这项工作得到了加拿大自然科学与工程研究委员会发现授权RGPIN36481-08给William G. Bendena的支持。

1.实验生物的制备<ol><li> 在开始测定前24小时挑选每个菌株10只L4分期线虫，以确保生物体是年轻成年人。对于每个突变体或对照线虫，选择10个L4（对照为10个，测定为10个）。 <ol><li>维持使用标准方法26，在标准琼脂平板27 24小时与OP50 大肠杆菌接种L4生物体。如果在随后的洗涤步骤中生物体丢失，则通过增加起始样品量来补偿（ 即挑选20个蠕虫而不是10个）。 注意:行为是一种天生变异的表型。在三个不同的日子对每个菌株执行一式三份的方法。包括新的菌株的附加控制菌株和条件，如讨论部分所强调的。 </li></ol></li><li> 在测定之前，转移实验组织在没有细菌的琼脂平板上，允许线虫自由移动1分钟以除去多余的细菌。 <ol><li>如果实验生物在测定前24小时内发生污染状况，请丢弃。 </li></ol></li><li>移取1 mL M9到无菌板上，将蠕虫洗至微量离心管中。 </li><li>以3,000 xg离心1分钟。蠕虫应在管的底部形成颗粒。吸出M9溶液而不破坏蠕虫颗粒。向蠕虫颗粒中加入1 mL M9，将反应管与溶液混合。 </li><li> 重复步骤1.4三次。 <ol><li>如果过多的细菌最初用蠕虫转移，重复总共5次。不要将细菌转移到铜食品赛道板上。如果将食物转移到测定板上，则会干扰准确的数据收集。 </li></ol></li><li>最终洗涤后，吸出上清至10081; M的M和蠕虫颗粒残留。 注意:饥饿相关行为30分钟后变得明显。因此，一旦洗涤步骤完成，蠕虫应立即从溶液中转移。 </li></ol> 2.测定板的制备<ol><li> 在测定前两天准备标准的NGM琼脂平板。 <ol><li>如果将板保持在潮湿的环境中，在测定前3天制备琼脂板。或者，取下板的盖子3-6小时以确保适当的干燥（如果在无菌环境中）。 </li></ol></li><li>使用厚的永久标记，沿着外边缘在板的下侧形成一条线，另一条形成中线屏障（ 图1 ）。中线屏障应与板的每个边缘等距。使用标尺来确保精确的测量。这些生产线将作为传递细菌和铜溶液的指南。只要那个大肠杆菌在铜溶液前转移，这些线将作为指标。 </li><li> 种子板与50μL的OP50 大肠杆菌仅在铜屏障的一侧形成均匀的草坪（ 图2 ）。细菌浓度在测定中应保持一致;然而，随着浓度的轻微差异，观察到很小的变异性。 <ol><li>使用板下面的标记线，以确保细菌不与铜溶液接触。只要铜溶液排列在板的边缘并形成中线屏障，转移细菌使得铜溶液不会与食物源接触。 </li></ol></li><li>标记第二组平板，不转移OP50 大肠杆菌 （ 图2 ）。这些板块将用于评估阴性对照。这些盘子也应该有一个标记在板的一半上，以表示初始转移来源。 </li><li>允许细菌干燥，然后在37℃孵育平板过夜。确保转移到37°C的孵化器或房间时细菌斑块不会受到干扰。过度的干扰可能会改变食物贴片的位置或形状。 </li></ol>趋化因子测定<ol><li>在测定开始时间前新鲜制备0.5M硫酸铜（II）溶液。如果每个板使用125μL的溶液，则根据使用的测定板的数量（ 例如 5个测定板，625μL）来放大该体积。 </li><li>吸取100μL硫酸铜（II）溶液在琼脂边缘，以产生外部铜屏障。铭牌的下侧应作为指导。 </li><li> 移取25μL硫酸铜（II）溶液以产生中线屏障。 <ol><li>确保硫酸铜（II）溶液不接触细菌贴片。使用斑点技术可能会因为琼脂上的凹痕/划痕而影响运动。 </li></ol></li><li>让铜溶液干燥到板上。时间段可能因板材和实验室条件而异。转移后每5分钟检查干燥。 注意:铜溶液显示蓝色色调，易于识别。使用实验室组织轻轻地吸取板边缘附近的溶液，以辨别干燥。 </li><li> 将20μL蠕虫颗粒从管底移取到无细菌检测板的一半。 <ol><li>确保将10个蠕虫转移到测定板上。如果意外包含额外的蠕虫，请用卤代烃油取出，以确保没有细菌添加到板上。在测定期间，每个测定应具有一致数量的线虫。 注意:如果传染虫数太少，在测定过程中发生错误，增加初始样品量将减轻洗涤和转移期间的潜在损失。 </li></ol></li><li>用实验室组织从板上清除多余的M9。 M9不应与硫酸铜（II）溶液接触。 注意:确保蠕虫和琼脂表面在执行此步骤时不受影响。如果使用过度苛刻，实验室组织可能会对板的琼脂表面产生压痕，并可以去除蠕虫。应该丢弃通过 KimWipe意外删除的蠕虫。 </li><li> 一旦M9溶液被去除并且所有的蠕虫已经开始非液体运动模式，启动测定秒表。 <ol><li>最佳地，在一分钟内取出M9溶液。基本参数是识别正弦运动。蠕虫在液体中不同， 例如颠簸而不是正弦波。每次实验器官开始测定停止观察战争停止颠簸。 </li></ol></li><li> 每30分钟检查一次测定板。 <ol><li>对于具有细菌斑块的测定板，如果在4小时内到达食物贴片，则对生物有积极的作用。对于阴性对照板，如果他们已经越过障碍物，则对生物有积极的作用。 </li></ol></li></ol>

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我们没有什么可以披露的。

该测定设计修改了食物种族测定24，以包括铜溶液以产生厌恶的中线屏障并围绕板的边缘以防止线虫的丢失。测试生物体在四小时内穿过障碍物并达到食物补丁的能力。在npr-9（GF）的上下文中，我们利用该测定来评估饥饿条件如何影响厌食反应和食物检测。如果我们以前将npr-9（GF）表征为对食物和厌恶线索的反应性有缺陷，我们将多个环境线索与营养状况相结合，...

秀丽隐杆线虫已经被用作研究神经生物学数十年的模型，因为分析仅由302个神经元组成的神经系统的电路相对容易1 。只要生物依赖于环境线索的响应，大部分神经系统都致力于调节环境信号的整合2 。尽管神经系统的简单， C.线虫可以检测和响应各种环境信号，包括驱避剂3 ，引诱剂4 ，温度5 ，甚至湿度6 。正确地整合环境信号的故障一直挂在哺乳动物模型系统7-9一些行为障碍和神经退行性条件。有一系列可用的神经疾病模型<html> 在线虫中的 10种和线虫药物筛选11的发展，这种生物体已被证明是用于研究神经生物学的有用系统。鉴于线虫连接群1的可用性和线虫基因组12中几乎每个基因的突变，我们对线虫神经系统的理解以及我们自己的扩展都受到创造性地适合测定的设计的限制。 一些趋化实验已经开发在过去40年的线虫响应评估，以多样化的厌恶刺激3，4，13，14，15。初步实验涉及引入急性环境刺激，而单个蠕虫在琼脂板上漫游= "外部参照"&gt; 3，14，16。记录了对运动反应的立即改变。例如，可以将挥发性气味辛醇施用于头发，并在线虫鼻前摆放，以刺激野生型蠕虫17中向后运动的开始。更复杂的实验也已经发展到包含多个变量作为评估行为选择18的手段。该测定的变化需要使用铜溶液以产生厌恶的中线屏障4 。引诱剂，即二乙酰基，被放置在化学屏障的一侧，蠕虫从二乙酰基源转移出去。蠕虫对铜的厌恶反应有缺陷，马上越过障碍达到二乙酰，而野生型蠕虫最初被屏障排斥。当蠕虫第一次接近铜屏障时，响应得分没有长期观察。 蠕虫在经历饥饿状况后进行评估，对环境刺激的敏感性降低19 。当厌恶的化学辛醇在线虫鼻前飘荡时，野生型生物体在食物中会在3-5秒内刺激向后的运动。在这些生物从食物中取出10分钟后，它们的延迟反应为8 - 10秒20 。因此，随着饥饿的增加，线虫对有害环境信号的反应显示出减少的反应，因为对食物的追求变得对生存更为重要。相反，过表达神经肽受体9（ npr-9）的线虫对食物上或食物上的辛醇没有反应，并且表现出无法应对许多厌恶刺激21 。这些npr-9（GF）生物体也不会在食物存在的情况下调节其逆转频率，但可以反对刺激刺激的刺激表示他们能够向后运动21 。鉴于它们表现出异常下降的食物反转频率，但可以在食物21的情况下调节其行为，因此我们还评估了npr-9（LF）突变体。耦合引进急性外界刺激的蠕虫的营养状况阐明由食品相关的途径大致可以调节感官信号传导机制已经资助途径22，23。食物在线虫环境中的存在也被用于评估乙醇提取反应24 。在该实验中，将蠕虫在不同浓度的乙醇中孵育，然后置于具有被称为"食物种族测定"的食品的琼脂平板上。食物贴片放置在盘子的一个边缘，而线虫w远离食物来源。通过测量蠕虫到达食物块所需的时间来评估乙醇抽出。 这种基于营养的铜避免测定建立在食物种族测定的基础上，以整合额外的环境变量，即食物和铜，同时评估随时间推移的行为变化。这是整个线虫群落中常用协议的改编4 。该方案已被用于评估厌恶反应和食物在四小时内的检测21 。由于蠕虫在食物剥夺30分钟后出现饥饿行为25 ，我们还可以评估营养状况的变化如何影响环境反应。该测定的条件测量实验生物随时间改变对厌恶刺激的反应性，因此评估行为变化生物进入饥饿状态（并持续测量长时间的饥饿）。由于npr-9（GF）动物不会改变他们对食物或许多厌恶线索的行为，我们试图确定这些行为缺陷是否会在饥饿的背景下持续存在。最终，该测定设计已经被配制为特异性地评估npr-9（GF）突变体，但是可以进一步适用于表征新菌株。

<table border="0" cellpadding="0" cellspacing="0" width="689">
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			<td height="71" style="height:71px;width:332px;">M9 Solution [3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O to 1 litre. Autoclave to sterilize before use.]</td>
			<td style="width:71px;"></td>
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			<td style="width:167px;">Produced in lab</td>
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			<td height="84" style="height:84px;width:332px;">Cupric Sulfate</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">C-1297</td>
			<td style="width:167px;">Use water to appropriately suspend to a concentration of 0.5M</td>
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a caenorhabditis elegans nutritional-status based copper aversion assay

为了确保生存，生物必须能够避免不利的栖息地，同时确保一致的食物来源。 秀丽隐杆线虫在检测到各种环境刺激后改变其运动模式，并可以调整其对饥饿状况的行为反应套件。当从食物源中取出超过30分钟时，线虫通常表现出降低的反应反应。观察响应于不断变化的营养状况的行为变化可以提供对调节从喂养到饥饿状态的过渡的机制的了解。 我们已经开发了一种测定线虫测量过渡障碍（ 即铜），然后在长时间内达到食物来源的能力。该协议基于以前的工作，通过以允许随着生物向a转移的持续数据收集的方式整合多个变量来构建n越来越饿了。此外，该测定允许增加的样品量，使得可以同时评估较大的线虫种群。 检测或响应铜的能力的生物有机体立即穿越化学屏障，野生型线虫最初被排斥。随着野生型蠕虫越来越缺乏，它们开始越过障碍并达到食物来源。我们设计了这种检测方法来评估一种不能应对各种环境线索的突变体，包括食物感觉或厌恶性化学物质的检测。当通过该方案进行评估时，有缺陷的生物体立即越过障碍物，但也不能检测食物来源。因此，尽管临时达到食物来源，这些突变体重复地跨越化学屏障。该测定可以直接测试蠕虫群体，以评估与厌恶和饥饿相关的潜在途径缺陷。

我们利用野生型（N2）， npr-9（tm1652）和npr-9过表达菌株， 即 npr-9（GF） （IC836- npr-9 :: npr-9; sur-5 :: gfp; odr -1 :: rfp），以评估对饥饿和铜厌恶的反应。野生型生物体能够检测并响应于所述厌恶铜阻挡，而NPR-9（GF）的突变体不发起超过4小时测定21至铜厌恶响应。饥饿30分钟后，大约50-60％的野生型（N2）生物体穿过铜?...

Watch this Scientific Journal Video about A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay at JoVE.com

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

一个<em秀丽隐杆线虫</em基于营养状态的铜反转测定

在这里，我们提出了一种秀丽隐杆线虫特异性测定法，用于评估铜厌恶行为的变化和定位常见食物来源的能力，因为生物体从充足的营养状态进入饥饿的营养状态。

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their ...

a-caenorhabditis-elegans-nutritional-status-based-copper-aversion

Research

JoVE Journal

Neuroscience

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

6.4K 次观看.  Queen's University. 在这里，我们提出了一种秀丽隐杆线虫特异性测定法，用于评估铜厌恶行为的变化和定位常见食物来源的能力，因为生物体从充足的营养状态进入饥饿的营养状态。 

视频: A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

突触荧光自动定量<em&gt; C。线虫</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

科研

神经科学

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

一<em&gt;线虫</em&gt;研究Amylopathy模型系统

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

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.  ...

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.

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 ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

琼脂糖微室的长期钙成像<em&gt;秀丽隐杆线虫</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

一个行为分析对机械感觉MARCM为基础的克隆<em&gt;果蝇</em

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. Embryonic elongation of the nematode Caenorhabditis elegans is a late developmental stage consisting of the elongation of the embryo along its longitudinal axis. This developmental stage is controlled by intercellular communication between hypodermal cells and underlying body-wall muscles. These signaling mechanisms control the morphology of hypodermal cells by remodeling the cytoskeleton and the cell-cell junctions. Measurement of embryonic lethality and developmental arrest at larval stages as well as alteration of cytoskeleton and cell-cell adhesion structures in hypodermal and muscle cells are classical phenotypes that have been used for more than 25 years to dissect these signaling pathways. Recent studies required the development of novel metrics specifically targeting either early or late elongation and characterizing morphogenic defects along the antero-posterior axis of the embryo. Here, we provide detailed protocols enabling the accurate measurement of the length and the width of the elongating embryos as well as the length of synchronized larvae. These methods constitute useful tools to identify genes controlling elongation, to assess whether these genes control both early and late phases of this stage and are required evenly along the antero-posterior axis of the embryo.

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo.

新颖的指标来表征线虫胚胎伸长率<em&gt;秀丽隐杆线虫</em

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

比率钙显像对单个神经元的行为线虫线虫

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic spines have been described for DD GABAergic neurons as the input sites from presynaptic cholinergic neurons in the motor circuit of Caenorhabditis elegans. This synaptic circuit can now serve as a powerful new in vivo model of spine morphogenesis and function that exploits the facile genetics and ready accessibility of C. elegans to live-cell imaging. 
This protocol describes experimental strategies for assessing DD spine structure and function. In this approach, a super-resolution imaging strategy is used to visualize the intricate shapes of actin-rich dendritic spines. To evaluate the DD spine function, the light-activated opsin, Chrimson, stimulates the presynaptic cholinergic neurons, and the calcium indicator, GCaMP, reports the evoked calcium transients in postsynaptic DD spines. Together, these methods comprise powerful approaches for identifying genetic determinants of dendritic spines in C. elegans that could also direct spine morphogenesis and function in the brain.

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are important cellular features of the nervous system. Here live imaging methods are described for assessing the structure and function of dendritic spines in C. elegans.&#160;These approaches support the development of mutant screens for genes that define dendritic spine shape or function.

秀丽隐杆线虫树突棘成像

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic ...

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed orientation. Here we describe methods to label, image, and analyze the microtubule dynamics and growth during the development and regeneration of touch neurons in C. elegans. Using genetically encoded fluorescent reporters of microtubule tips, we imaged the axonal microtubules. The local changes in microtubule behavior that initiates axon regeneration following axotomy can be quantified using this protocol. This assay is adaptable to other neurons and genetic backgrounds to investigate the regulation of microtubule dynamics in various cellular processes.

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

A protocol for imaging the dynamic microtubules in vivo using fluorescently labeled End binding protein has been presented. We described the methods to label, image, and analyze the dynamic microtubules in the Posterior lateral microtubule (PLM) neuron of C. elegans.

在体内卡诺哈布德炎电子神经元微管动力学和方向评估

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed ...