这项工作是由美国国立卫生研究院临床科学家发展奖K08NS002083，一个Shervert弗雷泽研究所格兰特和NARSAD青年研究者奖，以埃德加A.巴特纳支持。

1。发展迟缓/致命偷袭含量：野生型（N2）和两个突变（MUT1和MUT2）菌株在12孔板测试三中氯氮平浓度<ol><li>在第1天，倒入2毫升NGM培养基中10到12孔板的各孔中（每孔用直径2厘米），并允许过夜变硬的 ​​长凳上，在室温（RT）。同一天，挑大肠杆菌 OP50细菌菌落，感染了一瓶50毫升LB培养基溶液，它的文化在37℃摇床以220rpm旋转过夜。 </li><li>第2天，转移20微升OP50细菌培养到中心各NGM的好。培养板在37℃培养箱中或将它们在室温下过夜，使细菌草坪生长厚。 </li><li>通过在DMSO（二甲亚砜）的溶剂中溶解氯氮平作出80mM的氯氮平原液。然后作出80微升工作液用稀释的1.7 mM的醋酸b将原液ASED于表1中 。 注 ：感兴趣的特定药物的工作液必须通过做一系列的初步浓度测试，以找到最合适的浓度从区分突变体与野生型而定。 </li><li>转移80微升氯氮平工作溶液到种子的NGM板孔和旋动板以使溶液均匀地分布在表面上。等待该板干燥。使用板测定在同一天或将其放置在20℃培养箱中培养，第二天使用。 注 ：工作液是一种药物悬浮液由于氯氮平的低溶解度，尤其是在较高浓度。因此，将所述溶液的药物板，以保证药物浓度是准确之前旋涡振荡管。 </li><li>洗蠕虫关闭包含许多妊娠成人M9的缓冲区3.5公分板，然后旋转下来，在一个15毫升管在2000 rpm离心1分钟。 </li><li>吸出上清液。加入5 ml漂白剂溶液（氢氧化钠：次氯酸钠：双蒸2 O为4:1:5），并轻轻倒转试管破坏线虫。 </li><li>观察动物，直到其中一半溶解在大约5分钟。降速卵在2000 rpm下离心1分钟。 </li><li>吸出上清液，并添加14毫升M9缓冲液迅速。 </li><li>降速卵在2000 rpm下离心1分钟，吸出上清液，留下约100μl的溶液。旋涡暂停鸡蛋。 </li><li>在M9上暂停定期NGM板测试多少个蛋被转移转移2微升鸡蛋。调整悬浮卵的体积，以确保有大约30-35鸡蛋中的每个传输。转让30-35鸡蛋到分析板的每一个孔，然后孵育在20℃培养箱中培养24小时。 </li><li>在测定法的第一天，得分卵孵化的数目。调整阴影动物的总数由picki到25/well纳克去多虫如果必要的。 </li><li>在接下来的日子里，观察动物和它们的得分，每24小时。发育阶段是根据动物的大小和外阴11的形状进行评分。实验持续5-6天，最强大的效应通常出现在第三或第四天。 </li><li>输入的数据转换成一个Excel文件，并计算动物在每个发育阶段的百分率为实验的每一天。生成图表的功能&#39;100百分比堆积柱形图“在&#39;2-D列”菜单。 </li></ol> 2。咽抽水含量：年轻的成年动物的野生型和突变菌株进球对氯氮平检测板<ol><li>测定板是由下列用于发育延迟/致死率试验相同的协议。 </li><li>挑选50 L4动物对每个菌株接种NGM板24小时的检测前和孵化的蠕虫病毒在20°C </li><li>挑选10只动物到ASSAY板好。然后挑选10只动物到下一个孔每次15-20分钟。 </li><li>后在第一阱动物已暴露于药物30分钟后，通过观察咽泵启动测定解剖显微镜下和得分的泵，持续20秒9。一旦咽抽水是得分，挑虫脱盘。 </li></ol>

<ol>
	<li>Donohoe, D. R., Jarvis, R. A., Weeks, K., Aamodt, E. J., Dwyer, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+adaptation+in+C.+elegans+produced+by+antipsychotic+drugs+requires+serotonin+and+is+associated+with+calcium+signaling+and+calcineurin+inhibition.">Behavioral adaptation in C. elegans produced by antipsychotic drugs requires serotonin and is associated with calcium signaling and calcineurin inhibition.</a> Neurosci. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> C. elegans feeding. WormBook, ed. The C. elegans Research Community, doi:10.1895/wormbook.1.150.1. , (2012).</li><li>Chaudhuri, J., Parihar, M., Pires-daSilva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+introduction+to+worm+lab:+from+culturing+worms+to+mutagenesis.">An introduction to worm lab: from culturing worms to mutagenesis.</a> J Vis Exp. 47, (2011).</li><li>Lewis, J. A., Fleming, J. T., Estein, H. F., Shakes, D. 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> Carnorhabditis elegans Modern Biological Analysis of an Organism. 48, 3-29 (1995).</li><li>Davis, J. R., Li, Y., Rankin, C. 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作者宣称，有没有涉及利益冲突。

在这里，我们描述了用于测试的APD对C的发展和行为的影响的方法线虫 。 DMSO或乙醇来溶解氯氮平，因为药物是相对不溶于水。因为溶剂已报道影响C.线虫生物学12，DMSO单独或乙醇单独对照组是必不可少的。 DMSO在我们的实验中使用的最高浓度可达3％，这不会对下一个明显的效果线虫的发展。 DMSO可用于许多小分子，或什至一些大的分子， 例如脂氨?...

秀丽隐杆线虫是一种简单的遗传生物经得起大规模的正向和反向遗传筛选和化学遗传筛选。C。线虫是一个广泛的生物活性化合物的光谱敏感，并因此被成功地用于定义各种这样的化合物的作用机制。例如，生物活性物质利用蠕虫药理学包括乙酰胆碱受体激动剂（ 如左旋咪唑，尼古丁，噻烯氢嘧啶，噻嘧啶和），麻醉药（ 如氟烷），咖啡因，胆碱酯酶抑制剂（ 如涕灭威，lannate和敌百虫），氟化物，GABA相关研究化合物（ 如 γ-氨基丁酸和 ​​蝇蕈醇），伊维菌素，百草枯，佛波醇酯和五羟色胺有关的药物（ 如血清素和imiprimine）3。此外，C。线虫已经用于大型的小分子的屏幕，允许发现新的生物活性化合物s和鉴定新基因目标4。 该三线虫基因组包括潜在的抗精神病药物（APD）保守在人类的目标，包括基因编码所需的神经递质的合成和突触的结构和功能5蛋白。因此，C。线虫神经遗传学和神经生物学报价方法发现的APD作用的新的分子机制。线虫，APD的曝光处于发展初期产生发育迟缓，并在较高的浓度，杀伤力2,6。成年期的APD曝光产生的行为表型。例如，氯氮平曝光抑制运动和咽抽水，提高产蛋1,2,7。 APD引起的发育迟缓和杀伤力是强大的表型为大型化工遗传筛选。这些表型是复杂的，只要它们可能有一个以上的细胞和遗传巴斯秒。因此，这样的遗传筛选预期产生的各种间接的药物靶点。然而，我们的实验室已进行候选基因的屏幕和全基因组RNAi筛选为APD引起发育迟缓和杀伤力抑制器，并已成功恢复基因，可能编码直接目标，包括多巴胺，胰岛素和烟碱乙酰胆碱受体2,8。的基础上，成人APD诱导行为遗传筛选也导致了新的APD目标的确定，以及我们现在正在从哺乳动物7两个发育和行为屏幕验证目标。因此，无脊椎动物化学遗传学方法发现的APD作用的新的分子机制似乎是可行5,8。 该三线虫咽的是，包括20个神经元，20肌细胞，和20辅助细胞，包裹由基底膜的器官。类似于哺乳动物的心脏，咽是一个自治第二不断泵食品在来自外部环境的9。咽抽水率抑制妥协食物的摄取，从而突变或药物抑制咽抽水引起发育迟缓或逮捕9。 APD的抑制咽抽水率，占部分为他们的发展和生存能力1,2效果。在这里，我们用非典型APD氯氮平作为一个例子来证明药物检测线虫发育和咽抽水。

<table border="0" cellpadding="0" cellspacing="0" width="406">
	<tbody>
		<tr height="22">
			<td>Clozapine</td>
			<td>Sigma</td>
			<td>C6305</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">D8418</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Acetic acid (HAC)</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">BP2401</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Sodium hydroxide(NaOH)</td>
			<td style="width:71px;">EMD</td>
			<td style="width:119px;">SX0590-13</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Hypochlorite</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">425044</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:236px;">Centrifuge 5810</td>
			<td style="width:188px;">Eppendorf</td>
			<td style="width:200px;">5810 000.017</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:236px;">Incubator shaker</td>
			<td style="width:188px;">New Brunswick Scientific</td>
			<td style="width:200px;">M1246-0006</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:236px;">Low temperature incubator 815</td>
			<td style="width:188px;">Precision Scientific</td>
			<td style="width:200px;">J1790-1B</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:236px;">Stereo microscope</td>
			<td style="width:188px;">Olympus</td>
			<td style="width:200px;">SZX12</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:236px;">Petri dish 35 x 10 mm</td>
			<td style="width:188px;">Fisher Scientific</td>
			<td style="width:200px;">NC9434271</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:236px;">12-well tissue culture plate</td>
			<td style="width:188px;">BD Falcon</td>
			<td style="width:200px;">REF 353043</td>
		</tr>
	</tbody>
</table>

methods for studying the mechanisms of action of antipsychotic drugs in caenorhabditis elegans

秀丽隐杆线虫是一种简单的遗传生物经得起大规模的正向和反向遗传筛选和化学遗传筛选。该三线虫基因组包括潜在的抗精神病药物（APD）保守在人类的目标，包括基因编码所需的神经递质的合成和突触的结构和功能的蛋白质。 APD曝光产生以浓度依赖的方式发育迟缓和/或致死的线虫。这些表型引起的，部分是由APD-诱导抑制咽泵1,2的。因此，发展型有神经肌肉的基础上，使其成为有用的抗精神病药的药理学研究。在这里，我们展示了详细的程序，用于测试对线虫发育和咽抽水APD的影响。对于发育检测，同步胚胎放在线虫生长培养基含有的APD（NGM）板，和ANI发展的阶段MALS，然后每天进行评分。对于咽抽水速率法，上演年轻的成年动物都含有APD的NGM板测试。每单位时间的咽泵的数目被记录下来，并且该泵送率的计算方法。这些测定可用于研究许多其他类型的小分子，甚至大的分子。

1。发育迟缓/杀伤力测定结果一个典型的结果为发育延迟/致死率试验中演示了图1a和1b。当对照组野生型动物已发展到了妊娠成年阶段（ 图1a），暴露于氯氮平野生型动物仍然在年轻的幼虫阶段或已经死了（ 图1b）。图1c显示比较具有代表性的结果抑制基因的突变体（MUT1）和增强子的突变体（MUT2）与野生型在三个?...

Watch this Scientific Journal Video about Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans at JoVE.com

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

方法用于测试的抗精神病药物（APD）提供在秀丽隐杆线虫的效果表现出来。分析描述了用于测试的发展和生存能力和咽抽水率药物作用。这些方法也适用于药理学实验与比APD的其他类药物。

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.  ...

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Neuroscience

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

11.4K 次观看.  Harvard Medical School. 方法用于测试的抗精神病药物（APD）提供在秀丽隐杆线虫的效果表现出来。分析描述了用于测试的发展和生存能力和咽抽水率药物作用。这些方法也适用于药理学实验与比APD的其他类药物。 

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

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

在子宫内电穿孔：鼠标控制时空基因Transefection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

科研

神经科学

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

啮齿动物的中枢神经系统药物直接脑室交货

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

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 ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

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 ...

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. ...

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their locomotory patterns upon detection of diverse environmental stimuli and can modulate their suite of behavioral responses in response to starvation conditions. Nematodes typically exhibit a decreased aversive response when removed from a food source for over 30 min. Observation of behavioral changes in response to a changing nutritional status can provide insight into the mechanisms that regulate the transition from a well-fed to starved state.
We have developed an assay that measures a nematode&#39;s ability to cross an aversive barrier (i.e. copper) then reach a food source over a prolonged period of time. This protocol builds upon previous work by integrating multiple variables in a manner that allows for continued data collection as the organisms shift towards an increasingly starved condition. Moreover, this assay permits an increased sample size so that larger populations of nematodes can be simultaneously evaluated.
Organisms defective for the ability to detect or respond to copper immediately cross the chemical barrier, while wild type nematodes are initially repelled. As wild type worms are increasingly starved, they begin to cross the barrier and reach the food source. We designed this assay to evaluate a mutant that is incapable of responding to diverse environmental cues, including food sensation or detection of aversive chemicals. When evaluated via this protocol, the defective organisms immediately crossed the barrier, but were also incapable of detecting a food source. Hence, these mutants repeatedly cross the chemical barrier despite temporarily reaching a food source. This assay can straightforwardly test populations of worms to evaluate potential pathway defects related to aversion and starvation.

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

Here, we present a Caenorhabditis elegans-specific assay designed to evaluate changes in copper aversion behavior and the ability to locate a common food source, as the organism progresses from a well-fed to starved nutritional state.

一个<em秀丽隐杆线虫</em基于营养状态的铜反转测定

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

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 ...