C. briggsae （AF16）（见 材料表）。每次测定都使用dauers的混合性别群体。
1. 腔室准备
<ol><li>在通风橱中工作。使用本生燃烧器、1-2 个剃须刀片、钳子、镊子和塑料切割表面设置工作区（见 材料表）。</li>
	<li>对于每个腔室，收集两个 5 mL 血清移液器。用镊子从一个移液器上取?...

<ol>
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D., Eaton, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulus+discrimination+by+the+polymodal+sensory+neuron.">Stimulus discrimination by the polymodal sensory neuron.</a> Commun. Integrative Biology. 6 (2), 23469 (2013).</li><li>Mackowetzky, K., Yoon, K. H., Mackowetzky, E. J., Waskiewicz, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+evolution+of+the+vestibular+apparatuses+of+the+inner+ear.">Development and evolution of the vestibular apparatuses of the inner ear.</a> Journal of Anatomy. 239 (4), 801-828 (2021).</li><li>Eppsteiner, R. W., Smith, R. J. 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与先前方法的比较 与趋化性不同，使用传统的琼脂平板实验设计无法可靠地观察 Caenorhabditis 中的重症。标准培养皿的直径为 150 毫米，因此在任一方向上只有 75 毫米可供 dauers 展示对重力的偏好。尽管秀 丽隐杆线 虫的定向偏好可以在溶液12中测定，但这种方法的通量很低，因为必须一次分析一个蠕虫。此外，漂浮在介质中的蠕虫与在表面上爬行的蠕...

感知地球的引力对于许多生物的方向、运动、协调和平衡至关重要。然而，与其他感官相比，人们对重力感觉的分子机制和神经回路知之甚少。在动物中，重力感觉与其他刺激相互作用，并且可以被其他刺激所取代，从而影响行为。视觉线索、本体感觉反馈和前庭信息可以整合在一起，以产生相对于动物周围环境的身体意识感1，2。相反，引力偏好可以在存在其他刺激的情况下改变3，4，5。因此，引力行为是研究重力感觉和理解神经系统复杂的感觉统合和决策的理想选择。
秀丽隐杆线虫是一种特别有用的模式生物，用于研究重症，因为它具有多酚生命周期。当在发育过程中暴露于压力源时，包括高温、过度拥挤或缺乏食物，秀丽隐杆线虫幼虫会发育成具有高度抗压性的dauers6。作为dauers，蠕虫执行特征性行为，例如nictation，其中蠕虫“站立”尾巴并挥舞头部，这可能有助于扩散到更好的栖息地7。秀丽隐杆线虫和日本梭菌的重力测定表明，dauer幼虫的重力为负，并且这种行为在dauers中比在成虫中更容易观察到8，9。在其他Caenorhabditis菌株中测试重量性可能会揭示重力行为的自然变化。
重力感觉的机制已经在Euglena，果蝇，Ciona和其他各种物种中使用gravitaxis测定3，10，11进行了表征。同时，对Caenorhabditis的妊娠研究最初提供了不同的结果。一项对秀丽隐杆线虫定向偏好的研究发现，蠕虫在溶液中低头定向，表明正重力偏好12。同时，尽管C. japonica dauers很早就被确定为负引力8，但这种行为直到最近才在秀丽隐杆线虫9中得到描述。在蠕虫中开发具有代表性的妊娠测定时出现了一些挑战。Caenorhabditis菌株维持在琼脂平板上;出于这个原因，行为测定通常使用琼脂平板作为其实验设计的一部分13，14，15。最早报道的Caenorhabditis的重症测定是通过将板侧立与水平对照板成90°角来进行的8。然而，在这些条件下，趋重性行为并不总是稳健的。虽然可以在解决方案12中测定成虫的方向偏好，但这种定向偏好也可能与上下文相关，如果蠕虫在爬行而不是游泳，则会导致不同的行为。此外，秀丽隐杆线虫对其他刺激敏感，包括光和电磁场16，17，这些刺激会干扰它们对重力9的反应。因此，一种更新的重症测定法可以抵御其他环境变量，这对于剖析这种感觉过程的机制非常重要。
在本协议中，描述了用于观察妊娠 盲肠炎 的测定。这项研究的设置部分基于一种研究神经肌肉完整性的方法18，19。使用标准程序20培养和分离Dauer幼虫。然后将它们注入由两个装有琼脂的 5 mL 血清移液器制成的腔室中。这些腔室可以垂直或水平定向，并放置在黑暗的法拉第笼内12-24小时，以屏蔽光和电磁场。记录每个蠕虫在腔室中的位置，并与参考菌株（如 秀丽隐杆线虫 N2）的垂直分类进行比较。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1% Sodium Dodecyl Sulfate solution</td>
			<td></td>
			<td></td>
			<td>From stock 10% (w/v) SDS in DI water</td>
		</tr>
		<tr>
			<td>15 mL Centrifuge tubes</td>
			<td>Falcon</td>
			<td>14-959-53A</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mm Hex key</td>
			<td></td>
			<td></td>
			<td>Other similar sized metal tools may be used</td>
		</tr>
		<tr>
			<td>4% Agar in Normal Growth Medium (NGM) - 1 L</td>
			<td></td>
			<td></td>
			<td>Prior to autoclaving: 3 g NaCl, 40 g Agar, 2.5 g Peptone, 2 g Dextrose, 10 mL Uracil (2 mg/mL), 500 &#956;L Cholesterol (10 mg/mL), 1 mL CaCl2, 962 mL DI water; After autoclaving: 24.5 mL Phosphate Buffer, 1 mL 1 MgSO4 (1 M), 1 mL Streptomycin (200 mg/mL)</td>
		</tr>
		<tr>
			<td>5 mL Serological pipettes</td>
			<td>Fisherbrand</td>
			<td>S68228C</td>
			<td>Polystyrene, not borosilicate glass</td>
		</tr>
		<tr>
			<td>60% Cold sucrose solution</td>
			<td></td>
			<td></td>
			<td>60% sucrose (w/v) in DI water; sterilize by filtration (0.45 &#956;m filter). Keep at 4 &#176;C</td>
		</tr>
		<tr>
			<td>AF16 C. briggsae or other experimental strain</td>
			<td></td>
			<td></td>
			<td>Available from the CGC (Caenorhabditis Genetics Center)</td>
		</tr>
		<tr>
			<td>Bunsen burner</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cling-wrap</td>
			<td>Fisherbrand</td>
			<td>22-305654</td>
			<td></td>
		</tr>
		<tr>
			<td>Clinical centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable razor blades</td>
			<td>Fisherbrand</td>
			<td>12-640</td>
			<td></td>
		</tr>
		<tr>
			<td>Faraday cage</td>
			<td></td>
			<td></td>
			<td>Can be constructed using cardboard and aluminum foil; 30&#34; L x 6&#34; W x 26&#34; H or larger</td>
		</tr>
		<tr>
			<td>Ink markers</td>
			<td></td>
			<td></td>
			<td>Sharpie or other brand for marking on plastic</td>
		</tr>
		<tr>
			<td>Labeling tape</td>
			<td>Carolina</td>
			<td>215620</td>
			<td></td>
		</tr>
		<tr>
			<td>M9 buffer</td>
			<td></td>
			<td></td>
			<td>22 mM KH2PO4, 42 mM Na2HPO4, 86 mM NaCl</td>
		</tr>
		<tr>
			<td>N2 C. elegans strain</td>
			<td></td>
			<td></td>
			<td>Available from the CGC (Caenorhabditis Genetics Center)</td>
		</tr>
		<tr>
			<td>NGM plates with OP50</td>
			<td></td>
			<td></td>
			<td>1.7% (w/v) agar in NGM (see description: 4% agar in NGM). Seed with OP50</td>
		</tr>
		<tr>
			<td>Paraffin film</td>
			<td>Bemis</td>
			<td>13-374-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic cutting board</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pliers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rotating vertical mixer</td>
			<td>BTLab SYSTEMS</td>
			<td>BT913</td>
			<td>With 22 x 15 mL tube bar</td>
		</tr>
		<tr>
			<td>Serological pipettor</td>
			<td>Corning</td>
			<td>357469</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Laxco</td>
			<td>S2103LS100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tally counter</td>
			<td>ULINE</td>
			<td>H-7350</td>
			<td></td>
		</tr>
		<tr>
			<td>Thick NGM/agar plate media - 1 L</td>
			<td></td>
			<td></td>
			<td>See 4% Agar in NGM recipe; replace 40 g Agar with 20 g Agar</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

large-scale gravitaxis assay of caenorhabditis dauer larvae

重力感觉是一个重要且相对研究不足的过程。感知重力使动物能够驾驭周围环境并促进运动。此外，发生在哺乳动物内耳的重力感觉与听力密切相关 - 因此，了解这一过程对听觉和前庭研究具有影响。一些模式生物（包括 果蝇）存在重力测定。先前已经对单个蠕虫在溶液中沉淀时的方向偏好进行了测定。然而，尚未描述妊娠 盲肠 炎的可靠和稳健的测定方法。本协议概述了进行重症测定的程序，该程序可用于一次测试数百个 Caenorhabditis dauers。这种大规模、长距离检测允许详细的数据收集，揭示标准板检测中可能遗漏的表型。将沿垂直轴的Dauer运动与水平控制进行比较，以确保方向偏差是由于重力引起的。然后可以在菌株或实验条件之间比较重力偏好。该方法可以确定蠕虫中趋重物的分子、细胞和环境要求。

比较不同物种的重物 按照上述程序，可以将C . briggsae dauer gravitaxis与 秀丽隐杆线虫 和水平对照进行比较。 C. briggsae dauers的垂直分布（栗色）向室顶部倾斜，大部分蠕虫达到+7（图2A）。与水平控制（aqua）相反，其中dauers在腔室中心周围以大致钟形曲线分布，这种趋势表明负重力行为。这些数据可以与在同一实验日进行的秀 丽隐杆线虫<...

Watch this Scientific Journal Video about Large-Scale Gravitaxis Assay of Caenorhabditis Dauer Larvae at JoVE.com

Large-Scale Gravitaxis Assay of Caenorhabditis Dauer Larvae

本协议概述了用 Caenorhabditis dauer幼虫进行大规模重力测定的方法。与基于平板的测定相比，该协议可以更好地检测重症行为。

达乌尔 蝴蝶 幼虫的大规模趋重试验

Gravity sensation is an important and relatively understudied process. Sensing gravity enables animals to navigate their surroundings and facilitates ...

这种高通量测定可用于观察道尔盲肠幼虫的稳健引力行为。与在培养皿上进行的测定相比，在远距离上观察到蠕虫行为。这提高了测定的灵敏度，并允许进行详细的数据分析。研究趋重行为可以深入了解重力感觉在盲肠炎中是如何发生的，这与理解哺乳动物前庭系统有关。创建重力分析室，分离dauers并将蠕虫注射到室中需要练习。您可以通过提前一天隔离dauers并创建腔室来节省时间。首先设置一个本生燃烧器、一到两个剃须刀片、钳子、镊子、塑料切割表面和通风橱。要形成一个腔室，收集两个 5 毫升血清移液器，并用镊子从一个移液器上取下棉塞。用钳子将剃须刀片放在本生燃烧器上，直到变热。使用加热刀片切掉第二个移液器的锥形端，使整个移液器具有均匀的直径。现在，快速将两个改进的移液器末端靠近火焰并稍微融化它们。通过将它们牢固地压在一起来连接这些末端，确保两个移液器的壁是连续的。如果连接后有任何间隙可见，请将它们分开并重复此步骤。根据标准蠕虫程序用4%琼脂制备NGM，并在琼脂仍熔融时将其连接到血清移液器上来填充每个腔室。为了尽量减少由于冷却不均匀而导致琼脂稠度的任何变化，请在吸取琼脂时将移液器与工作台平行。然后慢慢抽取溶液并用封口膜密封吸头，然后从移液器中取出腔室。将腔室平放冷却，让琼脂变硬后再移动。冷却后，将三毫米的六角扳手加热到本生燃烧器上，并通过将其牢固地压入中线一侧约五毫米的每个腔室的壁上来创建一个小塑料开口。接下来，使用加热刀片去除腔室的棉花和锥形端部，并用石蜡膜密封末端。在实验前10至15天，将每种菌株与OP50细菌一起分块到两到三个大的NGM板上，并包裹石蜡膜。通过用M9缓冲液冲洗盖子和板并将溶液移液到15毫升离心管中来收集蠕虫。通过在室温下以 1600 x G 旋转 30 至 60 秒来沉淀蠕虫，并用移液管或真空吸液器吸出大部分 M9 缓冲液。在每个蠕虫颗粒中加入7毫升1%十二烷基硫酸钠溶液，并将蠕虫留在其中30分钟。为了允许曝气，在此期间保持连续旋转管子。然后，用M9冲洗三到五次，以除去洗涤剂。加入五毫升M9后，加入五毫升冷过滤的60%蔗糖溶液。充分混合，并在室温下以 1600 x G 离心五分钟以产生分离梯度。用两毫升M9缓冲液填充新的15毫升离心管。现在压碎玻璃巴斯德移液器的末端以扩大孔。并使用这些移液器将溶液的顶层从蔗糖梯度转移到新管中。用 M9 缓冲液冲洗 dauers 三到五次。在室温下以1600 x G 离心dauers五分钟，并用移液器或真空吸液器吸出大部分M9溶液。接下来，通过在解剖显微镜下手动计数三个单独的 1 微升液滴中的蠕虫数量来估计蠕虫密度，并使用这些计数的平均值来近似每微升的蠕虫数量。现在通过切割 10、20 或 200 微升移液器吸头的末端来加宽吸头孔。将微量移液器的体积设置为略大于预期的吸液体积。吸出约1至2微升的浓蠕虫溶液，并允许少量空气进入尖端。轻轻地将移液器吸头推入琼脂中，同时按下微量移液器。这将创建一个注射部位而不会堵塞尖端。将蠕虫释放到琼脂中，并使用石蜡膜密封开口。在室温下将腔室放置在深色法拉第笼内后，为了消除尽可能多的变量，标记每个腔室并将其垂直悬挂在法拉第笼内。通过在相同的环境条件下平放一些腔室来同时测试水平控制。然后使用垂直定向的N2蠕虫作为阳性对照，使用含有N 2 dauers的水平定向室作为对实验菌株的额外阴性对照。几个小时后，dauer蠕虫开始从起始部位分散，需要几个小时才能到达腔室的两端。在此期间，保持腔室不受干扰，并在注射后 12 至 24 小时内评分。接下来，一次移除一个腔室，并通过在解剖显微镜下寻找活的dauers并用墨水标记它们的位置来对重力进行评分。不要在注射部位两侧 2.5 厘米以内的蠕虫进行评分，因为这些蠕虫不太可能表现出定向偏好。此外，避免对看似死亡或被困在液体中的蠕虫进行评分，并丢弃任何含有超过 50% 死亡或游泳蠕虫的腔室。为了量化结果，使用记号笔将腔室的每一半分成七个3.5厘米，从距离注射部位2.5厘米开始。使用手动计数计数器来统计每个部分中观察到的蠕虫数量。在Briggsae盲肠中，大部分dauer蠕虫显示出向腔室顶部的迁移。然而，水平控制显示围绕腔室中心的分布，表明负重力行为。此外，布里格萨盲肠炎和秀丽隐杆线虫的垂直分布没有显示出任何差异，这表明布里格萨盲肠杆菌表现出类似于秀丽隐杆线虫道尔斯的负重症行为。该方案不使用麻痹剂，例如叠氮化钠。因此，重要的是在将重力室从法拉第笼中取出后立即对其进行评分。该测定导致在秀丽隐杆线虫中发现了负的趋重行为。通过测试几种突变菌株，它还揭示了蠕虫对妊娠的遗传和神经需求。

Research

JoVE Journal

Behavior

Large-Scale Gravitaxis Assay of Caenorhabditis Dauer Larvae

Asymmetric Walkway: A Novel Behavioral Assay for Studying Asymmetric Locomotion

非对称走道：一种新的行为检测方法的研究不对称步态

Behavioral assays are commonly used for the assessment of sensorimotor impairment in the central nervous system (CNS). The most sophisticated methods for ...

科研

行为学

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

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 ...

Profiling Anti-Neu5Gc IgG in Human Sera with a Sialoglycan Microarray Assay

使用唾液酸多糖微阵列测定在人血清中分析抗Neu5Gc IgG

Cells are covered with a cloak of carbohydrate chains (glycans) that is commonly altered in cancer and that includes variations in sialic acid (Sia) ...

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

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

A Burrowing/Tunneling Assay for Detection of Hypoxia in Drosophila melanogaster Larvae

A Burrowing/Tunneling Assay for Detection of Hypoxia in Drosophila melanogaster Larvae

用于检测果蝇幼虫缺氧的挖洞/隧道法

Oxygen deprivation in animals can result from exposure to low atmospheric oxygen levels or from internal tissue damage that interferes with oxygen ...

Swimming Induced Paralysis to Assess Dopamine Signaling in Caenorhabditis elegans

Swimming Induced Paralysis to Assess Dopamine Signaling in Caenorhabditis elegans

游泳诱导麻痹对线虫多巴胺信号的评价

The swimming assay described in this protocol is a valid tool to identify proteins regulating the dopaminergic synapses. Similar to mammals, dopamine (DA) ...

Calcium Imaging in Freely Behaving Caenorhabditis elegans with Well-Controlled, Nonlocalized Vibration

Calcium Imaging in Freely Behaving Caenorhabditis elegans with Well-Controlled, Nonlocalized Vibration

自由行为 秀丽隐杆线虫 的钙成像，具有良好控制，非局部化振动

Nonlocalized mechanical forces, such as vibrations and acoustic waves, influence a wide variety of biological processes from development to homeostasis. ...

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

测量 果蝇 拉瓦的机械结节的改进的检测和工具

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila ...

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

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

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 ...

A High Throughput Microplate Feeder Assay for Quantification of Consumption in Drosophila

A High Throughput Microplate Feeder Assay for Quantification of Consumption in Drosophila

用于果蝇消费量化的高吞吐量微板馈线分析

Quantifying food intake in Drosophila is used to study the genetic and physiological underpinnings of consumption-associated traits, their environmental ...

达乌尔 蝴蝶 幼虫的大规模趋重试验

达乌尔蝴蝶幼虫的大规模趋重试验