几个人本文中所描述的工作做出了贡献。 ，CVG建立了实验装置，并，LS，SHC，和CVG进行了实验。 CVG和，SHC写的稿件。随后所有的作者参加了在修订过程中，批准了最后一份手稿的复印件。我们感谢保罗·斯腾伯格的GCaMP应变。在这项工作中所用的一些线虫菌株的秀丽隐杆线虫的遗传学中心（CGC），这是由美国国立卫生研究院国家研究资源中心（NCRR）。在18改编而成，使用MATLAB图像分析程序。支持作者，美国波士顿大学和麻省理工生命科学中心。

1。光学装置<ol><li>使用标准的复合式显微镜，荧光显微镜的成像能力。我们使用了尼康的Eclipse的Ti-U倒置显微镜与一个Intensilight的HG照明。 </li><li>为了获得最佳的图像和信号质量，使用的高倍率，高数值孔径的目标。我们通常使用的尼康X60 1.4 NA浸油的目标。在某些情况下，它是可能的荧光团的表达水平和信号强度取决于使用低放大倍率（X40，X20）。 </li><li>使用高灵敏度的冷却CCD相机，如安道尔克拉拉相机，以最大限度地提高图像的灵敏度和降低背景噪音。 </li><li>对于单一颜色的荧光基团使用标准的显微镜，荧光过滤器设置特定波长的荧光。对于GCaMP使用标准的GFP过滤器设置优化，激发波长为488 nm，发射波长为525 nm。没有进一步的光学修改是必需的。 </li><li&gt;对于成比例的荧光基团，如卡默莱昂中，使用相同的图像，使得可以在两个不同的波长同时捕捉一个双成像系统。这是通过相机上投影的双重图像侧由侧。一个简单的设置要做到这一点，使用基本的光学元件，光学级面包板， 如图1所示。它由以下部分组成： <ol class="ualpha"><li>显微镜过滤器设置440±20 nm激发过滤器和455 nm的长传分色镜。 </li><li>图像遮罩初始图像放置在平面外，在显微镜成像端口（在摄像机的CCD传感器通常会坐）。这限制了字段使得它将填补只有一半的相机传感器的视图。把这个面膜的第一个图像平面形成了鲜明的图像边界，没有重叠的两个影像投射端。 </li><li>第一中继透镜焦距设定一个远离吨他最初的图像平面，它的光准直。 </li><li>的二色性反射镜的光分割成两个单独的成像波长或反射一种颜色（&gt; 515 nm处，黄色）的信道同时发送的​​其他的（&lt;515 nm处，青色）。 </li><li>发射滤光器（带通滤波器）的透射光的特定成像波长（青色，在485±40nm的和黄色的，在535±30nm的）进一步限制， </li><li>放置在使得它重新聚焦的光在CCD摄像机的第二图像平面上的光束路径的第二中继透镜（每个通道一个）。 </li><li>甲对反射镜和一个第二分色镜的光束导向路径，使得两个图像重组，并投射到相机的CCD传感器侧由侧， 图4A示出所得到的图像的一个例子。 </li></ol></li><li>初始光：启动设置和校准的光学系统具有高对比度的成像E使用低放大倍率透镜和发送的宽视场照明（在显微镜载玻片上绘制一个黑色夏普效果很好）。带来的图像集中通过显微镜目镜，然后切换为要使用的端口的显微镜。随着高亮度照明查看在最初的焦平面（一个数厘米外显微镜端口），索引卡上的图像。第一中继透镜（C）放置在光束路径，例如，它是一个从最初的焦平面的焦点距离，并对其进行准直的光，而没有偏转的光束路径（ 即直接集中在光束路径）。掩模放置在第一焦平面，调整，以限制的视场的一半，并在图像中产生一个尖锐的边境。位置的反射镜和过滤器（D，E，G）的 ​​光束路径的表表面保持平行，在两个平行的路径侧由侧运行，如在图1B中所示。中心的第二中继透镜（F）在其各自的电子束路径和来回移动它们沿着路径带来的重点放在相机的CCD传感器将在索引卡上的图像。如果做得正确的应该是齐焦显微镜的目镜和其他相机端口的两个图像。最后将相机放置在位置和微调的对齐方式。 </li><li>微调光学校正：使用双图像由CCD相机观察微调光学对准。使用高对比度的图像，并从低倍率，切换到更高的放大倍率进行最终对齐。透射图像（青色通道在图1B）的位置以覆盖通过移动相机，在X和Y位置的反射图像（黄色在图1B中的信道），以覆盖的领域中的另一半的一个相机的视场的一半查看由调整最后两个反射镜（G），在它的光束路径中。优化集中为每个通道通过移动第二中继透镜沿着光束路径来回。如果左不变的光学装置应该是稳定的，并且只需要罕见的微调调整。 </li></ol> 2。样品制备和数据采集。 <ol><li>收购在神经元中的利益表达钙敏感的荧光从秀丽隐杆线虫的遗传学中心（CGC）或其他来源的动物。可以使用标准 C或者转基因动物线虫技术。单细胞的表达是不是必要的（有时是不希望的，如果多个神经元以进行调查，参见图12），只要从多个细胞的图像和测量可以容易地分离。如果没有整合到基因组的荧光转基因的表达显着变化，可以从动物到动物发生。在这种情况下，预选的动物，使用荧光显微镜具有更高的表达水平增加的信号 - 噪声比，并提高了测量。荧光基团表达水平的东东d，来足以很容易地与相机成像根据需要通过实验的采集参数。 </li><li>进行所需的所需的固定化技术（见下面2.3）按两片玻璃之间的滑动由两块实验室磁带隔开一小滴的熔融琼脂糖琼脂糖焊盘。如果有必要，琼脂糖焊盘可以被转移到一个玻璃盖玻片，在安装前的动物。请参阅图2。 </li><li>固定C.线虫成像使用以下技术之一： <ol class="ualpha"><li>药理瘫痪：0.05％至2％琼脂糖凝胶垫（搅拌到熔化的琼脂糖凝胶垫在2.2前）左旋咪唑。选择5-10垫到动物，并盖上盖玻片。应用少量的热蜡的混合物（50％的石蜡和50％凡士林油）的盖玻片的角部，以保持恰当的位置。允许5-10分钟的左旋咪唑生效和动物到Become完全静止。 </li><li>僵硬的琼脂糖固定：C.线虫可以在物理上固定用生硬的10％琼脂糖凝胶垫和直径为0.05微米的聚苯乙烯纳米颗粒。 10％的琼脂糖垫（10％琼脂糖NGM缓冲，以重量计）为2.2。添增〜3μl的珠溶液（1:10到NGM聚苯乙烯微球以体积计）的垫的顶部。很快，挑选5-10 C.线虫入池垫珠溶液，轻轻地盖上盖玻片。使用熔化的蜡施加到盖玻片的角部，以保持恰当的位置。 （有关这项技术的更多详细信息，请参阅7以及以前的朱庇特“第8条） </li><li>胶：C.线虫可以胶合到位使用施加到一侧的动物的兽医级粘合剂的少量。 </li><li> ：许多微流控芯片的微流体装置被设计成物理按住 C 线虫在成像。 </li></ol> &lt;/ P&gt; <li>在显微镜下将准备好的幻灯片，并集中在所需的神经元。它往往是最容易找到的C.用明视场照明，低倍率的线虫 。然后切换到高放大倍率和荧光成像技术找到并专注于特定的神经元。 </li><li>刺激：如果一个特定的刺激神经元的响应是理想的（光，电场，味觉和嗅觉，温度等），刺激装置必须设计到显微镜的设置，这样，它可以施用于动物在而成像的样品制备。 </li><li>使用显微镜CCD摄像头采集图像。曝光时间和激发光的水平将取决于在基线上的荧光信号和表达水平，与较弱的信号，需要较长的曝光时间一致地更少的时间分辨率。我们通常使用〜300毫秒的曝光时间。 </li><li>单独或作为TI获取图像我的失效电影。由于C。线虫的神经元一般显示缓慢激活动力学（ 即他们缺乏钠的动作电位），速度相对较慢〜1秒的帧速率往往足以测量神经元动态。采集速率高达〜虽然这些弱荧光所需的积分时间可能是有限的，已被使用的每秒90帧。个人的电影的一个单一的神经元，可以很容易地扩展为几分钟的时间（约5-10分钟）或更长的时间，如果固定化制剂是稳定的，和帧速率相对缓慢。 </li></ol> 3。数据分析<ol><li>有许多ImageJ的NIS-Elements的分析功能，包括动态和比例不同的荧光成像软件套件。我们用一个简单的自定义MATLAB程序，测量平均荧光信号在选定的地区的利益。 </li><li>对于个别图像（或时间推移电影仍然存在完全静止）选择一个感兴趣的区域（ROI）内成像的神经元和一个单独的邻近地区为背景的测量。对于比例的图像，选择类似的ROI和背景区域的双重图像。 </li><li>成像的神经元内的时间推移电影的轻微移动而需要额外的分析自动选择在每个帧中的投资回报率，这可以通过使用大量的对象跟踪计划。我们的程序会显示一个压缩后的图像从一个粗略的边界（涵盖所有帧中的对象的利益），手动选择和一个独立的背景区域选择在影片中的所有图像。的第一帧，手动选择的ROI内的边界区域。对于每个随后的帧，程序选择的最亮的像素的边界区域内，以产生正确的尺寸（ 即相同数量的像素作为手动的第一帧的ROI）的ROI。对于图像的物镜t的利益有足够的亮，然后在协议选择一个合理的投资回报率每帧的边界地区周围的背景。 </li><li>的荧光信号，F，对于每个帧被计算为在ROI减去的背景区域中的平均强度的平均像素强度。的成比例的值的计算方法的两个信号的比值（YFP-YFP 背景 ）/（CFP-CFP 背景 ）-0.65。 0.65的因子补偿通过YFP的通道，这将取决于上的荧光基团，以及使用的特定的过滤器集（该值是通常〜0.6为卡默莱昂设置中）的CFP通道进入泄放。 </li><li>对于每个帧被计算为相对钙水平的百分比变化的强度归一化到一个初始基线测量值从第一帧（或一系列的帧）：ΔF/ F =（F（t）的F o的）/ Fò，其中，F（t）是在时间t 和 F o的荧光的初始基线值。 </li><li>甲的投资回报率，选择细胞体产生最强的最强大​​的信号，但它也可能选择和测量信号从ROI沿神经进程。 </li></ol> 4。问题解决<ol><li> 漂白可能会导致暴露于激发光漂白的荧光标记信号是依赖于暴露水平稳步下降。比例测量补偿。但是一个波长的选择性漂白仍时有发生（通常CFP漂白剂更快YFP）。如果白化现象减少曝光水平，通过减少曝光时间和/或激发光强度（在照明光路中的与中性密度过滤器）。如果有必要，可以通过运行模拟试验（与相同的激励曝光，但没有神经元的刺激）和量化的信号衰减的时间常数补偿用于漂白。 </li><li><stronG&gt;的固定化：对于简单图像分析中是非常重要的动物记录期间仍然保持相对。C.线虫曝光和实验刺激作出反应，使它们最有可能在记录时移动。轻微的运动可以显着测量到的噪音，尤其是当测量轴突的过程。成比例的分析，有助于减轻这些影响在一定程度上和从细胞体的测量更强大。认真执行和完善的固定程序将导致在完全静止的动物。 </li><li> 背景选择：成功的形象分析，精心挑选的背景区域是必要的。背景区域应该是合理均匀，然后成像的神经元更暗。它必须被充分除去，以避免散射光（从而拾取器的信号），从成像的神经元。我们找到一个统一的地区20-50微米的投资回报率在大多数情况下。 </li><li> 成比例的光学系统：光学的成像光学元件的正确对准是用于产生一个明确的双图像的关键。商业比例成像显微镜附件。另一种方法是采用快速连续成像，使用快速过滤器轮之间快速切换，发射波长为每个单独的图像，同时记录。 </li><li> 成比例分析：这里所描述的分析是一个相对简单的技术，凭借平均超过ROI生成鲁棒信号。它需要一个公认的明亮的物体选择的投资回报率。更复杂的的比例的分析是可能的精确对准和映射使得荧光的比例是可以计算的像素由像素的双重图像。 </li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+mediates+context-dependent+modulation+of+sensory+plasticity+in+C.+elegans.">Dopamine mediates context-dependent modulation of sensory plasticity in C. elegans.</a> Neuron. 55, 662-676 (2007).</li><li>Chalasani, S. H., Chronis, N., Tsunozaki, M., Gray, J. M., Ramot, D., Goodman, M. B., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+a+circuit+for+olfactory+behaviour+in+Caenorhabditis+elegans.">Dissecting a circuit for olfactory behaviour in Caenorhabditis elegans.</a> Nature. 450, 63-70 (2007).</li><li>Clark, D. A., Gabel, C. V., Gabel, H., Samuel, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+activity+patterns+in+thermosensory+neurons+of+freely+moving+Caenorhabditis+elegans+encode+spatial+thermal+gradients.">Temporal activity patterns in thermosensory neurons of freely moving Caenorhabditis elegans encode spatial thermal gradients.</a> J. Neurosci. 27, 6083-6090 (2007).</li><li>Biron, D., Shibuya, M., Gabel, C., Wasserman, S. M., Clark, D. A., Brown, A., Sengupta, P., Samuel, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+diacylglycerol+kinase+modulates+long-term+thermotactic+behavioral+plasticity+in+C.+elegans.">A diacylglycerol kinase modulates long-term thermotactic behavioral plasticity in C. elegans.</a> Nat. Neurosci. 9, 1499-1505 (2006).</li><li>Ghosh-Roy, A., Wu, Z. L., Goncharov, A., Jin, Y. S., Chisholm, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+and+Cyclic+AMP+Promote+Axonal+Regeneration+in+Caenorhabditis+elegans+and+Require+DLK-1+Kinase.">Calcium and Cyclic AMP Promote Axonal Regeneration in Caenorhabditis elegans and Require DLK-1 Kinase.</a> Journal of Neuroscience. 30, 3175-3183 (2010).</li><li>Bianchi, L., Gerstbrein, B., Frokjaer-Jensen, C., Royal, D. C., Mukherjee, G., Royal, M. A., Xue, J., Schafer, W. R., Driscoll, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurotoxic+MEC-4(d)+DEG/ENaC+sodium+channel+conducts+calcium:+implications+for+necrosis+initiation.">The neurotoxic MEC-4(d) DEG/ENaC sodium channel conducts calcium: implications for necrosis initiation.</a> Nat. Neurosci. 7, 1337-1344 (2004).</li></ol>

作者宣称，他们没有竞争的金融利益。

基因编码的钙指标已被广泛使用在 C 线虫神经生物学。许多组织已经采用这些技术来研究的初级感觉神经元对外界刺激的反应，这里展示的ASJ电场。著名的例子包括机械接触的感觉，具体的化学品，温度和电场12，16-19。活动的interneuron和肌肉细胞中也被监控，对刺激的反应，并在动物行为的控制。许多这些研究已推动这些技术的步骤进一步使用图像识别和跟踪技术，使从在?...

在这里，我们提出了切实可行的方法， 在体内钙成像C.线虫的神经元。基因编码的钙敏感的荧光团的发展与高signal-to-noise比使得C。线虫的神经生理学和活动测量一个相对简单和成本效益的系统。我们的成像是一个标准的复合式显微镜，采用宽视场常用的荧光基团的荧光成像。我们提出了几个不同的荧光基团和不同的样品制备技术，每个讨论的优点和缺点。从两个例子实验数据，然后提交。对这里所描述的技术的一个极好的额外资源可以发现WormBook，“成像”由R.克尔神经元和肌肉的活动，（ <a href="http://www.wormbook.org" target="_blank">http://www.wormbook.org</a> ）3。 两大类的genetiCALLY编码中常用的荧光钙记者，C.线虫 ：的单一通道GCaMP和基于FRET的卡默莱昂。我们将描述的方法和所产生的每一个数据的例子。 GCaMP是根据修改后的绿色荧光蛋白（GFP）的周围的钙离子浓度是敏感的。这是通过融合GFP和钙的高亲和蛋白钙调素，钙的钙调素的结合，使得成一个高效荧光确认2带来的GFP分子。在这些荧光的最新进展，产生特殊的信号大小的荧光强度增加高达500％，超过生理范围内的钙水平和合理的快速反应动力学〜95毫秒上升时间和〜650毫秒衰减时间4。在相对较短的时间周期（分钟），这些信号可以允许较低的分辨率成像（低倍率），一个乖巧的初始化IAL计量基准，为连续测量基线或比较否定的必要性。 卡默莱昂具有的优点是一个基于FRET的荧光基团，生成比较两个独立的通道或波长1的比率量度测量。它是由两个单独的荧光团（青色和黄色发光的荧光蛋白，CFP和YFP）链接由钙调素蛋白。复杂的是亮蓝色光（440 nm）的激发CFP。结合的钙带来荧光团更接近在一起，增加从CFP（供体）的荧光共振能量转移（FRET）送到YFP（受体），并造成在CFP发射（480 nm）的降低和YFP的发射（535 nm）的增加。相对钙水平的YFP / CFP强度的比率作为计量。卡默莱昂动力学比GCaMP，测定在体内有〜3秒5〜1秒的上升时间和衰减时间慢。但是，反向移动的信号的比率增加了的信号的大小和用于补偿由于在荧光基团的浓度，运动或重点不突出，漂移和漂白的变化的一些可能的工件。 需要用外源性给药探针和 C的样品制备多基因编码的荧光记者否定线虫透明的小身体允许在完整动物使用简单的宽视场荧光成像。因此，在样品制备的主要的技术挑战是安全地固定动物。有许多不同的常用的技术每个的优点和缺点。使用瘫痪的动物的药理活性剂是容易实现的，并允许安装一种制剂（左旋咪唑，胆碱能激动剂，导致肌肉组织抓紧通常用于6）上的多个动物。C.线虫也可以在物理上固定安装在僵硬的10％琼脂糖7，8。这最大限度地减少对动物生理的影响，使长期成像（小时）和多只动物的恢复，但技术上更困难。这两种技术限制物理访问的动物（这是下封面条），因此只能用于某些实验刺激（如光照，温度，电场，激光损伤）。对于刺激的物理访问是必需的，如触摸或化学品的管理，许多研究已经成功地粘C.线虫的地方（用兽医年级胶）9。这是技术上更具挑战性，是一个单一的动物准备，不使动物恢复。最后，许多微流控设备已在物理上抑制C.线虫 ，维护动物生理学，允许暴露于大多数类型的刺激（这取决于设备的设计），并且可以启用快速交换和恢复的动物<sup&gt; 10，11。然而，微流控芯片需要更多的技术技能和能力，在设计，制造和实施。在固定的动物活动和刺激反应，一般都可以感觉的interneurons测量。运动神经元的活动需要更先进的技术，成像技术在移动的动物。在这里，我们将介绍详细的方法，采用最简单的技术的药理瘫痪和固定僵硬的琼脂糖。 这里介绍的方法可用于测量神经活动和细胞生理学C.线虫 。我们给出一个例子的每个：使用GCaMP的的ASJ神经元的测量的感官响应外部电场，和使用卡默莱昂测量响应于激光损伤的神经元的生理钙。这些例子显示了两种类型的荧光基团的优点和缺点，并说明什么是可能的系统。

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Eclipse Ti-U inverted 
 microscope</td>
 <td>Nikon</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Intensilight HG Illuminator</td>
 <td>Nikon</td>
 <td>C-HGFI</td>
 <td>Fluorescent light source</td>
 </tr>
 <tr>
 <td>CFI Plan Apo VC 60X Oil</td>
 <td>Nikon</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Optical table or 3'X3' optical 
 grade breadboard</td>
 <td>Thor Labs</td>
 <td>&nbsp;</td>
 <td>If an optical table is not used an 
 optical grade breadboard on a 
 solid laboratory bench should 
 suffice.</td>
 </tr>
 <tr>
 <td>Clara Interline Camera</td>
 <td>Andor 
 Technology</td>
 <td>&nbsp;</td>
 <td>High-sensitivity CCD camera</td>
 </tr>
 <tr>
 <td>wtGFP Longpass Emission</td>
 <td>Chroma Technology 
 Corp.</td>
 <td>41015</td>
 <td>GFP filter set for imaging GCaMP</td>
 </tr>
 <tr>
 <td>Filter 440 +/- 10 nm</td>
 <td>Chroma</td>
 <td>D440/20x EX</td>
 <td>excitation filter for cameleon</td>
 </tr>
 <tr>
 <td>Dichroic mirror &gt; 455 nm 
 longpass</td>
 <td>Chroma</td>
 <td>455DCLP BS</td>
 <td>microscope dichroic for cameleon 
 imaging</td>
 </tr>
 <tr>
 <td>Dichroic mirror &gt; 515 nm 
 longpass</td>
 <td>Chroma</td>
 <td>515DCLP BS</td>
 <td>dichroic mirror for cameleon 
 imaging</td>
 </tr>
 <tr>
 <td>Filter 535 +/- 15 nm</td>
 <td>Chroma</td>
 <td>D535/30m EM</td>
 <td>YFP emission filter</td>
 </tr>
 <tr>
 <td>Filter 480 +/- 20 nm</td>
 <td>Chroma</td>
 <td>D485/40m EM</td>
 <td>CFP emission filter</td>
 </tr>
 <tr>
 <td>Lens, 200 mm, Achromat</td>
 <td>Thor Labs</td>
 <td>AC508-200-A1</td>
 <td>Relay lens for FRET optics (3)</td>
 </tr>
 <tr>
 <td>Silver broadband mirror</td>
 <td>Thor Labs</td>
 <td>ME2S-P01</td>
 <td>FRET optics (2)</td>
 </tr>
 <tr>
 <td>NGM buffer</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Levamisole</td>
 <td>Sigma</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Polybead Microspheres</td>
 <td>Polysciences, Inc.</td>
 <td>08691-10, 2.5% by 
 volume, 50 nm 
 diameter</td>
 <td>polystyrene nanoparticles for C. 
 elegans immobilization</td>
 </tr>
 <tr>
 <td>Transgenic strain, Strain 
 gpa-9::GCaMP3(in pha-1; 
 him-5 bkg) </td>
 <td>Sternberg Lab</td>
 <td>Strain PS6388</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Transgenic strain, mec- 
 4::YC3.60</td>
 <td>Gabel Lab</td>
 <td>Strain CG1B</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

in vivo neuronal calcium imaging in c. elegans

线虫C.线虫是一种理想的模式生物比较简单，成本低， 在体内的神经成像。透明的小身体和简单的特点，以及神经系统允许任何神经元在完整的动物识别和荧光成像。简单的固定化技术对动物的生理影响最小允许延长时间的推移成像。基因编码的钙敏感的荧光基团（如卡默莱昂1和GCaMP 2）的发展，使细胞的生理和神经元的活动有关的神经元钙在体内成像。表达这些荧光团在特定神经元的大量的转基因株系很容易得到，或者可以使用既定的技术构造。这里，钙动态测量在一个单一的神经元在体内，同时使用GCaMP和卡默莱昂的程序，我们将详细介绍。我们讨论的优点和disadvant年龄以及样品制备（动物固定）和图像分析的各种方法。最后，我们提出两个实验的结果：1）使用GCaMP测量一个特定的神经元的感官响应到外部的电场和2）使用卡默莱昂测量的生理创伤性激光损伤的神经元的钙应答。如这些钙成像技术被广泛使用在 C 线虫和已经扩展到测量遗传背景之间自由活动的动物，同时多个神经元和比较。C.线虫在体内的神经成像技术的简单性和成本的优势比其他模型系统提供了一个强大而灵活的系统。

在这里，我们从两个不同的实验结果。首先采用GCaMP测量到一个定义的外部刺激响应一个特定的感觉神经元，给一个很好的例子是如何可以使用荧光钙记者进行光学监测神经元的电活动的完整C.线虫 。第二个采用，变色龙来衡量细胞内钙瞬变响应特定的激光损伤的神经元内触发，从而说明在一个单一的细胞在体内钙的生理可测。把重点放在每个测量技术方面的单项试验结果，并详细?...

Watch this Scientific Journal Video about In vivo Neuronal Calcium Imaging in C. elegans at JoVE.com

In vivo Neuronal Calcium Imaging in C. elegans

它的小的透明体，证据充分的解剖学和主机适合遗传技术和试剂，<em&gt; C.线虫</em是一个理想的模式生物<em在体内</em&gt;神经成像使用相对简单的，低成本的技术。在这里，我们描述了单个神经元内保存完好的成年动物的基因编码的荧光钙指标的成像。

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, ...

in-vivo-neuronal-calcium-imaging-in-c-elegans

Research

JoVE Journal

Neuroscience

In vivo Neuronal Calcium Imaging in C. elegans

24.7K 次观看.  Boston University School of Medicine. 它的小的透明体，证据充分的解剖学和主机适合遗传技术和试剂， C.线虫

视频: In vivo Neuronal Calcium Imaging in C. elegans

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

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 ...

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

在犁鼻器官的切片准备工作表示一个基因编码的钙传感器成像神经元的反应

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

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 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 ...

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

在基于生物发光钙指标GFP的水母体内脑功能成像方法

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

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 ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

脑切片神经元树突的双光子钙成像

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

比率钙显像对单个神经元的行为线虫线虫

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

Toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and neurodegenerative disease across species. Although these neurotoxic challenges have long been considered to be cell-intrinsic, considerable evidence now supports that misfolded human disease proteins originating in one neuron can appear in neighboring cells, a phenomenon proposed to promote pathology spread in human neurodegenerative disease.
C. elegans adult neurons that express aggregating proteins can extrude large (~4 &#181;m) membrane-surrounded vesicles that can include the aggregated protein, mitochondria, and lysosomes. These large vesicles are called &#8220;exophers&#8221; and are distinct from exosomes (which are about 100x smaller and have different biogenesis). Throwing out cellular debris in exophers may occur by a conserved mechanism that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, relevant to processes by which aggregates spread in human neurodegenerative diseases.
While exophers have been mostly studied in animals that express high copy transgenic mCherry within touch neurons, these protocols are equally useful in the study of exophergenesis using fluorescently tagged organelles or other proteins of interest in various classes of neurons.
Described here are the physical features of C. elegans exophers, strategies for their detection, identification criteria, optimal timing for quantitation, and animal growth protocols that control for stresses that can modulate exopher production levels. Together, details of protocols outlined here should serve to establish a standard for quantitative analysis of exophers across laboratories. This document seeks to serve as a resource in the field for laboratories seeking to elaborate molecular mechanisms by which exophers are produced and by which exophers are reacted to by neighboring and distant cells.

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

This protocol describes approaches for detection and quantitation of large aggregate and/or organelle extrusions (~4 &#181;m) produced by C. elegans cells in the form of membrane-bound exophers. We describe strains, growth conditions, scoring criteria, timing, and microscopy considerations needed to facilitate dissection of this debris expulsion mechanism.