我们要感谢帮助开发协议，在这里，特别是杜松子酒Fonte酒店，谁开发的原程序的链接实验室的现任和前任成员。这项工作已经从美国国立卫生研究院（AG12423）和阿尔茨海默氏症协会（ZENITH奖）的奖项，以民盟的支持

1）准备瘫痪检测线虫生长介质板。 麻痹检测执行标准线虫的生长介质（NGM）常规的 C板线虫的繁殖和遗传学（Stiernagle 2006年）。 <ol><li>高压灭菌的NGM的溶液中含有氯化钠，蛋白胨琼脂和一个锥形瓶中，并添加适当的数额无菌氯化钙 2，尿嘧啶，胆固醇，硫酸镁1M 1M KPO 4 。分装10毫升的液体NGM到每60毫米× 15毫米培养皿。允许NGM，以巩固在室温下过夜。 </li><li>如果测试化合物/提取，分装提取的影响到每个板块和使用吊具均匀地分布在板面的提取物。允许提取物在室温下过夜干燥。超过1毫升的量将需要更多的时间来干。 </li><li>每盘现货250μL 大肠杆菌菌株 OP5O生长在LB培养基过夜光密度为0.4-0.6。使细菌在室温下干燥过夜。 </li></ol>相关变量板块的时代已经瘫痪检测的一个显着的影响，年纪较大的板块往往要干出。使用板浇瘫痪检测一周内。对于理想的效果，倒板前3-4天开始同步蠕虫人口以下（2）]。对于任何实验，只能使用同一批次浇板。 改变大小的草坪（即发现与传播）和/或细菌的类型，也将改变蠕虫的行为。可以使用替代性大肠杆菌麻痹检测作为食物来源（例如，用于喂养RNAi实验的HT115株），但可以改变瘫痪的动力学，因此需要适当的内部控制大肠杆菌 。 2）年龄同步蠕虫种群的制备应变CL4176（SMG - 1（cc546 TS）我; dvIs27 [MYO - 3 /Aβ的小基因+ ROL - 6（su1006）标记基因] X是最常用的化验Aβ引起的瘫痪，这种菌株和其他转基因模型可用。为一个象征性的费用从线虫遗传学中心 （http://www.cbs.umn.edu/CGC/）的学术研究。 <ol><li>为了最大限度地测试人群的年龄，最好是开始一个同步的亲代同步。一周前发起的试验人群的麻痹检测，使用铂金蠕虫选择器将其转移到了10厘米NGM的传播与OP50板为2小时蛋20-30妊娠的成年人躺在在16℃（CL4176宽松的温度）。取出妊娠的成年人，并让后代成长为7天，届时他们将“第二天”妊娠成人。 </li><li> （第一天）第二天妊娠成人准备的年龄同步测试人群。对于每一个实验条件的对象是50-75年龄同步蠕虫生成一式三份板。使用铂金蠕虫选择器，转移到60毫米（10毫升体积）与OP50发现NGM板的第2天的妊娠成人10-12。允许蠕虫在16 ° C打下鸡蛋2个小时，然后摘掉成年人，让鸡蛋孵化，成长在16 ° C。在这一点上是最好的人谁也不会得分板代码，所以射手将是盲目的实验条件。 </li></ol>相关变量产卵时，胚胎发育阶段（〜26细胞阶段的最佳条件下的野生型蠕虫）是一个成人的年龄和营养的功能。如果用于编写测试人口的妊娠成年人老年人或饿死，稍后阶段鸡蛋将被解雇，减少了人口的年龄和重现性麻痹动力学同步。这是比单无菌（即，只有OP50细菌）用于文化（微生物污染物会严重影响这个实验），重现性是最高的，如果一式三份板有类似的蠕虫。 3）转基因诱导和瘫痪检测<ol><li> （3天）至25日的上移板打下° C 48小时后同步鸡蛋;蠕虫应在第三幼虫阶段（L3）。板应安排无堆叠在25℃孵化器，以允许所有板块在同一时间达到25℃。 </li><li> （4天）开始的蠕虫瘫痪（株CL4176）得分在18-20小时后开始的升档。此时，在人群中的所有蠕虫应该已经达到第四幼虫（L4）的阶段。继续在两个小时的的增量得分，直到每个板块上的所有蠕虫瘫痪。原因不明，瘫痪甚至整个车身长度：头部区域的最后一部分是蠕虫停止移动。因此，W最近发起瘫痪奥姆斯不能转化跨板，但可以将他们的头上，从而左右他们的前清除细菌和清除细菌留下了“光环”。这些蠕虫都会成为完全瘫痪，因此晕蠕虫被归类为瘫痪。有些蠕虫不会有光晕，但也不会显示自发的运动，这些都是刺激与蠕虫选择器进行测试。如果打了招呼的蠕虫无法接受一个完整的体波传播后打回原形，也取得了瘫痪。为了有效地得分，这是最简单的方法，使他们不会被无意rescored未来的得分期间，将瘫痪的蠕虫板unspotted部门。 （这也可以让错误分类的蠕虫展示运动，让得分校正）。 </li><li>要生成一个瘫痪曲线，在每个时间点的蠕虫病毒在每个尚未瘫痪板的一小部分是转换为百分比，以及平均“unparalyzed”的比例是绘制免受升档启动时间。 （阴谋以这种方式的理由是，由此产生的曲线是正式类似的生存曲线，从而可以使用标准的非参数生存统计分析。） </li></ol>相关变量必须发生之前，他们到达瘫痪中期L4阶段发起，MYO - 3启动子的发育调控，并大大减少了在后期L4或成虫表达的转基因myo-3/Aβ蠕虫升档。同样，这种基因的表达被阻塞，如果蠕虫成为饿死，因此至关重要的是，蠕虫是整个实验过程中精心喂养。我们从来没有看到一个瘫痪的蠕虫病毒，在任何条件下恢复，和升档12-16小时后，所有CL4176蠕虫会瘫痪，即使它们下移至16 ° C。 CL4176应变，足够的转基因表达的上调导致瘫痪可以发生在较低温度下（即，在20-25℃），虽然瘫痪的时间将被改变。瘫痪率取决于使用特定的转基因株（我们已经构建了更快和更慢的CL4176率myo-3/Aβ株），所以替代株得分窗口可能需要进行调整。 4）代表性的成果在图1所示为CL4176麻痹曲线的情节，都未经处理和暴露6.27毫米咖啡因（NGM板终浓度）。在一个高度统计学意义延迟约4小时，我们在此模型中，抑制Aβ的毒性指示瘫痪的治疗效果。在图2所示是一个独立的实验，化验cafestol，咖啡中发现的另一种化合物的影响。此图是典型的治疗，不影响Aβ的毒性。请注意，在两个实验中的一半左右未经处理的CL4176人口瘫痪24小时，并瘫痪28小时完成。这是典型的时限控制CL4176人口瘫痪。从这个时机的重大偏差的环境条件的改变或自发的转基因拷贝缺失的指示。 （CL4176包含一个集成的染色体myo-3/Aβ基因的多个副本的转基因阵列，尽管这种转基因阵列通常是相当稳定，任何传播的应变温度&gt; 16 ° C将选择经历了罕见的蠕虫自发的转基因删除，导致变种，会减少Aβ的表达和较慢的瘫痪结果。） <img src="/files/ftp_upload/2252/2252fig1.jpg" alt="图1" /> 图1。麻痹应变CL4176蠕虫未经处理或暴露到6.27毫米咖啡因（Sigma公司）的曲线 。表明次启动温度上移。误差线=扫描电镜<img src="/files/ftp_upload/2252/2252fig2.jpg" alt="图2" /> 图2。麻痹CL4176蠕虫接触到0.48毫米cafestol（Sigma公司）的曲线 。误差线=扫描电镜

<ol>
	<li>Arya, U., Dwivedi, H., Subramaniam, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reserpine+ameliorates+Abeta+toxicity+in+the+Alzheimer's+disease+model+in+Caenorhabditis+elegans.">Reserpine ameliorates Abeta toxicity in the Alzheimer's disease model in Caenorhabditis elegans.</a> Exp Gerontol. 44, 462-466 (2009).</li><li>Cohen, E., Bieschke, J., Perciavalle, R. M., Kelly, J. W., Dillin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposing+activities+protect+against+age-onset+proteotoxicity.">Opposing activities protect against age-onset proteotoxicity.</a> Science. 313, 1604-1610 (2006).</li><li>Dosanjh, L. E., Brown, M. K., Rao, G., Link, C. D., Luo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+phenotyping+of+a+transgenic+C.+elegans+expressing+neuronal+amyloid-beta.">Behavioral phenotyping of a transgenic C. elegans expressing neuronal amyloid-beta.</a> J Alzheimers Dis. , (2009).</li><li>Drake, J., Link, C. D., Butterfield, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+precedes+fibrillar+deposition+of+Alzheimer's+disease+amyloid+beta-peptide+(1-42)+in+a+transgenic+Caenorhabditis+elegans+model.">Oxidative stress precedes fibrillar deposition of Alzheimer's disease amyloid beta-peptide (1-42) in a transgenic Caenorhabditis elegans model.</a> Neurobiol Aging. 24, 415-4120 (2003).</li><li>Fay, D. S., Fluet, A., Johnson, C. J., Link, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+aggregation+of+beta-amyloid+peptide+variants.">In vivo aggregation of beta-amyloid peptide variants.</a> J Neurochem. 71, 1616-1625 (1998).</li><li>Florez-McClure, M. L., Hohsfield, L. A., Fonte, G., Bealor, M. T., Link, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+insulin-receptor+signaling+promotes+the+autophagic+degradation+of+beta-amyloid+peptide+in+C.+elegans.">Decreased insulin-receptor signaling promotes the autophagic degradation of beta-amyloid peptide in C. elegans.</a> Autophagy. 3, 569-580 (2007).</li><li>Fonte, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+in+vivo+beta-amyloid+peptide+toxicity+by+overexpression+of+the+HSP-16.2+small+chaperone+protein.">Suppression of in vivo beta-amyloid peptide toxicity by overexpression of the HSP-16.2 small chaperone protein.</a> J Biol Chem. 283, 784-7891 (2008).</li><li>Hassan, W. M., Merin, D. A., Fonte, V., Link, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AIP-1+ameliorates+beta-amyloid+peptide+toxicity+in+a+Caenorhabditis+elegans+Alzheimer's+disease+model.">AIP-1 ameliorates beta-amyloid peptide toxicity in a Caenorhabditis elegans Alzheimer's disease model.</a> Hum Mol Genet. 18, 2739-2747 (2009).</li><li>Link, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+human+beta-amyloid+peptide+in+transgenic+Caenorhabditis+elegans.">Expression of human beta-amyloid peptide in transgenic Caenorhabditis elegans.</a> Proc Natl Acad Sci U S A. 92, 9368-9372 (1995).</li><li>Link, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+analysis+in+a+transgenic+Caenorhabditis+elegans+Alzheimer's+disease+model.">Gene expression analysis in a transgenic Caenorhabditis elegans Alzheimer's disease model.</a> Neurobiol Aging. 24, 397-413 (2003).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook. , 1-11 (2006).</li><li>Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid-beta-induced+pathological+behaviors+are+suppressed+by+Ginkgo+biloba+extract+EGb+761+and+ginkgolides+in+transgenic+Caenorhabditis+elegans.">Amyloid-beta-induced pathological behaviors are suppressed by Ginkgo biloba extract EGb 761 and ginkgolides in transgenic Caenorhabditis elegans.</a> J Neurosci. 26, 13102-13113 (2006).</li></ol>

我们所描述的协议，可以有效地用于检测Aβ的毒性化合物影响。发布的例子包括：银杏叶提取物成分的影响（吴等 ，2006）和利血平（Arya等人，2009年）。已对在其他系统中的Aβ的毒性保护的化合物也显示，在此模型中的保护（例如，美金刚，我们未发表的结果）。建立这种模式的一个重要理由是，发达的遗传和分子工具在 C 线虫可以用来调查对Aβ毒性机制。因此，Aβ的毒性作用的特定的突变可以被检测（例如，减少胰岛素样信号引入DAF - 2功能丧失突变，FLOREZ - 麦克卢尔等 。2007年生效），而这种模式也可以用来检查过度表达的转基因（Fonte酒店等，2008）的影响。最近，我们已经取得了广泛使用这种模式，检查单中Aβ的氨基酸替换（未发表数据）的毒性的影响。 这里描述的转基因模型分析的急性Aβ的表达肌肉细胞功能的影响。在麻痹肌肉细胞和它们的肌的时候是完好的;瘫痪的表型是不是肌肉细胞死亡的结果。然而，（〜48小时后最初的升档）后瘫痪肌肉的完整性和最终死亡的蠕虫有一个击穿。我们没有调查这下游的Aβ表达影响，或用作毒性标志。 这里描述的诱导Aβ的表达模式是没有适当的调查治疗方法，调节β-淀粉样蛋白形成本身。虽然同构的Aβ表达形式易被察觉淀粉样蛋白沉积的转基因蠕虫（ 仙子等人1998年; Link 等人，2001年），与诱导的Aβ表达的转基因蠕虫很少有淀粉样蛋白沉积，并出现瘫痪的表型淀粉样蛋白沉积 （德雷克等独立。 2003年）。我们的研究结果是一致的观点，急性毒性诱导的Aβ表达的可溶性低聚物Aβ的积累，而不是淀粉样蛋白沉积的结果。

<table border="1"><tbody><tr><td>试剂</td><td> Quantity/2L </td><td>公司/目录＃ </td><td>评论</td></tr><tr><td>氯化钠</td><td> 6克</td><td> Sigma - Aldrich公司/ S9888 </td><td></td></tr><tr><td>洋菜</td><td> 40克</td><td> Sigma - Aldrich公司/ A7002 </td><td></td></tr><tr><td>蛋白胨</td><td> 5克</td><td> Sigma - Aldrich公司/ P0556 </td><td></td></tr><tr><td> DDH 2 Ø </td><td> 2L </td><td></td><td></td></tr><tr><td> 1M氯化钙2 </td><td> 2ML </td><td></td><td> 147.02mg/mL在DDH 2 O </td></tr><tr><td>尿嘧啶</td><td> 2ML </td><td></td><td> 2mg/mL在DDH 2 O </td></tr><tr><td>胆固醇</td><td> 2ML </td><td></td><td>在乙醇5mg/mL（没有反应釜） </td></tr><tr><td> 1M硫酸镁4 </td><td> 2ML </td><td></td><td> 246.5mg/mL在DDH 2 O </td></tr><tr><td> 1M KPO 4 </td><td> 50毫升</td><td></td><td> 98mg KH 2 PO 4毫升，48mg K2HPO4/mL DDH 2 O，pH值</td></tr><tr><td>锥形瓶中</td><td></td><td> VWR </td><td></td></tr></tbody></table>

assaying &beta;-amyloid toxicity using a transgenic c. elegans model

积累的β-淀粉样肽（Aβ）一般认为是阿尔茨海默氏病的诱导，但目前仍不清楚有关的机制（S）的毒性。 Aβ是还交存肌注包涵体肌炎，严重的人类性肌病。激烈的研究线虫线虫可以transgenically设计，以表达人类的Aβ。根据对组织或对Aβ表达的时机，转基因蠕虫可以很容易衡量的，对Aβ毒性的一个读出的表型。例如，泛神经元Aβ的表达的转基因蠕虫的缺陷是联想学习（杜新志等人，2009年），而同构肌肉特异性表达的转基因蠕虫显示一个渐进的，年龄依赖的麻痹表型（Cohen等链接，1995年; 2006年）。一个特别有用的C。线虫模型采用了一个温度敏感突变的mRNA监视系统工程师肌肉温度诱导Aβ的转基因表达，在重现瘫痪表型后，温度升档（ 链接等，2003） 。治疗计数器在此模型中Aβ的毒性[例如，一个保护性基因（ 哈桑等人，2009年）的表达，或置身于银杏叶提取物（吴等 ，2006）]再现改变温度升档，这些转基因引起的瘫痪率蠕虫。在这里，我们描述了我们的协议，在这种转基因 C.测量瘫痪率线虫模型，特别注意实验变量，可以影响这个测量。

Watch this Scientific Journal Video about Assaying Î²-amyloid Toxicity using a Transgenic C. elegans Model at JoVE.com

Assaying &amp;#946;-amyloid Toxicity using a Transgenic C. elegans Model

强研究的线虫<em&gt;线虫</em&gt;可transgenically的设计，以表达人类β-淀粉样肽（Aβ）。诱导表达的Aβ<em&gt; C。线虫</em&gt;肌肉导致一个快速，重现性瘫痪的表型，可用于监测治疗，调节Aβ的毒性。

含量测定β-淀粉样蛋白的毒性，使用转基因 C。线虫模型

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

assaying-amyloid-toxicity-using-a-transgenic-c-elegans-model

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Neuroscience

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

25.3K 次观看.  University of Colorado. 强研究的线虫线虫可transgenically的设计，以表达人类β-淀粉样肽（Aβ）。诱导表达的Aβ C。线虫肌肉导致一个快速，重现性瘫痪的表型，可用于监测治疗，调节Aβ的毒性。

视频: Assaying &#946;-amyloid Toxicity using a Transgenic C. elegans Model

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal's endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila manifesting altered circadian or sleep properties.

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

We describe procedures for recording daily locomotor activity rhythms of Drosophila and subsequent data analysis. Locomotor activity rhythms are a reliable behavioral output of animal circadian clocks and are used as the standard readout of clock function when studying circadian mutants or examining how the environment regulates the circadian system.

含量自发活动，研究昼夜节律和睡眠参数果蝇</em

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) ...

科研

神经科学

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

在 C 在体内激光干切断线虫

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

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 ...

C. elegans Tracking and Behavioral Measurement

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the surface of an agar plate. For the study of the genetic, biochemical, and neuronal basis of behavior, C. elegans is an ideal organism because it is genetically tractable, amenable to microscopy, and shows a number of complex behaviors, including taxis, learning, and social interaction1,2. Behavioral analysis based on tracking the movements of worms as they crawl on agar plates have been particularly useful in the study of sensory behavior3, locomotion4, and general mutational phenotyping5. Our system works by moving the camera and illumination system as the worms crawls on a stationary agar plate, which ensures no mechanical stimulus is transmitted to the worm. Our tracking system is easy to use and includes a semi-automatic calibration feature. A challenge of all video tracking systems is that it generates an enormous amount of data that is intrinsically high dimensional. Our image processing and data analysis programs deal with this challenge by reducing the worms shape into a set of independent components, which comprehensively reconstruct the worms behavior as a function of only 3-4 dimensions6,7. As an example of the process we show that the worm enters and exits its reversal state in a phase specific manner.

C. elegans Tracking and Behavioral Measurement

We have developed a video-rate tracking microscope system that can record and quantify C. elegans behavior at high resolution and high speeds. We have also developed computational methods to reduce the dimensionality of the worm images to a fundamental set of measurements that completely describe the shape of the worm.

线虫跟踪和行为测量

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

长期的嗅觉适应性的分子读数<em&gt; C.线虫</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

彗星试验测定DNA损伤的海马神经元

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

C.在体内神经元钙成像技术线虫

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

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 ...

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this protocol focuses on how to image dividing neuroblasts, which are found underneath the epidermal cells and may be important for epidermal morphogenesis. Tissue formation is crucial for metazoan development&#160;and relies on external cues from neighboring tissues. C. elegans is an excellent model organism to study tissue morphogenesis in vivo due to its transparency and simple organization, making its tissues easy to study via microscopy. Ventral enclosure is the process where the ventral surface of the embryo is covered by a single layer of epithelial cells. This event is thought to be facilitated by the underlying neuroblasts, which provide chemical guidance cues to mediate migration of the overlying epithelial cells. However, the neuroblasts are highly proliferative and also may act as a mechanical substrate for the ventral epidermal cells. Studies using this experimental protocol could uncover the importance of intercellular communication during tissue formation, and could be used to reveal the roles of genes involved in cell division within developing tissues.

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes how to image dividing cells within a tissue in Caenorhabditis elegans embryos. While several protocols describe how to image cell division in the early embryo, this protocol describes how to image cell division within a developing tissue during mid late embryogenesis.

可视化的神经细胞胞质分裂过程中<em&gt; C。线虫</em&gt;胚胎

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this ...

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of stress-induced plasticity is the dauer stage of C. elegans. Dauers are an alternative developmental larval stage formed under conditions of low concentrations of bacterial food and high concentrations of a dauer pheromone. Dauers display extensive developmental and behavioral plasticity. For example, a set of four inner-labial quadrant (IL2Q) neurons undergo extensive reversible remodeling during dauer formation. Utilizing the well-known environmental pathways regulating dauer entry, a previously established method for the production of crude dauer pheromone from large-scale liquid nematode cultures is demonstrated. With this method, a concentration of 50,000 - 75,000 nematodes/ml of liquid culture is sufficient to produce a highly potent crude dauer pheromone. The crude pheromone potency is determined by a dose-response bioassay. Finally, the methods used for in vivo time-lapse imaging of the IL2Qs during dauer formation are described.

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant &#8216;dauer&#8217; juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer.

在永久性幼虫特有的神经重构的C语言活体成像线虫

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of ...