Name: in vivo imaging of dauer-specific neuronal remodeling in c. elegans
Uploaded: 2014-09-04T15:00:00.000+00:00
Duration: 9 min 39 s
Description: Watch this Scientific Journal Video about In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans at JoVE.com

I thank Dr. Maureen Barr, under whose supervision the dauer IL2 remodeling phenotype was initially characterized. Thanks to Dr. C. Britt Carlson for critical reading of the manuscript. Funding in the Barr lab was from the NIH (5R01DK59418) and postdoctoral fellowships from the USDA (2010-65106-20587) and the New Jersey Commission on Spinal Cord Research (CSCR12FEL004). Current funding for this work is from a University of Illinois Urbana-Champaign College of ACES FIRE grant.

1，原油永久性幼虫信息素产生<ol><li>成长OP50 大肠杆菌从大肠杆菌单菌落O 2 / N在37°C，而在100毫升的LB肉汤200rpm振荡。注:此O / N培养可事先保存在4℃。 </li><li>成长氮气C.线虫于6月15日至20日毫米培养皿中以NGM琼脂（见配方在表1）接种50μl的OP50 大肠杆菌的大肠杆菌 ，直到细菌几乎耗尽19。 </li><li>请在1升锥形5X250毫升的媒体中（见配方见表1）烧瓶19。注意:此溶液可预先制成。 </li><li>成长OP50 E.文化大肠杆菌在4升的LB培养基的O / N以1毫升OP50的大肠杆菌接种大肠杆菌 ，并在37℃下振荡16小时。 </li><li>从平板（步骤1.2）与1ml为S基础溶液（见配方见表1）和从4-5板成每4 1升锥形烧瓶中，在步骤1.3中制备的在S媒体转移线虫洗线虫（4- 5板每个瓶）19。 </li><li>离心从步骤1.4的OP50，在650×g离心10分钟，除去上清液。悬浮每个细菌沉淀在10ml为S基础的解决方案。悬浮细菌的等量转移到线虫的每个烧瓶。 </li><li>将培养瓶在定轨摇床上，在室温（20 - 25℃）和100 - 150转4 - 5天，直到溶液开始清除，表明食物的消耗。 </li><li>一旦食物被消耗殆尽，增长OP50 大肠杆菌的另一个O / N培养大肠杆菌在4X 1升LB培养基中。离心细菌，在650×g离心10分钟，并从一个烧瓶中加入沉淀到每一S介质瓶与线虫。返回在S媒体瓶中的轨道摇床中，如在步骤1.7中，额外3-4天，直至食物被再次耗尽。 </li><li>取约1毫升一份的从每个烧瓶中的溶液，并估计线虫浓度由子采样从每1ml等份1微升和计数线虫的数目。注意:线虫种群应该是50,000 - 75,000线虫/毫升。在此浓度下的大多数线虫是dauers。 </li><li>放弃任何污染瓶中。注:污染经常表现为菌丝在步骤1.9所示。 </li><li>线虫离心机在4000 XG在4℃下10分钟。丢弃线虫沉淀，并保留上清液。可选地，看到讨论了使用这些动物做一个低效力的信息素。 </li><li>筛选通过＃1 Whatman过滤纸上清液。注意:保温瓶将加速这一进程。 </li><li>此外，通过过滤0.45微米的无菌真空过滤单元上清。注:滤液可以保持在o / n 4°C或进入1.14。 </li><li>滤液中加入2L烧杯中搅拌棒。放置在加热搅拌台通风柜。煮沸边搅拌，蒸发至约200毫升将溶液转移到500ml烧杯中。继续煮，直到约50ml的乐英尺注:应在该步骤中被视为是可能的解决方案，以沸腾。最后将50ml颜色为黑褐色。在烹调过程中的任何时间，该热可以被关闭，并且将滤液存储的O / N在室温或4℃。 </li><li>放凉后，离心5分钟以1000×克倒出上清液的100毫升的烧杯中，并弃去沉淀。 </li><li>熬上清下降到棕色外壳。从火上移开，并打破了锅铲地壳。添加足够200标准乙醇以覆盖外壳。用封口膜封住烧杯中，并让它坐在O 2 / N在室温。倒掉乙醇到一个干净的烧杯中，并放置到一边。加入新鲜的乙醇覆盖地壳。重复O 2 / N提取3次。池中提取物。 </li><li>使用真空泵干的乙醇提取物。如果真空泵不可用，乙醇提取物添加到一个干净的烧杯和温暖至50°C，而在通风橱中搅拌。注意:一旦乙醇蒸发升渣的粘飞行棕色应该保持。如果加热的情况下，应当小心以不燃烧的溶液，加热时间过长。 </li><li>在10ml的蒸馏水的无菌水溶解残余物。过滤灭菌该溶液用1毫升等分0.22μm的注射器过滤器，并储存在-20℃。注:1ml水来溶解残余物，每100毫升原液体线虫培养物。例如，如果该液体培养烧瓶中的一个由于丢弃污染，则调整所用的水以溶解残余物的量。 </li></ol> 2，确定信息素剂量反应<ol><li>准备永久性幼虫信息素通过使6X5毫升NGM媒体与高贵的琼脂，蛋白胨没有，并补充50微克/毫升硫酸链霉素和一系列永久性幼虫信息素（见配方见表1）4，19，在13×毫米混合成分玻璃培养管，沸腾本生灯，然后倒入切成3厘米的培养皿。 </li><li>称取分icrocentrifuge管。加入1 ml 大肠杆菌的O / N培养的大肠杆菌 OP50到管和离心〜17000 XG的离心管。删除所有的上清液。再次称重该管和确定将细菌沉淀的重量。悬浮用M9缓冲液（见配方在表1）的粒料为4％的最终浓度（ 重量/体积）。等分试样将10μl细菌悬液到每一信息素板4的中心。商店板在o / n RT。 </li><li>蜕皮到成年到信息素板4后接10-15妊娠N2野生型雌雄同体的一天。存储该板在25℃下进行3 - 4小时。 </li><li>摘掉所有的成人雌雄同体的。记录产卵的各板的数量。用封口膜密封板，并恢复至25℃，3天17。 </li><li>算dauers的百分比（ 图2）。注意:对于这两种方法是要注意，重要的是dauers（和较小的前10吨非dauers）会爬上墙壁和到培养皿的盖子。因此重要的是要检查的培养皿为线虫的所有表面。 </li><li>适合数据的剂量-反应曲线并估计欧盟90浓度信息素（足够的信息素诱导90％的动物形成dauers）， 如图3注:我们通常使用逻辑回归模型来估计欧盟90 ，但不同的统计软件包可能会产生类似的估计可供选择的方法。 </li></ol> 3，现场永久性幼虫IL2神经影像学<ol><li>作为协议2使用步骤2.6确定欧盟90值准备永久性幼虫信息素板。 </li><li>诱导使用方法dauers在协议2与应变携带一个综合IL2记者转基因（myIs13 [P KLP-6 :: GFP]，myIs14 [P KLP-6 :: GFP]，myIs16 [P <eM&gt; KLP-6 :: tdTomato]或qIs56 [P 滞后2 :: GFP]）12，20，21注意:当Q Is56时，GFP的表达不会在IL2Qs可见，直到几个小时后永久性幼虫蜕皮和一些初始重塑事件将不可见。另外，虽然从P 滞后2 :: GFP转基因的荧光信号是在IL2s最终明亮永久性幼虫比P KLP-6荧光报告期间，在P 滞后-2 :: GFP转基因也表示弱的头部肌肉的可掩盖细IL2分支机构。 </li><li>下面产卵，取出成人雌雄同体，用封口膜密封板孵育34小时，在25℃17。注:时间为永久性幼虫蜕皮的长度略有不同个体之间。树突树枝状最迅速的时期始于4 - 5小时以下的永久性幼虫蜕皮的发生（39 - 44小时后产蛋在25°C）12。 </li><li>成像的前一天，弥补了10％的溶液（W / V）琼脂糖M9的缓冲区与永久性幼虫信息素在欧盟90浓度。分装约100μl琼脂糖迈上了一个显微镜载玻片和快速，但轻轻地，在上面放置另一张幻灯片创建一个平坦的表面琼脂糖。经过琼脂糖凝固，包裹在保鲜膜和储存的幻灯片在湿度室（用湿纸巾枪头箱）。 </li><li>在34小时的潜伏期后，检查信息素板对已停止抽水咽线虫。单独的幻灯片，使得一个滑动的仅一个表面具有琼脂糖上。选择一个或多个线虫到10％的琼脂糖凝胶垫和1μ升0.1微米的聚苯乙烯珠22。注:使用一个良好的同步人口，多数平板上的线虫会进入蜕皮从L2d的到永久性幼虫。不过，也有可能是其中í变化ndividuals。因此重要的是要注意的附加功能（造口闭塞，咽重构）来估计时间的动物已经在永久性幼虫蜕皮内（见代表性结果为例）的量。 </li><li>确定线虫的方向（左/右，背/腹）。捕获IL2Q树突Z堆叠图像在放大100倍以尽可能低的荧光强度，每15分钟或视需要为特定的实验。继续拍摄图像，直到线虫已经从L2d的角质层分离，固定化是不可能的，或者动物从永久性幼虫恢复（咽开始抽水）。如果不积极成像，放置在一个恒温恒湿试验箱的幻灯片。注:我们使用啶显微镜机动阶段，微分干涉对比（DIC）的光学元件。 </li></ol>

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永久性幼虫特异性神经可塑性和其他永久性幼虫相关的形态学变化的检查需要控制和可靠的形成dauers要么通过基因突变（ 即，DAF-C突变体）或由野生型动物的接触信息素。而使用的基因突变的诱发dauers方便，它可能会混淆的结果。因此，与DAF-C突变收集的数据应该被随后确认与诱导暴露于永久性幼虫信息素进入持久期野生型动物。 的粗永久性幼虫信息素的生产相...

表型可塑性，包括发育和行为的可塑性，可以适应恶劣的环境条件很重要，被认为是动力，发展1。℃。线虫是一种模式生物经常使用的开发和神经生物学研究。在理想情况下，C。在进入成年繁殖期通过四个幼虫阶段线虫的发展。然而，低粮食供应和人口密度高，长的条件下， 线虫可以接受的发育开关，进入一个长寿命和抗逆持久期2。 Dauers显示非dauers几个形态和行为上的差异而可能介导这一抗逆性。的环境线索和遗传途径调节永久性幼虫条目已经广泛地描述3，4，5，6，7，但控制个体的表型变化的分子机制发生ðuring持久形成是不太容易理解。 永久性幼虫特定表型的检测要求生产的永久性幼虫动物。这可以通过以下三种方式:1）从C的饥饿文化采摘线虫 ，2）使用的持久形成组成（DAF-C）突变体，3）诱导持久形成经过纯化信息素。这是比较简单的，从一个标准的NGM板有三挑dauers 线虫的人口已经耗尽了它的细菌的食物供应。在我们手中，一个标准的NGM板最初接种用50μlOP50 大肠杆菌 ，使其生长3天，随后接种3 - 4成人雌雄同体保 ​​持在25℃，将用尽的一个星期内的食物供应和产生几千内的3天dauers（除了众多饥饿非dauers）。然而，这种方法是不令人满意的蜕皮到d中期间出现的审查过程奥尔。因此很难确定哪个几千动物在一个典型的饥饿板的正进入永久性幼虫。此外，使用饿死板提供了可能性进行同步。采用DAF-C突变允许同步和可靠的代dauers的。然而，没有保证特定的daf-C突变不会影响其他永久性幼虫特异性表型单独或与其他突变组合。 à永久性幼虫信息素的存在，最初是由Cassada和罗素暗示，后来被黄金和里德尔证实。该持久性信息素已被化学特点2，8，9，10，纯化信息素是由一个复杂的混合ascarosides，它以不同的形式规范双方的行为和发育过程9,11。使用粗永久性幼虫信息素的提取物可以用于可靠的控制异步同步dauers在野生型背景。 </P&gt; 对神经系统和相关的神经胶质细胞在永久性幼虫幼体形成12，13，14进行改造，其中一些变化是不可逆的，如神经胶质细胞的永久性幼虫13，14在重塑，但其他的重塑事件后，恢复到良好的可逆性环境条件。枝晶树枝状，从单一的树突切换到multidendritic神经元和轴突重塑12:例如，一组4 IL2Q神经元中持久形成包括经过快速，可逆的神经重构。本文的方法允许可靠的体内成像应激诱导的神经可塑性的模式生物。提出了生产和粗永久性幼虫信息素的检测方法被先前描述和编译从几个来源4，8，15，16，17，18。

<table><tbody><tr><td>N2 &lt;em&gt;C. elegans&lt;/em&gt; Bristol strain</td><td>Caenorhabditis Genetics Center</td><td></td><td></td></tr><tr><td>PT2660 myIs13[P&lt;sub&gt;klp-6&lt;/sub&gt;::gfp] III</td><td>Upon request from Schroeder lab</td><td></td><td></td></tr><tr><td>PT2762 myIs14[P&lt;sub&gt;klp-6&lt;/sub&gt;::gfp] V</td><td>Upon request from Schroeder lab</td><td></td><td></td></tr><tr><td>JK2868 qIs56[P&lt;sub&gt;lag-2&lt;/sub&gt;::gfp] V</td><td>Caenorhabditis Genetics Center</td><td></td><td></td></tr><tr><td>OP50 E. coli</td><td>Caenorhabditis Genetics Center</td><td></td><td></td></tr><tr><td>Petri dishes</td><td>Tritech Research</td><td>60 mm T3308, 35 mm T3501</td><td></td></tr><tr><td>NGM agar</td><td>Various</td><td></td><td>3 g NaCl, 17 g agar, 2.5 g peptone in 975 ml H&lt;sub&gt;2&lt;/sub&gt;O autoclaved cooled to 55&amp;deg;C followed by 1 ml of 5 mg/ml cholesterol in ethanol, 25 ml of 1 M KPO&lt;sub&gt;4&lt;/sub&gt; buffer (pH 6.0), 1 ml of 1 M CaCl&lt;sub&gt;2&lt;/sub&gt;, 1 ml of 1 M MgSO&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>S Media</td><td>Various</td><td></td><td>S media consists of S basal solution (1.46 g NaCl, 0.25g K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 1.5 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 0.25 ml of 5 mg/ml cholesterol in ethanol and H&lt;sub&gt;2&lt;/sub&gt;O to 250 ml. Autoclaved.) To the S basal solution add 2.5 ml of potassium citrate pH 6.0 (2 g citric acid monohydrate, 29.4 g tri-potassium citrate monohydrate&lt;sub&gt;, &lt;/sub&gt;H&lt;sub&gt;2&lt;/sub&gt;O to 100 ml, autoclaved), 2.5 ml of Trace Metals Solution (1.86 g disodium EDTA, 0.69 g FeSO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;&amp;bull;7 H&lt;sub&gt;2&lt;/sub&gt;O, 0.2 g MnCl2&amp;bull;4 H&lt;sub&gt;2&lt;/sub&gt;O, 0.29 g ZnSO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;&amp;bull;7 H&lt;sub&gt;2&lt;/sub&gt;O, 0.025 g CuSO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;&amp;bull;5 H&lt;sub&gt;2&lt;/sub&gt;O, H&lt;sub&gt;2&lt;/sub&gt;O to 1 L. Autoclaved and stored in the dark), 0.75 ml of 1 M CaCl&lt;sub&gt;2&lt;/sub&gt;, and 0.75 ml of 1 M MgSO&lt;sub&gt;4&lt;/sub&gt;.</td></tr><tr><td>Dauer pheromone plates for dose-response assay</td><td>Various</td><td></td><td>Mix 0.085 g Noble agar, 0.5 ml of 0.513 M NaCl and 5 &amp;micro;l of 5 mg/ml cholesterol dissolved in ethanol in each of 6x 15 ml culture tubes. Add sufficient crude pheromone to each tube to create a range of concentrations. Add H&lt;sub&gt;2&lt;/sub&gt;O to bring the volume to 4.725 ml.&amp;nbsp; Boil over a Bunsen burner until the agar is dissolved and then cool to 55 &amp;deg;C. Add 125 &amp;micro;l 1 M KPO&lt;sub&gt;4&lt;/sub&gt; buffer (pH 6.0), 50 &amp;micro;l 0.1 M CaCl&lt;sub&gt;2&lt;/sub&gt;, 50 &amp;micro;l 0.1 M MgSO&lt;sub&gt;4&lt;/sub&gt; and 50 &amp;micro;l of 5 mg/ml streptomycin sulfate to each tube.&amp;nbsp; Pour the contents of each tube into a 35 mm diameter Petri dish. Let the agar solidify at room temperature overnight (if longer, store in enclosed container to prevent excessive drying).</td></tr><tr><td>Microsphere beads 0.1 micron</td><td>Polysciences Inc.</td><td>00876-15</td><td></td></tr><tr><td>M9 buffer</td><td>Various</td><td></td><td>Mix 3 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 6 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 5 g NaCl, 1 ml 1 M MgSO&lt;sub&gt;4&lt;/sub&gt;, H&lt;sub&gt;2&lt;/sub&gt;O to 1 liter. Sterilize by autoclaving</td></tr></tbody></table>

in vivo imaging of dauer-specific neuronal remodeling in c. elegans

控制应激诱导的表型可塑性在动物的机制是频繁复杂和困难的体内研究。应力诱发塑性的一个典型的例子是C的持久期线虫 。 Dauers是低浓度的细菌性食物和高浓度永久性幼虫信息素的条件下形成的一种替代发育的幼虫期。 Dauers显示广泛的发育和行为的可塑性。例如，一套四内唇象限（IL2Q）神经元中持久形成进行了广泛可逆的重塑。利用众所周知的环境途径调节永久性幼虫条目，先前建立的方法，用于生产由大型液体线虫培养物粗永久性幼虫信息素被证实。用这种方法，对50000的浓​​度 - 液体培养物75000线虫/ ml的是足以产生一种高效的粗永久性幼虫信息素。粗素效价对射探测器由剂量 - 反应的生物测定rmined。最后，在持久形成用于体内的时间推移的IL2Qs的成像方法进行描述。

生产的粗永久性幼虫信息素产生一种黄色液体（ 图1）即热和冷的稳定。的粗素等分试样储存在-20℃下无限期地与活性无明显损失。下列生产信息素，单剂量 - 反应的生物测定是足够的效力的一个粗略的估计。然而，对于大多数实验是必要的，以重复的生物测定2 - 3次，取平均值，从每个试验。从两个信息素生物试验的代表性结果列于图3中 ，从这些数据的EC 50和...

Watch this Scientific Journal Video about In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans at JoVE.com

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 ...

in-vivo-imaging-of-dauer-specific-neuronal-remodeling-in-c-elegans

Research

JoVE Journal

Neuroscience

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

11.2K 次观看.  University of Illinois Urbana-Champaign. Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant ‘dauer’ juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer. 

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

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

简单的微流控芯片的<em在体内</em&gt;成像<em&gt; C.线虫</em&gt;<em&gt;果蝇</em&gt;和斑马鱼

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

科研

生物工程

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for the targeted expression of genetically encoded calcium sensors. This protocol outlines the use of these sensors for the in vivo imaging of calcium dynamics in targeted cells, specifically the body wall muscles of the worms. By utilizing the co-expression of presynaptic channelrhodopsin, stimulation of acetylcholine release from excitatory motor neurons can be induced using blue light pulses, resulting in muscle depolarization and reproducible changes in cytoplasmic calcium levels. Two worm immobilization techniques are discussed with varying levels of difficulty. Comparison of these techniques demonstrates that both approaches preserve the physiology of the neuromuscular junction and allow for the reproducible quantification of calcium transients. By pairing optogenetics and functional calcium imaging, changes in postsynaptic calcium handling and homeostasis can be evaluated in a variety of mutant backgrounds. Data presented validates both immobilization techniques and specifically examines the roles of the C. elegans sarco(endo)plasmic reticular calcium ATPase and the calcium-activated BK potassium channel in the body wall muscle calcium regulation.

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

This method provides a way to couple optogenetics and genetically encoded calcium sensors to image baseline cytosolic calcium levels and changes in evoked calcium transients in the body wall muscles of the model organism C. elegans.

在 Vivo 钙成像中，在 C. elegans身体壁肌肉

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

生物化学

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, ...

神经科学

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 ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

成像 C。线虫胚胎使用啶显微镜和开放源码软件

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

生物学

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days.

用于秀丽隐杆线虫长期生长和成像的简单微流控芯片

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding ...

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the neurotransmitter glutamate, and the excessive stimulation of its postsynaptic receptors. However, information on the subsequent molecular events that culminate in the distinct neuronal swelling morphology of this type of neurodegeneration is lacking. Other aspects, such as changes in specific subcellular compartments, or the basis for the differential cellular vulnerability of distinct neuronal subtypes, remain under-explored. Furthermore, a range of factors that come into play in studies that use in vitro or ex vivo preparations might modify and distort the natural progression of this form of neurodegeneration. It is therefore important to study excitotoxic necrosis in live animals by monitoring the effects of interventions that regulate the extent of neuronal necrosis in the genetically amenable and transparent&#160;model system of the nematode Caenorhabditis elegans. This protocol describes methods of studying excitotoxic necrosis in C. elegans neurons, combining optical, genetic, and molecular analysis. To induce excitotoxic conditions in C. elegans, a&#160;knockout of a glutamate transporter gene (glt-3) is combined with a neuronal sensitizing genetic background (nuls5 [Pglr-1::G&#945;S(Q227L)]) to produce glutamate receptor hyperstimulation and neurodegeneration. Nomarski differential interference contrast (DIC), fluorescent, and confocal microscopy in live animals are methods used to quantify neurodegeneration, follow subcellular localization of fluorescently labeled proteins, and quantify mitochondrial morphology in the degenerating neurons. Neuronal Fluorescence Activated Cell Sorting (FACS) is used to distinctly sort at-risk neurons for cell-type specific transcriptomic analysis of neurodegeneration. A combination of live imaging and FACS methods as well as the benefits of the C. elegans model organism allow researchers to leverage this system to obtain reproducible data with a large sample size. Insights from these assays could translate to novel targets for therapeutic intervention in neurodegenerative diseases.

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

In a C. elegans excitotoxicity model, this protocol employs in vivo imaging to analyze the regulation of necrotic neurodegeneration, the effect of genes encoding candidate mediators, and involvement of mitochondria. Cell dissociation and sorting is used to specifically obtain at-risk neurons for cell-specific transcriptomic analysis of neurodegeneration and neuroprotection mechanisms.

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the ...

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 ...

Quantifying Levels of Dopaminergic Neuron Morphological Alteration and Degeneration in Caenorhabditis elegans

Dopamine neuron loss is involved in the pathology of Parkinson&#39;s Disease (PD), a highly prevalent neurodegenerative disorder affecting over 10 million people worldwide. Since many details about PD etiology remain unknown, studies investigating genetic and environmental contributors to PD are needed to discover methods of prevention, management, and treatment. Proper characterization of dopaminergic neuronal loss may be relevant not only to PD research, but to other increasingly prevalent neurodegenerative disorders.
There are established genetic and chemical models of dopaminergic neurodegeneration in the Caenorhabditis elegans model system, with easy visualization of neurobiology supported by the nematodes&#39; transparency and invariant neuronal architecture. In particular, hermaphroditic C. elegans&#39; dopaminergic neuron morphological changes can be visualized using strains with fluorescent reporters driven by cell-specific promotors such as the dat-1 dopamine transporter gene, which is expressed exclusively in their eight dopaminergic neurons.
With the capabilities of this model system and the appropriate technology, many laboratories have studied dopaminergic neurodegeneration. However, there is little consistency in the way the data is analyzed and much of the present literature uses binary scoring analyses that capture the presence of degeneration but not the full details of the progression of neuron loss. Here, we introduce a universal scoring system to assess morphological changes and degeneration in C. elegans&#39; cephalic neuron dendrites. This seven-point scale allows for analysis across a full range of dendrite morphology, ranging from healthy neurons to complete dendrite loss, and considering morphological details including kinks, branching, blebs, and breaks. With this scoring system, researchers can quantify subtle age-related changes as well as more dramatic chemical-induced changes. Finally, we provide a practice set of images with commentary that can be used to train, calibrate, and assess the scoring consistency of researchers new to this method. This should improve within- and between- laboratory consistency, increasing rigor and reproducibility.

Quantifying Levels of Dopaminergic Neuron Morphological Alteration and Degeneration in Caenorhabditis elegans

In this article, we showcase how to use a seven-point scoring system to consistently quantify changes to dopaminergic neuron dendrite morphology in C. elegans. This system is intended for analyses of dopaminergic neurodegeneration assays utilizing genetic, chemical, and age-based models of neurodegenerative disorders.

卡诺哈布迪炎电子甘的多巴胺神经元形态变化和退化的量化水平

Dopamine neuron loss is involved in the pathology of Parkinson&#39;s Disease (PD), a highly prevalent neurodegenerative disorder affecting over 10 million ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

体内神经元钙处理的亚细胞成像

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

在永久性幼虫特有的神经重构的C语言活体成像线虫

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简单的微流控芯片的

在 Vivo 钙成像中，在 C. elegans身体壁肌肉

C.在体内神经元钙成像技术线虫

琼脂糖微室的长期钙成像

成像 C。线虫胚胎使用啶显微镜和开放源码软件

用于秀丽隐杆线虫长期生长和成像的简单微流控芯片

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

在体内卡诺哈布德炎电子神经元微管动力学和方向评估

卡诺哈布迪炎电子甘的多巴胺神经元形态变化和退化的量化水平

体内神经元钙处理的亚细胞成像