我们想感谢斯科特·哈密尔顿，奥哈洛伦实验室的成员，仔细阅读这篇稿子。我们也感谢匿名审稿人很好的建议和有见地的意见。

1。建设GFP标记EGL-4表达动物<ol><li> ODR-3基因（使用2678 bp的直接上游的起始密码子）的启动子下克隆的翻译融合GFP :: EGL-4：（对）ODR-3 :: GFP :: EGL-4。在化学传感器的神经元对ODR-3启动子驱动表达：AWA; AWB; AWC，以及在ASH弱。 </li><li>进样的质粒（对）ODR-3 :: GFP :: EGL-4成野生型（N2）的动物在50纳克/微升的共注射的标记物，使用标准的种系的转化技术8：（对）OFM-1： ：GFP的9（25纳克/微升），和（p）ODR-1 :: 10红色荧光蛋白（25纳克/微升）。 ODR-1启动子驱动器的的AWC和AWB化学传感器神经元对表达的，和OFM-1启动子驱动器中的体腔细胞中的表达。 </li><li>主题表达的转基因动物外的阵列详细的步骤1.2中的紫外线（UV）/三甲基（TMP）集成协议11。 </li><li>漂白剂的动物12同步，并培养他们对土壤线虫生长培养基（NGM）板OP50 E.大肠杆菌 L4阶段。关闭NGM板清洗L4动物与M9缓冲删除OP50 E.大肠杆菌的细菌。 </li><li>删除M9缓冲区和30μg/ ml的TMP解决方案的蠕虫病毒颗粒上的1.5 ml离心管中加50-100μl。在铝箔包裹的1.5 ml离心管，阻挡环境光，在旋转器上，并轻轻搅动15分钟箔纸包裹的1.5 ml离心管中。在一个大的非种子选手NGM板的TMP /蠕虫的解决方案，传输的蠕虫病毒。 </li><li> NGM板UV 350μJ（X100），使用Stratalinker 2400上公开的蠕虫。然后添加蠕虫的OP50的NGM板，在黑暗中孵育5小时。 </li><li>选择50个转基因蠕虫约10种子板（每块板）2-5蠕虫。转让P0的新盘每3天24小时。 </li><li>挑选250个克隆F1一nimals（每盘）1只动物。克隆了2-4 F2动物每个F1板的。检查F3动物100％的传输线（P）OFM-1 :: GFP的表达模式荧光显微镜下解剖，以寻找。 </li><li>异交的最终集成线（s），与野生型N2动物5倍。我们会参考到最后的集成线路pyIs500，GFP标记的EGL-4分子GFP :: EGL-4。 </li></ol> 2。动物的培育和维护核转位分析<ol><li>在25°C对标准的10厘米NGM板（见下面的配方）种子OP50 E.培养pyIs500动物大肠杆菌 13（ 图1）。 </li><li>在白天1，中挑四点五十六L4 pyIs500动物从板培养在25℃下到五个单独的10厘米OP50 E.大肠杆菌接种板和孵育在25℃（即，4-5 L4动物每板）。 </li><li>第4天，洗成人p关闭大NGM板使用S-基底缓冲pyIs500动物opulations（见下面的配方）。使用一次性的玻璃吸液管转移的动物，因为它们会粘到塑料吸头的侧。 </li></ol> 3。长期气味诱导核易位分析<ol><li>洗净pyIs500动物，S-基底去除细菌的3倍。不要离心洗涤;动物之间，而让动物在一个固定的1.5 ml离心管重力沉降。这是至关重要的，以确保所有的细菌被删除，NGM馅饼不受污染，残留的细菌会产生负面影响的易位检测的结果。此外，我们还发现，高压灭菌的1.5 ml离心管，管内壁上，使动物要坚持的两侧，使用非蒸压1.5毫升的离心管中，产生了“粘性”属性。 </li><li>虽然动物之间的清洗解决，使吨他适应的解决方案。基底的S-缓冲液中加入100毫升到100毫升的毕业班缸。加入到S-基底适应的气味，并用石蜡膜的带状密封的圆筒。如果执行，苯甲醛适应7.5微升苯甲醛加入到100毫升的S-基底，丁酮适应到100毫升的S-基底中添加11微升2 - butaone。轻轻地反转（不摇）毕业缸S-基底和气味的30倍，创造均匀的乳液。 </li><li>在最后一次洗涤S-基底，让动物定居，并删除所有液体。出版商一管“+ ve”的或“适应”和其他管“负”或“unadapt”。然后加入1毫升的适应组合，以适应离心管（+ VE），并添加1毫升的S-基底的不适应控制的离心管中（VE）。尽量保持每个管中在100和200之间的动物数目。一些实践中，它是可以由眼来估计近似的颗粒的动物数量。 </li><li>将1.5 ml的微量离心管帽保护到每个管（防止从弹出打开盖子），将管旋转器上的80分钟。我们发现，气味暴露60分钟和80分钟之间产生相似的结果（包括在长期的适应反应）。然而，80分钟曝光提供了更一致的结果，在我们的手中。因此，我们所有的长期适应的检测标准的使用80分钟的气味曝光（ 图2）。 </li><li> 80分钟后，洗净动物在S-基底的3倍。每次洗间的引力使动物能够解决。 </li></ol> 4。短期气味引起的核易位检测<ol><li>在短期气味适应的情况下，使用相同的协议作为每长期臭味诱导易位测定（步骤3.1-3.4），然而，而不是在适应80分钟气味孵育pyIs500动物，使用30分钟曝光。下面概述的气味诱导核易位测定的定量（步骤5）是相同的长期和短期之三米的风险。 </li></ol> 5。评分气味引起的核易位事件<ol><li> 2％琼脂糖垫含有5 mM叠氮化钠（NaN 3）中，溶解在卫生署2 0琼脂糖凝胶电泳。包含NaN的一个玻璃载玻片上放置一个熔化的琼脂糖下降。立即将另一显微镜载玻片上的第一张幻灯片扁平化的熔化的琼脂糖下降。静置30秒，然后轻轻地梳理出了一台琼脂糖凝胶垫〜1毫米的显微镜玻片上留下一个显微镜幻灯片。 </li><li>在最后一次洗涤S-基底，让蠕虫，解决在1.5 ml离心管中，并删除所有的液体。然后，添加动物逐滴几个显微镜载玻片上的琼脂糖凝胶垫。重要的是要保持每张幻灯片的动物的数量在50和100之间。太多的动物每张幻灯片将复杂的得分和蓝色光激发后，通常会创建一个散射光芒。过量的液体可恶人从琼脂糖垫使用小剑擦拭。一个0.5毫米的玻璃盖玻片放置到焊盘上，静置2分钟，以允许为NaN 3的固定的动物。请注意，动物可以从幻灯片中后得分。动物会恢复从南3麻醉〜1小时后，如果转移到M9缓冲下降的种子NGM板。 </li><li>在这一点上是非常重要的，每一个实验者设计的关键跟踪滑动试验（+ VE）的幻灯片和幻灯片是控制（VE）的幻灯片，通过幻灯片，另一个人会盲目地得分GFP： EGL-4定位模式。这将控制任何实验者的偏见。 </li><li>分数50和100之间的每张幻灯片的动物直立使用荧光显微镜的放大倍数10X，40X和63X的目标和GFP / RFP过滤器。首先，找到动物用明视场照明的10倍。其次，找到AWC神经元关闭brigh，中叔场照明和使用RFP滤波器。 （P）的的ODR-1 ::红色荧光蛋白驱动器表达在AWB和AWC神经元。 AWC神经元与AWB细胞体是小的，圆形的形状（ 图2）相比，有独特的椭圆形细胞体。 </li><li> AWC细胞体一旦被找到，切换到GFP过滤器，并记录本地化的GFP：EGL-4核或胞质。使用反使实验者观察目镜，而得分幻灯片。注意：步骤5.1-5.5至少重复3次以产生统计学显着的数据（ 图1-2）在不同的日子。 </li></ol> 6。监测GFP标记的EGL-4从长期恢复过程中的气味适应<ol><li>步骤3.5后，除pyIs500动物的种群分成多个等份和得分核与细胞质GFP :: EGL-AWC在时间点零（即，80分钟后曝光）和Multiple随后的时间点（图3）。 </li><li>传输控制（VE）和实验（+）NGM板使用一次性的玻璃吸液管上的动物。 </li><li>让动物恢复不同的时间长度，重新审视定位在每个时间点GFP :: EGL-4英寸AWC在5.1至5.5的步骤。 </li></ol> 7。物料 NGM板：1升增加至1 L DDH 2 0：21克细菌的琼脂3克氯化钠（NaCl）的2.5克细菌蛋白胨。高压灭菌媒体。高压灭菌后琼脂，冷却，直到温度显示54°C.添加以下解决方案：25毫升1M磷酸钾缓冲液1毫升1 M的硫酸镁（ 硫酸镁 ）1毫升1 M氯化钙（ 氯化钙 ），1毫升乙醇中的胆固醇（5毫克/毫升股票）。 1 M二元K表2 HPO 4174.2克/摩尔：500毫升：在400毫升DDH 2 O，溶解87.1克DibasiC K 2 HPO 4。音量调节至500 L DDH 2 O。过滤消毒使用的500毫升无菌过滤器单元。 1 M一元KH 2 PO 4136.1克/摩尔：1 L：在800毫升DDH 2 O，溶解136.1克一元KH 2 PO 4。音量调节至1 L DDH 2 O。过滤消毒使用的500毫升无菌过滤器单元。 1 M磷酸钾（K 3 PO 4）缓冲液pH 6.0：1升1升的无菌过滤单元，加入868毫升的1 M一元KH 2 PO 4。允许单位来筛选，再加入132毫升的1 M二元K 2 HPO 4。让单位来过滤所有的液体。取出过滤器单元，并调节pH值至6.0。在室温下储存。 S-基肥：1 L：加入50毫升1M K 3 PO 4缓冲液，pH值约为6.0和20毫升5M NaCl溶液。填写1 LDDH 2 O。高压灭菌解决方案。 M9：对于1升：3克KH 2 PO 4，6克Na 2 HPO 4，5克NaCl，1毫升1M的用MgSO 4，H 2 O的填写DDH 2 0 1 L。高压灭菌消毒。

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没有利益冲突的声明。

这里描述的气味诱导核项目的GFP标记的EGL-4分子提供了一个强大的分子C.嗅觉适应中读出的线虫 。气味引起的核易位检测很简单，只需要几天的准备时间。 pyIs500，我们已经建立了这些实验动物，表达了标记，这说明了的AWC神经元以及表达GFP标记的EGL-4蛋白。因此，我们认为，实验者可以非常迅速，几乎没有工作经验这一类的神经元（也许是经检查几十动物）后开始感觉很舒服?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> Bacto琼脂</td><td> Difco公司</td><td> DF0140-07-4 </td><td> NGM板</td></tr><tr><td>氯化钠</td><td>费舍尔化工</td><td> S671-10 </td><td> NGM板</td></tr><tr><td>细菌用蛋白胨</td><td> Difco公司</td><td> DF0118-07-2 </td><td> NGM板</td></tr><tr><td>磷酸氢二钾</td><td>费舍尔化工</td><td> S375-500 </td><td> S-基底缓冲和NGM板</td></tr><tr><td>磷酸二氢钾</td><td>费舍尔化工</td><td> P285-500 </td><td> S-基底缓冲和NGM板</td></tr><tr><td> Kimwipes - 小</td><td> Kimberly-Clark公司</td><td> LS2770 </td><td></td></tr><tr><td>乙醇100％ </td><td>金盾化工有限公司有限公司</td><td> 43196-115 </td><td>稀释气味的趋化实验</td></tr><tr><td>氯化钙</td><td> Sigma-Aldrich公司</td><td> C8106-500G </td><td> NGM板</td></tr><tr><td>硫酸镁</td><td> MP Biomedicals </td><td> 150136-500G </td><td> NGM板</td></tr><tr><td>叠氮化钠99％ </td><td> Fisher Scientific则</td><td> ICN10289180 </td><td>麻醉剂</td></tr><tr><td>琼脂糖 - 超</td><td> Invitrogen公司</td><td> 16500-500 </td><td>琼脂糖凝胶垫</td></tr><tr><td>苯甲醛</td><td> Sigma-Aldrich公司</td><td> B1334-100G </td><td> AWC气味</td></tr><tr><td>丁酮，ACS级</td><td> Sigma-Aldrich公司</td><td> 360473-500ML </td><td> AWC气味</td></tr><tr><td>微量离心管 - 1.5毫升彩色</td><td>丹维尔</td><td> LS8147 </td><td></td></tr><tr><td>巴斯德移液管一次性玻璃5-3/4“ </td><td> Fisher Scientific则</tD&gt; <td> 13-678-20B </td><td></td></tr><tr><td> Stratalinker </td><td> Stratagene公司</td><td> Stratalinker 2400 </td><td> UV整合</td></tr><tr><td>过滤器真空瓶 - 500毫升</td><td>的Nalgene </td><td> 09-740-25B </td><td></td></tr></tbody></table>

a molecular readout of long-term olfactory adaptation in c. elegans

在持续的刺激感官的神经元减少其对信号的灵敏度，以适应他们的反应。适应的反应有助于形状的重视和保护细胞过度刺激。 C.适应内的嗅觉电路线虫最早是由Colbert和巴格曼1,2。在这里，作者定义的参数的嗅觉适应的范例，这是他们设计一个隔离的能力，以适应挥发性气味的化学传感器翼细胞C型（AWC）感觉神经元检测缺陷的突变体的遗传筛选。当野生型C.线虫动物暴露于一个有吸引力的AWC-检测到的气味3为30分钟，他们会适应他们的气味的响应，然后将忽略适应中的趋化行为〜1小时测定气味。当野生型C.线虫动物暴露于一个有吸引力的AWC-〜1小时检测到的气味，他们将忽略在交流的适应气味hemotaxis行为分析〜3小时。这两个阶段的嗅觉适应性C.线虫被描述为短期嗅觉适应30分钟后，气味暴露引起的，长期的嗅觉适应60分钟后，气味暴露引起的。 L&#39;Etoile酒店等。后来的工作，发现了一种蛋白激酶G（PKG）EGL-4的短期和长期的嗅觉适应性AWC神经元所需要的。 EGL-4蛋白含有核定位序列，有必要为长期的嗅觉适应性反应，但可有可无的短期嗅觉适应性反应的AWC 4。绿色荧光蛋白标记EGL-4，这是可能的可视化本地化的EGL-4在AWC暴露在长时间的气味。使用此功能齐全的GFP标记的EGL-4（GFP :: EGL-4）分子，我们已经能够开发出分子读出长期的嗅觉适应性AWC 5。使用这个米olecular读出的嗅觉适应，我们已经能够进行正向和反向遗传筛选，以确定突变的动物表现出有缺陷的亚细胞定位模式GFP :: EGL-AWC 6,7。在这里，我们描述：1）建造的GFP :: EGL-4表达动物; 2）的协议，用于培养动物长期臭味诱导核易位测定;和3）的得分长期臭味诱导核易位核GFP :: EGL-4状态的事件和恢复（重新过敏）。

本地化模式的GFP :: EGL-4英寸的AWC长时间气味曝光之前和之后的一个例子示出在图2中。在此之前长时间的气味曝光，GFP :: EGL-4定位于细胞质中的AWC（图2B），80分钟后，气味暴露GFP :: EGL-4定位于细胞核内的AWC（图2D）。在行为层面上，与胞内GFP :: EGL-4 AWC动物被吸引到一个点源的气味（ 图2C图形代表的趋化实验结果的不适应动物-注意的趋化指数接近1?...

Watch this Scientific Journal Video about A Molecular Readout of Long-term Olfactory Adaptation in C. elegans at JoVE.com

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

在这里，我们描述了一个长期的嗅觉适应性的分子读出<em&gt;秀丽隐杆线虫</em&gt;。蛋白激酶G，EGL-4，稳定的适应措施是必要的，在初级感觉神经元对AWC。在长时间的气味暴露EGL-4转位从细胞质到细胞核的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 ...

a-molecular-readout-of-long-term-olfactory-adaptation-in-c-elegans

Research

JoVE Journal

Neuroscience

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

10.7K 次观看.  George Washington University. 在这里，我们描述了一个长期的嗅觉适应性的分子读出秀丽隐杆线虫。蛋白激酶G，EGL-4，稳定的适应措施是必要的，在初级感觉神经元对AWC。在长时间的气味暴露EGL-4转位从细胞质到细胞核的AWC。

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

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment through neuron-specific detection of food and chemical odors1, 2, and can associate nutritive states with chemical odors3, temperature4, and the pathogenicity of a food source5.
Here, we describe assays of C. elegans associative learning and short- and long-term associative memory. We modified an aversive olfactory learning paradigm6 to instead produce a positive response; the assay involves starving ~400 worms, then feeding the worms in the presence of the AWC neuron-sensed volatile chemoattractant butanone at a concentration that elicits a low chemotactic index (similar to Toroyama et al.7). A standard population chemotaxis assay1 tests the worms' attraction to the odorant immediately or minutes to hours after conditioning.
After conditioning, wild-type animals' chemotaxis to butanone increases ~0.6 Chemotaxis Index units, its "Learning Index". Associative learning is dependent on the presence of both food and butanone during training. Pairing food and butanone for a single conditioning period ("massed training") produces short-term associative memory that lasts ~2 hours. Multiple conditioning periods with rest periods between ("spaced training") yields long-term associative memory (&lt;40 hours), and is dependent on the cAMP Response Element Binding protein (CREB),6 a transcription factor required for long-term memory across species.8
Our protocol also includes image analysis methods for quick and accurate determination of chemotaxis indices. High-contrast images of animals on chemotaxis assay plates are captured and analyzed by worm counting software in MatLab. The software corrects for uneven background using a morphological tophat transformation.9 Otsu's method is then used to determine a threshold to separate worms from the background.10 Very small particles are removed automatically and larger non-worm regions (plate edges or agar punches) are removed by manual selection. The software then estimates the size of single worm by ignoring regions that are above a specified maximum size and taking the median size of the remaining regions. The number of worms is then estimated by dividing the total area identified as occupied by worms by the estimated size of a single worm.
We have found that learning and short- and long-term memory can be distinguished, and that these processes share similar key molecules with higher organisms.6,8 Our assays can quickly test novel candidate genes or molecules that affect learning and short- or long-term memory in C. elegans that are relevant across species.

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

Here we describe methods to test C. elegans associative learning and short- and long-term associative memory. These population assays employ the worms abilities to chemotax toward volatile odorants, and form positive associations upon pairing food with the chemoattractant butanone. Increasing the number of conditioning periods induces long-term memory.

线虫的积极丁酮学习，短期，长期联想记忆检测

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment ...

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 ...

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, ...

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and social interactions can be best studied by observing unrestrained, freely behaving animals. Weakly electric fish (WEF) display readily observable exploratory and social behaviors by emitting electric organ discharge (EOD). Here, we describe three effective techniques to synchronously measure the EOD, body position, and posture of a free-swimming WEF for an extended period of time. First, we describe the construction of an experimental tank inside of an isolation chamber designed to block external sources of sensory stimuli such as light, sound, and vibration. The aquarium was partitioned to accommodate four test specimens, and automated gates remotely control the animals&#39; access to the central arena. Second, we describe a precise and reliable real-time EOD timing measurement method from freely swimming WEF. Signal distortions caused by the animal&#39;s body movements are corrected by spatial averaging and temporal processing stages. Third, we describe an underwater near-infrared imaging setup to observe unperturbed nocturnal animal behaviors. Infrared light pulses were used to synchronize the timing between the video and the physiological signal over a long recording duration. Our automated tracking software measures the animal&#39;s body position and posture reliably in an aquatic scene. In combination, these techniques enable long term observation of spontaneous behavior of freely swimming weakly electric fish in a reliable and precise manner. We believe our method can be similarly applied to the study of other aquatic animals by relating their physiological signals with exploratory or social behaviors.

We describe a set of techniques for studying spontaneous behavior of freely swimming weakly electric fish over an extended period of time, by synchronously measuring the animal&#39;s electric organ discharge timing, body position and posture both accurately and reliably in a specially designed aquarium tank inside a sensory isolation chamber.

自由游泳弱电鱼的长期行为跟踪

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and ...

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 ...

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has proven to be an invaluable model organism in helping to tackle this challenge, from inspiring technological approaches,&#160;building the human brain connectome, to actually shedding light on the specific molecular drivers of basic functional patterns. The bulk of the behavioral studies in C. elegans have been performed on solid substrates. In liquid, animals exhibit behavioral patterns that include movement at a range of speeds in 3D, as well as partial body movements, such as a posterior curl without anterior shape change, which introduce new challenges for quantitation. The steps of a simple procedure, and use of a software that enables high-resolution analysis of C. elegans swim behavior, are presented here. The software, named CeleST, uses a specialized computer program that tracks multiple animals simultaneously and provides novel measures of C. elegans locomotion in liquid (swimming). The measures are mostly grounded in animal posture and based on mathematics used in computer vision and pattern recognition, without computational requirements for threshold cut-offs. The software tool can be used to both assess overall swimming prowess in hundreds of animals from combined small batch trials and to reveal novel phenotypes even in well-characterized genetic mutants. The preparation of specimens for analysis with CeleST is simple and low-tech, enabling wide adaptation by the scientific community. Use of the computational approach described here should therefore contribute to the greater understanding of behavior and behavioral circuits in the C. elegans model.

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

An efficient and simple methodology for computer-based analysis of nematode swimming behavior in liquid is described. The method requires little to no investment for C. elegans laboratories. The hardware used is standard, and the computer software for the behavioral analysis (CeleST) is an open source one.

的自动分析<em&gt;℃。线虫</em&gt;游泳行为使用天书软件

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

嗅球的四次免疫染色用于嗅觉感觉轴突分子识别码的可视化

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its completely mapped nervous system and transparent anatomy, presents an ideal model for understanding real-time neural dynamics using calcium indicators. In combination with microfluidic technologies and experimental designs, calcium-imaging studies using these indicators are performed in both free-moving and trapped animals. However, most previous studies utilizing trapping devices, such as the olfactory chip described in Chronis et al., have devices designed for use in the more common hermaphrodite, as the less common male is both morphologically and structurally dissimilar. An adapted olfactory chip was designed and fabricated for increased efficiency in male neuronal imaging with using young adult animals. A turn was incorporated into the worm loading port to rotate the animals and to allow for the separation of the individual neurons within a bilateral pair in 2D imaging. Worms are exposed to a controlled flow of odorant within the microfluidic device, as described in previous hermaphrodite studies. Calcium transients are then analyzed using the open-source software ImageJ. The procedure described herein should allow for an increased amount of male-based C. elegans calcium imaging studies, deepening our understanding of the mechanisms of sex-specific neuronal signaling.

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of an adapted &#34;olfactory chip&#34; for the efficient calcium imaging of C. elegans males is described here. Studies of male exposure to glycerol and a pheromone are also shown.

应用一种适应微流控嗅觉芯片的神经活性成像对雄性线虫神经元的信息素反应

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its ...

Generation and Long-term Maintenance of Nerve-free Hydra

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The interstitial cells can be eliminated through two consecutive treatments with colchicine, a plant-derived toxin that kills dividing cells, thus erasing the potential for renewal of the differentiated cells that are derived from the interstitial stem cells. This allows for the generation of Hydra that lack nerve cells. A nerve-free polyp cannot open its mouth to feed, egest, or regulate osmotic pressure. Such animals, however, can survive and be cultured indefinitely in the laboratory if regularly force-fed and burped. The lack of nerve cells allows for studies of the role of the nervous system in regulating animal behavior and regeneration. Previously published protocols for nerve-free Hydra maintenance involve outdated techniques such as mouth-pipetting with hand-pulled micropipette tips to feed and clean the Hydra. Here, an improved protocol for maintenance of nerve-free Hydra is introduced. Fine-tipped forceps are used to force open the mouth and insert freshly killed Artemia. Following force-feeding, the body cavity of the animal is flushed with fresh medium using a syringe and hypodermic needle to remove undigested material, referred to here as "burping". This new method of force-feeding and burping nerve-free Hydra through the use of forceps and syringes eliminates the need for mouth-pipetting using hand-pulled micropipette tips. It thus makes the process safer and significantly more time efficient. To ensure that the nerve cells in the hypostome have been eliminated, immunohistochemistry using anti-tyrosine-tubulin is conducted.

Generation and Long-term Maintenance of Nerve-free Hydra

Through a double treatment with colchicine, a plant-derived toxin that kills dividing cells, nerve-free Hydra vulgaris can be generated. These Hydra cannot feed or egest on their own. This paper describes an improved method for long-term maintenance of nerve free Hydra vulgaris in the laboratory.

无神经的一代和长期维持<em&gt;水润</em

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The ...