笔者想感谢贝纳姆A.协助协议的发展。这项工作是由NIH资助NS06747 BAB

1。成像蠕虫的制备这部分协议的基础上发表C.线虫培养技术1,2， 图1中概述。 <ol><li>生长在高的蛋白胨NGM的琼脂板（10厘米）播种与NA22 ... E.蠕虫大肠杆菌的细菌，直到近饿死。染色，如果一个板块将可靠地生产出足够的蠕虫1个人污点，一路上，考虑损失和用于选择成像蠕虫（见第2部分）的严格标准。 </li><li> DDH 2 0几毫升倾吐板丰年虫，简要纷飞，液体倒入15毫升锥形管（如果必要的转移大部分的蠕虫管重复）。颗粒为3分，1000倍Ğ临床离心机。 DDH 2 O的清洗颗粒1-3X，直到上清液是明确的，以除去残留的细菌。使用聚丙烯转移移液器去除上清，蠕虫坚持以玻璃（从这个步骤开始，这是关键，以尽量减少步骤之间的时间，以避免在卵子活力的损失）。 </li><li>最后一次洗涤后，悬浮的蠕虫病毒颗粒5毫升碱性次氯酸钠溶液溶解蠕虫和释放卵子。轻轻摇动，并检查管解剖显微镜下每分钟一次。当约50％的蠕虫裂解（他们会出现弯曲和破开），终止灌装管顶端鸡蛋缓冲，翻转几次，3分钟，1000XĞ造粒裂解。溶解时间不得超过5分钟。 </li><li>用移液管取出上清液，用鸡蛋缓冲颗粒3X。 </li><li>分开碎片鸡蛋，漂浮在DDH 2 O的 30％蔗糖垫：从1.4步的最后一次洗涤后，小心地取出5毫升DDH 2 0上清，重悬沉淀。加入5毫升无菌DDH 2 O的 60％的蔗糖，拌匀。在临床离心机旋转6分钟，Ğ1000倍。鸡蛋会收集半月板（他们将哈VE的外观阴天），而碎片会沉淀。 </li><li>使用塑料移液管，吸鸡蛋在一个最小的体积，并转移到非种子选手NGM的平板。坚持管侧的任何鸡蛋，可冲洗下来轻轻地转移以及。一个10厘米非种子选手，每盘分析应变是足够的。 </li><li>鸡蛋隔夜（16小时）在20°C孵育的非种子选手板发泄板的前几个小时，干它留下了几个小时的盖子稍微偏离，但一定要覆盖过夜。 </li><li>孵化后，转让的L1幼虫在S基底的圆锥形管15毫升（添加一个几毫升S基底板，漩涡，倒入管）。佩莱在临床离心机（3分，1000倍Ğ）悬浮在体积最小的S基底，并转移至10厘米NGM的琼脂板NA22 ...细菌接种。这里每个步骤1.1原板使用2板。孵育蠕虫，直到达到所需的年龄。 </li><li>监测每天一次或两次的文化。如果他们是在危险Øf挨饿，转移到新鲜NA22 ...板。对于野生型N2蠕虫，饥饿之前，整个板块移动作为动物迁移大批远离食品耗尽区形成了一条线或波蠕虫。食品一旦这种形式，将在几个小时内完全耗尽。 </li><li>蠕虫现在准备染色或实时成像。悬浮液中的固定和染色蠕虫是最理想的，很容易因为大量完整的蠕虫可以成像。染色方法的基础上芬尼和Ruvkun的协议3-6冻结的破解程序是可取的，因为需要大量的蠕虫成像。 </li></ol> 2。共聚焦成像<ol><li>装入上聚焦成像幻灯片的蠕虫病毒。蠕虫密度调整到每微升几百。使在2％琼脂糖垫的S-基底四周环薄薄的凡士林，以防止在成像蒸发（通过3毫升装上了截止25号针头注射器适用）。吸取1蠕虫悬挂几微升到盖玻片（使用麻醉剂，如果成像活虫），降低到盖玻片的琼脂糖垫，遍布垫的蠕虫均匀。 </li><li>选择蠕虫成像。蠕虫利益的突触是面向物镜，没有更多的径向位移，超过±45°（0°是指直接朝着目标导向）选择。快速执行此评估（几秒钟），以避免漂白。初步培养规模进行了优化，以确保足够的蠕虫将满足这些几何条件。 </li><li>完成标本共聚焦成像。相对平坦的样品，可在约14个0.4微米厚的部分包含产生最好的结果。高速，低分辨率图像（512 x 512像素）有足够的快速准确的数据采集。避免饱和过度的信号强度探测器，因为这将导致低估荧光信号。 </li></ol> 3。个体突触集群的自动识别和分析<ol><li>开放式多TIF共聚焦显微镜使用，Volocity 4.0（或更高）软件（珀金埃尔默）的输出文件。 </li><li>作物以外地区的形象，不包含利益结构（图片- &gt;扩展聚焦- - &gt;动作- &gt;使用“写意的投资回报率的工具的兴趣选择面积&gt;作物以选择; 图2A）。注意，扩展重点在电脑屏幕上产生的z平面的投影来帮助你分析，但不影响基础数据;同样是真实的亮度和对比度调整，如果必要的话）。 </li><li>使用直线工具分析测量的长度。将计算该行的长度，并添加到数据表（ 图2B）。 </li><li>识别物体使用强度对象的过滤器（测量模式）。突触集群指定一个阈值强调和非突触的背景是不是（ 图2C）。列入用另一种颜色标记一个独立的突触标记可能有助于明确确定的突触。典型的突触范围从几个到几百个像素。设置阈值，一旦使用控制样品，并用它为所有后续的样品。阈值每个样品分别是不可接受的，因为它引入了实验者偏见的潜力。对象被选中自动汇总（ 图2D）。 </li><li>安排和导出数据。消除2个或更少的像素的对象，因为这些通常是背景斑点。这些都可以通过进一步筛选淘汰，或在随后的分析。 “对象大小”，以方便他们搬迁下游，重新排序的数据文件，所以他们将所有组。在CSV格式，可以通过Microsoft Excel或其他电子表格程序打开导出的数据。每个图像产生一个输出文件。 </li><li>如果Volocity软洁具是不可用，替代的方法，Z平面聚焦数据分析预测，可利用（如文献。7，或使用ImageJ的手动分析），虽然3维信息丢失（见讨论）。 </li></ol> 4。代表结果量化的方法应该是能够区分不同亮度和不同的卷集群种群。 图3中的代表图像和相应的定量数据表明这些参数的基础上分化的例子。作为一般规则，结果应符合什么是显而易见的眼睛。在UNC-49 GABA的免疫受体的腹神经索C 的情况下线虫 ，所有的动物通常出现在规模和成熟度非常相似，一组五个蠕虫（归分析神经线的长度）的总突触的荧光值显示标准错误<html>值约10％的平均4。基因突变和其他试验性治疗（如药物暴露）可能会改变，不仅数量和强度值和突触集群频率分布，但可能发展的时间进程和同步的蠕虫，从而导致更高的变异。然而，定量结果应该总是反映什么可以欣赏视觉的图像，Volocity确定应该显示发生错误的位置，并提出纠正措施，如阈值或去除不真实对象的对象，如果没有，检查。 <img alt="图1" src="/files/ftp_upload/4090/4090fig1.jpg" /> 图1。准备同步C.从这个过程中得到的文化。同步文化，因为C。线虫逮捕和发展的L1幼虫阶段在没有食物的情况下，恢复食物时介绍。 <html> ove_content“&gt; <img alt="图2" src="/files/ftp_upload/4090/4090fig2.jpg" /> 图2。定量突触puncta。 <ol class="ualpha"><li>外来地区的形象出现，以减少文件大小和增加特异性。左手面板显示图像开始，中间面板显示选择区域，右侧面板显示图像后种植。红色信号是UNC-49 GABA受体免疫，绿色信号是突触的绿色荧光蛋白（突触标记）表示GABA的神经元在突触前4。 <a href="http://www.jove.com/files/ftp_upload/4090/4090fig2alarge.jpg" target="_blank">点击这里查看大图</a> 。 </li><li> A线的神经线部分进行分析得出的长度，其长度将被自动记录在结果表中。这个信息是必要的正常化由于突触数据分析的神经线的长度，它可以改变数倍，如果蠕虫变得支离破碎，在染色。www.jove.com/files/ftp_upload/4090/4090fig2blarge.jpg“目标=”_blank“&gt;点击这里查看大图。 </li><li>阈值用于识别单个突触集群。左侧面板显示图像前阈值;不同的阈值水平的例子是在单独的面板（绿色信号略）所示。每个标识的对象是作为一个彩色的地区形象的描绘。注意最左边的面板在视觉上明显的突触集群和最右边的面板上的彩色地区之间的对应关系。 <a href="http://www.jove.com/files/ftp_upload/4090/4090fig2clarge.jpg" target="_blank">点击这里查看大图</a> 。 </li><li>在结果表中，每一个确定的对象，单独列出。可以选择单个对象，对图像进行检查，如果需要强调的。注意：该表中包含的信息为红色（罗丹明）和绿色（绿色荧光蛋白“）的渠道;绿色通道信息可能是误导，因为对象是根据严格红色荧光。也可以进行这种分析，在单一的彩色图像。 <a href="http://www.jove.com/files/ftp_upload/4090/4090fig2dlarge.jpg" target="_blank">点击这里查看大图</a> 。 </li></ol><img alt="图3" src="/files/ftp_upload/4090/4090fig3.jpg" /> 图3。代表结果。代表显微镜，从野生型C. UNC-49 GABA受体染色前线虫 （A）和（B）与治疗，GABA受体激动剂蝇蕈醇导致受体下调后成为长期暴露后。面板显示前（左）后（右）阈值和拆除的小背景斑点的裁剪图像。 （三）图所示的标本定量突触参数（A）和（二）：总荧光归到神经线长（左），和累积概率直方图个别突触荧光内容（中）和突触的体积（和规格的T），显示出统计学显着减少突触的内容和数量激动剂曝光（N = 60为未经处理的，N = 115蝇蕈处理突触的突触诱导，P &lt;0.001，Kolmogorov-Smirnov检验<a href="http://www.physics.csbsju.edu/stats/KS-test.html" target="_blank">http://www.physics.csbsju .edu /统计/ KS-test.html的</a> ）。

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没有利益冲突的声明。

这里介绍的方法设计到突触的大量人口在 C中提取的多参数的定量数据线虫 ，同时最大限度地提高治疗组内的一致性。三个特点有助于实现这些目标。首先，免疫组化进行同步蠕虫种群，以确保所有的动物都是一样的年龄。这一步是至关重要的，因为发育调控的表达水平可能掩盖的实验性治疗的效果（如UNC的49 GABA受体免疫变化的若干倍在发展（光戴维斯等人，在准备中））。...

<table border="1"><tbody><tr><td> 该软件的名称 </td><td> 公司 </td><td></td><td> 评论（可选） </td></tr><tr><td> volocity V4.0或更高</td><td>珀金埃尔默/ Improvision </td><td></td><td>检查你访问这个软件的本地影像核心设施。演示软件可在珀金埃尔默网站。这种方法只需要Volocity定量模块。 </td></tr><tr><td></td><td></td><td></td><td> 表2。特定的试剂和设备。 </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> 鸡蛋缓冲区 </td><td>氯化钠 氯化钾 氯化钙 MgCl 2的 肝素钠 （调整pH值至7.3） </td> &lt;TD&gt; 118毫米 48毫米 2毫米 2毫米 24毫米</td></tr><tr><td> 碱性次氯酸钠（5毫升） </td><td>新鲜的家用漂白水（丢弃后30天内开瓶） 10N的氢氧化钠 DDH 2 O的 </td><td> 1.0毫升0.25毫升 3.75毫升</td></tr></tbody></table></td></tr><tr><td></td><td></td><td></td><td> 表1。解决方案。 </td></tr></tbody></table>

automated quantification of synaptic fluorescence in c. elegans

突触的强度是指突触突触前神经递质释放的事件的反应幅度，并有一个整体的神经电路功能产生重大影响。突触强度严重依赖于大量聚集在突触的突触后膜部位的神经递质受体。受体水平的发展，建立和受体之间贩运表面本地化，突触，细胞内池代表突触可塑性和神经调节的重要机制，可以通过改变。严格的方法来量化本地化的突触神经递质受体的丰度是必不可少的，以研究突触的发展和可塑性。荧光显微镜是一个最佳的方法，因为它保留了空间信息，区分从非突触池的突触，和本地化为不同类型的突触受体人口之间的歧视。遗传模式生物线虫elegans是特别适合这些研究中，由于体积小，相对简单的神经系统，其透明度，并提供强大的基因技术，使本地完整的动物突触检查。 在这里，我们提出了一个定量荧光标记C.突触神经递质受体的方法线虫 。其主要特点是多面的共聚焦显微镜输出文件，制表位置，体积，荧光强度，并为每个突触的总荧光三个维度的单个突触的自动识别和分析。这种方法有两个超过手册z平面聚焦数据预测分析的主要优点。首先，因为每一个聚焦数据集平面，没有数据丢失通过Z-平面上的投影，通常是基于像素的强度平均值或最大值。其次，识别突触是自动化的，但可以通过前检查perimenter作为数据分析所得，允许快速，准确地从大量的突触中提取数据。数百数千突触每个样品可以很容易地获得，产生大量的数据集，以最大限度地提高统计能力。注意事项准备C。进行分析，并进行聚焦成像，以尽量减少动物在治疗组之间的变异线虫进行了讨论。尽管分析C。线虫突触后受体，这种方法通常是有用任何突触本地化的蛋白质，事实上，任何荧光信号，定位于离散集群，puncta，或细胞器的类型。 执行过程中的三个步骤：1）制备样品，2）聚焦成像，和3）图像分析。具体到 C步骤1和2 线虫 ，而第3步是普遍适用于任何点状共聚焦显微镜的荧光信号。

Watch this Scientific Journal Video about Automated Quantification of Synaptic Fluorescence in C. elegans at JoVE.com

Automated Quantification of Synaptic Fluorescence in C. elegans

强烈的大量聚集在突触的神经递质受体影响突触强度。这种方法量化单突触决议的荧光标记的神经递质受体在三个层面<em&gt; C。线虫</em&gt;，让数百个突触被迅速的特点是在一个单一的样本没有推出Z-平面上的投影的扭曲。

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 ...

automated-quantification-of-synaptic-fluorescence-in-c-elegans

Research

JoVE Journal

Neuroscience

Automated Quantification of Synaptic Fluorescence in C. elegans

10.2K 次观看.  University of Toledo. 强烈的大量聚集在突触的神经递质受体影响突触强度。这种方法量化单突触决议的荧光标记的神经递质受体在三个层面 C。线虫，让数百个突触被迅速的特点是在一个单一的样本没有推出Z-平面上的投影的扭曲。

视频: Automated Quantification of Synaptic Fluorescence in C. elegans

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

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

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

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

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 ...

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 ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

横向扩散和培养的神经元膜蛋白的胞吐评估使用荧光恢复和荧光损失漂白

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

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 ...

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

嵌入后突触蛋白的免疫标记海马切片培养

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

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, ...

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 ...

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 ...