张玉华发展由美国国立卫生研究院的支持授予R21AG027513和U01AG045864。数据和一些简短的视频表示是改编自Restif 等。 （2014年）8，这是根据知识共享署名（CC BY）牌照http://creativecommons.org/licenses/by/4.0/出版。我们感谢里卡多Laranjeiro稿件帮助。

1.线虫生长与处理<ol><li> C.生长在含NGM发现与OP50-1 大肠杆菌作为食物来源1-3标准培养皿线虫 。 </li><li>保持培养物中维持所需温度的恒温箱。 线虫生长良好，从15至25℃，以20℃为标准生长温度。 注:游泳是一个强大的表型，但游泳的活力可能是由于环境因素，包括在平板上的污染和过热的文化和幻灯片的影响。强努力保持稳定的条件应该作出。 </li><li>使用由铂丝阻燃密封入玻璃棒处理单动物4转移的一个自制的线虫挑。 </li></ol> 2.设置游泳分析<ol><li>使用由立体显微镜，数码CCD摄像头和数字视频录制软件的集成系统。天书软件CAN为一个单独的计算机上使用。 <ol><li>盖上暗布的显微镜 （ 如毡），以防止眩光在显微镜载玻片游泳降面积，提高图像质量。 </li><li>调整显微镜的工作距离和放大倍率的游泳区的全视图，基镜获得的映衬下动物的鲜明对比。将照明到暗视野可视化的成年人的身体作为白色对黑色的背景，这非常适用于计算可视化和与游泳分析软件跟踪。 </li><li>注意保持工作区域，显微镜载玻片，和游泳降得很干净。杂物可通过堵塞成年人的自由游动的干扰分析，和纤维的细丝可以作为跟踪过程中不运动的动物进行检测。 </li></ol></li></ol> 3.动物的制备为液体体质分析<ol><li>吸取60μLØ˚F1X M9缓冲区中预先打印载玻片10毫米环。确保滴完全覆盖环的内侧区域。该软件提取使用包含在幻灯片上会比利用，如果多孔板这个墨滴大小的更多信息，游泳。 </li><li>接单成人线虫，并将它们传送到1×M9缓冲液中滴，最大限度地减少细菌，可以云中的液体的运输。首先将动物转移到不包含很多细菌草坪上板的一侧，让他们爬行，然后轻轻提起他们，尽量减少细菌。 <ol><li>当线虫转移到下降，用接近的分析站解剖范围，以减少的游泳时间的长度，这可能会影响动物25的行为。 </li><li>将只有4个成年人在下降，如果他们大力移动和交叉路径频繁。重要思想为指导，以减少重叠的游泳者（这混淆了计算分析），考虑将4只动物每一滴水的时候都那么积极，年轻，健康成人WT每下降5只动物，当他们年长显示较少的活力。 注:健康WT线虫本能地开始被放置在液体后不久游泳。随着线虫挑的帮助下，轻轻地大人在下降，如果他们聚集分开。 </li></ol></li></ol> 4.体质分析 - 液 - 游泳录像<ol><li>装有显微镜来记录他们的游泳的基础上成人的滑动位置。 30秒在液体中的个体动物的行为的视频捕获细节（ 视频1），以及大量的这种记录时由CELEST软件8,26计算机辅助是很容易实现的采样;然而，较长的时间游泳可能是理想的其他特定行为研究25。重要的是，一定要保持整个所有记录相同的放大倍率的放大倍率，因为不同affecŧ游泳分析和意志偏差比较。 注:提供与使用的材料表中列出的数字视频录制软件的指导，这些步骤; VirtualDub的开放式访问软件可以替代，虽然我们还没有测试过这一点。 <ol><li>使用以下设置作为成功的游泳分析指导:696点¯x520像素的图像尺寸，0.02毫米/像素的图像分辨率和18帧/ s的速率。这些设置更高分辨率的版本可能有利于跟踪，但不会影响程序计算的措施。 注:1倍M9的缓冲区上的显微镜载玻片60μL下降所提供的游泳区没有太多的深度，这最大限度地减少向上和向下运动游泳。 </li></ol></li><li>在设置下，进入"录音"，载"限价序列大小"到"磁盘上的545架"。在相同的标题下，选中"使用录像管理器"对话框。点击"修改当前的录音功丁脚本..."按钮，然后在"命令"，选择"抓取窗格"，并根据"停止条件"，输入"停止后，0:00:30.00"。 </li><li>要录制，选择家庭标题下的"在RAM新建序列"，然后按下红色的"录制"按钮。该软件将在抢30秒545帧，数码相机和保持他们的RAM内存。 </li></ol> 5.体质液体分析 - 视频处理<ol><li>通过点击标题的家，并在视频录制软件，选择"导出全序列为...&#39;片段视频为图像帧序列。 注:数字视频录制软件可以保存帧作为.BMP，.JPG，.TIF或.png，所有这一切都可以通过这里介绍的游泳分析程序读取。需要注意的是.TIF文件使用更多的存储空间，但一些格式如.jpg可能会降低图像质量。 </li><li>启动从MATLAB B中的游泳分析程序Ÿ点击运行按钮。 </li><li>在游泳分析程序的主屏幕上，使用"添加一个视频"或"添加多个视频"按钮来上传图片序列帧一次或同时附上相关的标签，如基因型，日期，审判和持续时间组。该软件创建标记序列的数据库。 </li><li>通过点击&#39;1启用上传序列多个动物的同步跟踪。过程的视频"。在新的屏幕中，选择在左边框中输入所需的视频，然后单击"添加到列表"。对于每一个视频，在右上角的列表中单击其名称右下角显示视频的第一张图像。通过选择环内3分划定游泳区，并通过单击"进程上面列出的所有视频"启动处理。查看在中心底部的处理的进展情况。 </li><li>一旦完成，"关闭"视频处理画面。 </li></ol> 6。体质液体分析 - 测量<ol><li>点击"2。计算措施&#39;，打开一个新的屏幕，其中所处理的视频可以上传，一次一个，以验证或拒绝单个动物的跟踪。已处理的视频会出现在在哪里可以点击视频名称上传该视频左侧的列表中。 </li><li>使用本手册一步把重点放在对比成功跟踪（绿色街区）错过了跟踪（红色块）区域。 注意:可替换地，人们可以依靠有效跟踪的自动输出，因为该软件具有（如在2020动物中观察到从404影片8 94.1％效力）一个鲁棒跟踪系统。 <ol><li>找到"分段体的有效性"中的下半部分在屏幕上的第一个颜色条。 "有效框架"的比例为绿色，而"拒绝框架"的比例为红色。有效帧的≥80％意味着该跟踪我载文信息用于计算措施来自于一个30秒至少432帧出540游泳记录在18帧/秒的审判。 </li><li>轻松浏览和修改的有效块/拒绝使用"下一步块"，"开关有效性"，"分割块"和"隔离框"选项框。在极少数情况下2只动物在拥挤的游泳区交叉路径，切换其身份证号码在特定帧范围的"开关"按钮。 </li><li>查看在屏幕的下半部分的条形图上的跟踪和重叠的表演的附加信息。分割体（每帧动物的体长）的长度重叠发生率相关。 </li></ol></li><li>如果该视频的任何亮点干扰分析，通过选择"眩光区"功能调整的游泳区明亮区域。 </li><li>点击"保存并计算措施"保存有效期输入之前最多加载另一个处理的视频。在这个阶段，软件计算，在后台（未示出给用户），动物个体的游泳性能的曲率图和如下参数（ 视频2）。 注:如果集中在这里测得的参数在表1中描述。波引发速率，体波数，不对称，拉伸和卷曲基于该软件计算用于动物的曲率图计算出的;行进速度，笔触和活动指数参数不是基于曲率的地图。 </li><li>点击"关闭"退出"计算的措施"屏幕。 </li></ol> 7.体质分析 - 液 - 结果输出注:该软件可以在游泳运动的多种措施，从公开到很细微的行为特征用肉眼不容易打进封面报道（ 视频3和4）。在这里，重点是8个参数，通常表现出良好的动态范围:波引发速率，身体波数，不对称，拉伸，卷边，行驶速度，笔触和活动指数。 <ol><li>在程序的主界面，点击"3。显示结果"来获取分析输出。 </li><li>在左边的框中选择想要的画面序列进行分析，并将其移动到新的或现有的样本组通过单击"添加到所选样本"或"添加到新样品"按钮创建进行统计学处理样本组。 </li><li>点击"图形显示这些样品"去，通过选择显示在屏幕顶部的曲线图和样品为每8个参数的关键统计数据的第二个屏幕。 <ol><li>如果需要的话，使用"颜色"，"直方图＃&#39;，和选择"蠕虫＃&#39;调整条形图的表示在屏幕的左上角。 </li></ol></li><li>使用"2D直方图&#39;通过与"测量在Y轴上"下拉在屏幕的左边的菜单的"在X轴上测度绘制任何两个参数的组合。单击"关闭"退出并返回到"显示结果"屏幕。 </li><li>使用屏幕左上方的"导出..."按钮来详细的数值数据保存为.csv文件打开并在电子表格程序进一步操纵。 </li><li> &#39;关闭&#39;的&#39;显示结果&#39;屏幕回到主屏幕，然后单击"退出"，以确保包含分析视频序列数据库的保存。 </li></ol>

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The authors have nothing to disclose.

作为模型系统的使用线虫的继续，由于其遗传延展性，实验易处理性和解剖学注释以细致详细地增加。例如， 线虫雌雄同体的神经元结构和连接清楚地映射出34-36，极大地方便了控制特定行为的特定神经元回路的调查。 302的神经元构成了成人雌雄同体的神经系统，其处理范围广泛的感觉输入到像运动基本行为输出。更复杂的男性的神经系统的结构也被描述37，使性别特异性电路加以处理。 37 线虫行为已被广泛地研究了含有固体培养基的标准培养板中。由于WT 线虫在可预见的正弦波琼脂填充板从移动，偏差整体格局可以通过眼睛进行检测和人工评分。这种方法，然而，是受实验者的标准，是劳动密集型的。硬件和软件工具，用于跟踪和测量线虫在固体培养基上运动删除的主观性偏差和允许大规模的研究，允许更多复杂的生物学问题加以解决。最近由谢弗实验室16创建的行为数据库的分析扩展和深度已与上板运动的计算系统来实现的一个很好的例子。 当WT 线虫放入液体，动物很快适应了新的环境运动，开始游泳。 线虫游泳利用运动的更大的范围比爬行和可以更不规则8。像张玉华软件旨在填补C.详细分析差距线虫液体，permi行为的那些不容易通过的未受助眼测定，或可以比手动得分更迅速地完成运动相关的参数拟合定量。在8硬H成个人可以处理多达200影片，1000〜记录每天。 软件定义游泳充当体能和行为进行了全面评估指纹参数。除了富集在液体线虫行为和它们的基础的分子途径的复杂的小面的理解，该软件可用于探索生物学的多个方面，包括药理学应答，老化，和不同的行为。这里介绍的，那发生在C的物理性能的量化变化概述线虫成年人随着年龄的增长是该软件的这种应用的一个例子（更详细的介绍，请参阅Restif 等8）。在老龄化的背景下，一些测量参数下降，而更为增加或没有在野生型一致地变化。趋势是由长寿突变体的计算轮廓证实，在很大程度上，和同年龄人群的优雅和坏几岁同伙的相对配置文件保存在统一的环境条件。该软件的高分辨率也可以揭示细微的表现型先前在广泛表征突变体（ 例如， 年龄-1（hx546）在图1中）未知。 有描述的协议的几个特别关键的步骤。维护游泳环境应变片文化的一个恒定的温度环境，是游泳重复性重要的，所以实验者我们非常鼓励去煞费苦心，避免随意的温度变化。游泳媒体应在相同的温度下将板。同样地，仔细注意墨滴大小为游泳将有助于确保重现性。最后，谨慎的做法是在思考推进有关积累大量的视频文件的卸载。建议处理从视频采集计算机除了一个网站的图片。 利用这里提出供游泳分析软件的具有一定的局限性。首先，虽然程序可以同时跟踪多个动物，如果超过五只动物被联合分析，有一个增加的机会，该动物将在视频图像跨越彼此游泳。当程序不能明确地确定哪些动物是它，它审查这些数据帧。虽然此方案功能确保个别动物数据是高质量的，它限制了吞吐量。其次，图像应该还算干净，就是无灰尘，污迹和灯光刺眼，因为相关的信号也可以混淆图像分析。作为协议第2.1.1节，一个非常低的高科技投入，可以通过消除并发症的波动大大有助于图像捕捉注意到我Ñ环境照明是覆盖用深色布不允许环境光到达阶段舞台区。第三，该程序用于在成年阶段的动物进行了优化。低龄幼虫游泳速度非常快，有小的机构，这增加了程序错误。第四，一些软件利用MATLAB中，并且当存在版本升级和/或操作系统升级，一些程序链接可能会中断。目前，该软件对MATLAB 2015B和Mac OS版10.10进行了优化，但我们预计很快就会发布一个软件版本，反对这种变化更加健壮。最后，视频数据文件可以变得很大快，并且需要的存储空间被分配。 总之，这里介绍的是一种简单的方法，可以通过任何实验室容易地实现没有太多投资创建线虫游泳CELEST分析的影片。软件包的功能包括广泛的自动化，从通过跟踪分析，同时多动物跟踪，以及使用的数学基础（ 即，曲率措施）对于大多数运动参数的定量。该软件是开源的，其代码和演示公开可作为Restif 等详细说明。 8。虽然该方案特点进行跟踪先进的计算机视觉分析，其他已发布跟踪系统（ 例如 ，Greenblum 等人 ，2014年38），与这里介绍的软件的参数分析兼容。未来的改进将朝向的软件转换成一个更强大的包，不限制使用上面提到的操作系统（在材料的表也表示）的特定版本被引导。

定义遗传学，表观遗传学，经验和环境的影响的行为是如何在现代生物学的一个重大挑战。操作简单，适合遗传模型，可以通过计算跟踪和测量可以实现这一目标作出重要贡献。该线虫就是这样的一个模式。本文的目的是演示线虫游泳运动如何可以跟踪和定量提取八与生物相关性新颖特征的信息。 线虫具有约2相对短的寿命- 3瓦特和大约4天在20℃ 的1,2-生殖周期。在标准的实验室培养，这种微小线虫生长在含线虫生长介质（NGM）培养皿正在流传着一种细菌的食物来源3,4。 WT N2动物琼脂填充板优雅正弦波积极动;他们改变移动鼠漫游时（食品求），住宅（浏览），或从餐（无效饱腹感静止期）回收ES 5。减值6或7-12岁还可以显着地改变上板的运动。 基因调节行为，长期保健或老化，可以功能特点，通过分析线虫的运动模式或运动。衡量长期保健方法之一是到老年人分为根据自己的平板上运动三大类（ 如 A，B和C），剧烈体力活动是A类和麻痹是C类7,8,13。虽然这样的定性排序是揭示体质的差异是有用的，分类是没有明确的界限笼统，因而他们的得分是受实验者的偏见。 越来越多的纤夫有升高的客观性，复杂性，和C的分析精度线虫运动在固体培养基上14-18。 线虫在平板上运动主要是受限制的，其中该动物使得与媒体的固体表面接触的平面。 （ 线虫也可以通过从身体仍然与固体表面接触的其余部分的抬起头离开，定位体在多个平面诱导探索。然而，这种行为是不寻常的。）当放置在液体中，这线虫发起波动的运动，或游泳，比在固体表面的动物，具有更广泛的空间运动，速度和运动的深度更大范围，并与前从后移动不同年龄的发病率增加。由于体能和应对新环境的快速分析，实验者可以将动物个体成液滴和得分它的身体弯曲频率下的解剖范围。录像可以方便的游泳活力的现在和未来的刻痕动物。然而，手动方法限制了可被打进特征的数目，并完全约束在一次得分一只动物。 运动在液体比固体培养基上运动少了探讨。实际上，有一些是健壮和容易在实验室中实施以测量液体19-24运动几个软件选项。该软件张玉华（ 线虫游泳测试）提供的，可提供数据（曲率分数）的议案8的特性直接相关的使用和数学为基础的措施简单; （在Restif 等人 8的特征和优点的详细描述）。此外，计算分析使表型特征是不可能的人眼的得分澄清。这里，体现这种分析方法的分辨率数据呈现和易于实现协议描述后续天书分析记录游泳试验。

<table border="0" cellpadding="0" cellspacing="0" width="1764">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:484px;">REAGENT</td>
			<td style="width:256px;"></td>
			<td style="width:375px;"></td>
			<td style="width:649px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:484px;">N2</td>
			<td style="width:256px;">Caenorhabditis Genetics Center (CGC)</td>
			<td style="width:375px;"></td>
			<td>C. elegans wild type (ancestral).</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:484px;">OP50 Escherichia coli</td>
			<td style="width:256px;">Caenorhabditis Genetics Center (CGC)</td>
			<td style="width:375px;"></td>
			<td>Biosafety Level: BSL-1.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:484px;">OP50-1 Escherichia Coli&#160;</td>
			<td style="width:256px;">Caenorhabditis Genetics Center (CGC)</td>
			<td style="width:375px;"></td>
			<td>Streptomycin resistant strain of OP50.&#160; Biosafety Level: BSL-1.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:484px;">Streptomycin sulfate salt</td>
			<td style="width:256px;">Sigma-Aldrich</td>
			<td style="width:375px;">S6501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:484px;">Printed Microscope Slides&#160;</td>
			<td style="width:256px;">Thermo Fisher Scientific</td>
			<td style="width:375px;">Gold Seal Fluorescent Antibody Microslides: 3032-002</td>
			<td>have two etched 10mm diameter circles delineated by white ceramic ink</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:484px;">Nematode Growth Medium (NGM)</td>
			<td style="width:256px;">For 1L: 17g Agar, 3g NaCl, 2.5g Peptone, 1mL 1M CaCl2, 1mL 5mg/mL Cholesterol in ethanol, 25mL 1M KPO4 buffer, 1mL 1M MgSO4, H2O to 1 L. Sterilize by autoclaving.</td>
			<td style="width:375px;"></td>
			<td>Stiernagle, T. Maintenance of C. elegans. WormBook, 1-11, doi:10.1895/wormbook.1.101.1 (2006)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:484px;">M9 buffer</td>
			<td style="width:256px;">For 1L: 3g KH2PO4, 6g Na2HPO4, 5g NaCl, 1mL 1 M MgSO4, H2O to 1 L. Sterilize by autoclaving.</td>
			<td style="width:375px;"></td>
			<td>Stiernagle, T. Maintenance of C. elegans. WormBook, 1-11, doi:10.1895/wormbook.1.101.1 (2006)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:256px;"></td>
			<td style="width:375px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:484px;">EQUIPMENT</td>
			<td style="width:256px;"></td>
			<td style="width:375px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:484px;">CeleST</td>
			<td style="width:256px;">Driscoll Lab, Rutgers University</td>
			<td style="width:375px;">C. elegans Swim Test</td>
			<td>Open Source, see http://celestmod.github.io/CeleST/ and http://celest.mbb.rutgers.edu/</td>
		</tr>
		<tr height="189">
			<td height="189" style="height:189px;width:484px;">MATLAB</td>
			<td style="width:256px;">www.mathworks.com/downloads</td>
			<td style="width:375px;">MatLab version 2015b (best)</td>
			<td style="width:649px;">The CeleST version demonstrated here has best functionality with Mac OS 10.10 and MatLab 2015b. MATLAB 2015B introduced changes to how MATLAB handled graphics, including a new coding convention and syntax. These changes resolved an issue that couldn&#39;t be resolved elegantly (primarily because the internals of MATLAB really needed the major graphics overhaul implemented in MATLAB 2015B). For this reason, CeleST should always be run on MATLAB 2015B or later versions. However for users without access to MATLAB version 2015B or later (or MATLAB at all), we have created a CeleST program that doesn&#39;t need MATLAB on the computer at al. An installer is downloaded by the prospective user and then it installs itself onto the computer through a couple prompts like most programs.&#160;&#160;</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:484px;">Mac OS</td>
			<td>www.apple.com</td>
			<td style="width:375px;">Version 10.10</td>
			<td style="width:649px;">Currently, CeleST has been ported to the major operating systems (Windows, Mac, and Linux). The current code can be run on any of the operating systems and there are versions for each operating system that don&#39;t even require users to have MATLAB to use CeleST (this version requires a large download). The Windows version has been tested the least and is most prone to bugs as such. Linux has been moderately tested. And Mac has been and continues to be tested extensively (primarily because it&#39;s the operating system in our lab).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:484px;">Stereomicroscope&#160;</td>
			<td style="width:256px;">Zeiss</td>
			<td style="width:375px;">Stemi 2000-C&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:484px;">Transmitted Light Base</td>
			<td style="width:256px;">Diagnostic Instruments</td>
			<td style="width:375px;">TLB 3.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:484px;">Digital CCD Camera</td>
			<td style="width:256px;">QImaging</td>
			<td style="width:375px;">Rolera-XR Mono Fast 1394 (ROL-XR-F-M-12)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:484px;">Digital Video Recording Software</td>
			<td style="width:256px;">Norpix</td>
			<td style="width:375px;">Streampix Version 3.17.2</td>
			<td></td>
		</tr>
	</tbody>
</table>

automated analysis of c. elegans swim behavior using celest software

解剖调节行为的神经元和神经肌肉电路仍然是生物学的一个重大挑战。该线虫已被证明是在帮助解决这一难题，从激励的技术方法，构建人脑连接组，实际上基本功能模式的具体分子司机脱落光了宝贵的模式生物。已在固体基质进行线虫的行为学研究的大部分。在液体中，动物表现出的行为模式，包括在一个范围在三维速度运动，以及局部的身体动作，如无前形状变化的后卷曲，这引入定量的新挑战。一个简单的程序的步骤，以及使用使线虫的高分辨率分析游泳行为的软件，这里提出。该软件名叫张玉华，将使用跟踪一个专门的电脑程序同时对多个动物，并提供C的新措施线虫液体（游泳）运动。这些措施大多是植根于动物的姿势和基于计算机视觉和模式识别用于数学，没有阈临界值计算需求。软件工具可以用来既在数百只动物从合并的小批量试验评估总体游泳实力和揭示新的表型，即使在充分表征的基因的突变体。的标本适乐时分析，制备简单，技术含量低，被科学界能够适应面广。因此此处所描述的计算方法的使用应有助于行为和行为电路中的线虫模型的更多的理解。

通过在液体（游泳）分析运动，表型不是在固体培养基上显而易见（爬行）可以被阐明。定量游泳运动我们开发了测量的游泳行为八十人的新参数的特定软件。八个最有用的这些参数进行详细在表1中说明。这些参数被命名为波起始速度，身体波数，不对称，拉伸，卷边，行驶速度，笔触和活动指数。研究已经例示了软件的功率限定数百与WT，行为或长寿突变体背景8老化成人的功能下降，并分析了充分研究的长寿突变体年龄-1（hx546）和daƒ-16（mgDƒ50），其中滋生扰乱了正常的胰岛素信号通路的突变。基因年龄-1编码的磷脂酰肌醇3-激酶（PIK3）催化亚基，并且，当它怀有突变hx546，使寿命延长和胁迫抗性27-29。该基因daƒ-16编码的寿命缩短或删除时30-33损害的应激反应一个叉头框O（FOXO）转录因子。 如波起始速度，行驶速度，笔触和活动指数游泳这样的一些参数逐渐随着年龄的增长，即使在良好的遗传背景（ 图1）有所下降。在当前的知识线，长寿命的AGE-1（hx546）突变表明在先进的和极其古老的年龄比WT更剧烈的身体表现。也如预期，短期daƒ-16（mgDƒ50）突变体显示的折衷性能，尤其是在极其古老的年龄。值得注意的是，这是只有在天书计算机视觉和数学算法包，该SUPERIO的审查AGE-1（hx546）突变体中的R游泳演出是在成年期发病检测。 年龄-1（hx546）的结果在年轻成人生活增强的物理性能表明，该突变影响正常发育和/或年轻成人表型在以前没有认识到（ 图1）的方式的事实。 体波数，不对称，舒展，和冰壶参数呈上升趋势随着年龄的野生型和突变体老化成人（ 图2）。有趣的是，该软件的分辨率级别显示更精细的行为特征，如年龄-1（hx546）在其整个寿命和极端旧daƒ-16（mgDƒ50）突变体的无力突变体的持续对称伸展和蜷到相同的程度-age WT和年龄-1（hx546）大人。 除了物理PERFO的整体必然损失rmance由于年龄，每个个体成年通过老化过程显示了独特的级数模式，即使当遗传学和环境几乎同质7。 （通过控制遗传学和环境，这些因素的可能的混杂影响减至最小，拉开随机性的年龄相关变性显著贡献。）相似的遗传背景的同步线虫种群保持在受控环境中仍含有的混合根据自己的特点老龄化不同类别的个体。虽然所有的启动健康成人，有的迅速失去其体质（坏腾空类，C类），而其他人保持旺盛的时间较长时期内（婉约腾空，A级）。因此坏腾空高度似乎比曼妙腾空一个相当短长期保健。 在我们的研究中进一步8详细，优雅的腾空高度保持青春的身体fitnESS通过与更年轻的成年人的游泳曲线比较所观察到的（ 图3,4和5）。这个持续健身相当于在后育龄长寿命年龄-1（hx546）突变体的物理性能（D 11）（ 图1和2）。相反，坏的腾空高度明显失去了他们的身体能力再现之后不久，在执行类似的极端老progericdaƒ-16（mgDƒ50）成人（ 图1 - 4）的水平。这些相似性可以通过比较总可以得出，但是特别签名仔细看看容易引人注目。例如，虽然有两拉伸和卷曲在极端旧野生型和老化的突变体的程度之间的一些相关性（ 图2），这个关系不坏不停观察（ 图4和5），其表现出较高的倾向卷曲了，但不测试的样品中的拉伸。我们目前的软件从而通过提供工具，更复杂的研究，不听话之前添加维体质或运动能力的分析。综上所述，张玉华提供了在这里强调8新措施，其中定义了特定的基因，表观遗传学行为指纹和环境背景的形式全面读数，使独特而通用参数模式的识别，可以根据具体情况的签名（环境，药理，营养），生物过程或生物体状态如长期保健。 <img alt="图1" src="/files/ftp_upload/54359/54359fig1.jpg" /> 图1:波引发速率（A），活动性指数（B），笔触（C）及旅行速度（D）为WT， 年龄-1（hx546）和daƒ-16天书软件报告（mgDƒ50）<html>第D 4（青年期），11（-复制后）和20（极端旧）成人。在y轴上的手段&#39;号&#39;&#39;＃&#39;。的WT在绿色和daƒ-16中的红色着色灰色， 年龄-1。误差条是平均值（SEM）的标准误差。同龄WT和老化的突变体，使用单向方差分析，接着进行Dunnett的多重对比试验统计学意义进行比较。 **，p值= 0.001 - &lt;0.01; ***，P = 0.0001 - &lt;0.001。 62例中的每个数据点从四个独立的试验。注意，这里，和图2中 ，每个单独的30秒的视频与4只动物制成，以及每个试验中，我们得分总共4游泳视频16的动物，这是用于示出每个数据点4生物学重复进行。 <a href="http://ecsource.jove.com/files/ftp_upload/54359/54359fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <html> ntent"FO:保together.within页="1"&gt; <img alt="图2" src="/files/ftp_upload/54359/54359fig2.jpg" /> 图2:体波数（A），非对称性（B），拉伸（C），和冰壶（D）为WT， 年龄-1（hx546）和daƒ-16第D 4（mgDƒ50）成人软件报告（年轻成年），11（后再现）和20（极端老）。在y轴上的手段&#39;号&#39;&#39;＃&#39;。 WT在灰色， 年龄-1在绿色和DAF-16中的红色着色。误差条是平均值（SEM）的标准误差。同龄WT和老化的突变体，使用单向方差分析，接着进行Dunnett的多重对比试验统计学意义进行比较。 *，P = 0.01 - &lt;0.05; **，p值= 0.001 - &lt;0.01; ***，P = 0.0001 - &lt;0.001。 N =在每个数据点62从四个独立的，30秒游泳试验。e.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/54359/54359fig3.jpg" /> 图3:波引发速率（A），活动性指数（B），笔触（C），及旅行速度（D）为年轻WT大人（D 4），和同龄优美和坏腾空（D10软件报告和11）。在y轴上的手段&#39;号&#39;&#39;＃&#39;。年轻的工作小组是彩色灰色，A级优美的腾空高度的绿色和C类不良腾空高度为红色。误差条是平均值（SEM）的标准误差。 A级优美的腾空高度和C类不良腾空采用单因素方差分析，接着进行Dunnett的多重对比试验相比，到D 4青壮年。 ****，P &lt;0.0001。 N =在来自两个独立的每个数据点27，30秒游泳试验。图稍微偏离Restif 等进行修改。 （2014年）8，这是创意下公布共享署名（CC BY）牌照http://creativecommons.org/licenses/by/4.0/。 <a href="http://ecsource.jove.com/files/ftp_upload/54359/54359fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54359/54359fig4.jpg" /> 图4:体波数（A），非对称性（B），和冰壶（C）软件报告为年轻成人WT（D 4），和同龄优雅和坏腾空（D 10和11）。在y轴上的手段&#39;号&#39;&#39;＃&#39;。年轻的工作小组以灰色，有色类绿色和C类不良腾空从容腾空高度为红色。误差条是平均值（SEM）的标准误差。 A级优美的腾空高度和C类不良腾空采用单因素方差分析，接着进行Dunnett的多重对比试验相比，到D 4青壮年。 **，p值= 0.001 - &lt;0.01; ****，P &lt;0.0001; <s仲&gt; N / A，不适用，因为只有一个动物出来的总样本量的卷曲。 N =在来自两个独立的每个数据点27，30秒游泳试验。图稍微偏离Restif 等进行修改。 （2014年）8，这是在知识共享署名（CC BY）牌照HTTP下出版:/creativecommons.org/licenses/by/4.0/。 <a href="http://ecsource.jove.com/files/ftp_upload/54359/54359fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54359/54359fig5.jpg" /> 图5:拉伸软件报告为年轻的成年人WT（D 4），和同龄优美和坏腾空（D 10和11）。年轻的工作小组以灰色，有色类绿色和C类不良腾空从容腾空高度为红色。误差条是平均值（SEM）的标准误差。 A类优美腾空类和C类不良腾空进行了比较到D 4ÿ翁荣南成人使用单向ANOVA，然后通过Dunnett的多重对比试验。 N =在每个数据27指向来自两个独立试验。图稍微偏离Restif 等进行修改。 （2014年）8，这是在知识共享署名（CC BY）牌照HTTP下出版:/creativecommons.org/licenses/by/4.0/。 <a href="http://ecsource.jove.com/files/ftp_upload/54359/54359fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="视频1" src="/files/ftp_upload/54359/54359video1.jpg" /> 视频1:C的一组有代表性的游泳线虫成虫。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54359/Video1.mov" target="_blank">请点击此处观看该视频。 （右键点击下载）。</a> <img alt="视频2" src="/files/ftp_upload/54359/54359video2.jpg" /> 视频2:测试动物的游泳表演的各个曲率地图天书软件计算。曲率映射计算的背景;它们不会出现在与用户的软件接口。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54359/Video2.mov" target="_blank">请点击此处观看该视频。 （右键点击下载）。</a> <img alt="视频3" src="/files/ftp_upload/54359/54359video3.jpg" /> 视频3:根据个人的曲率地图游泳办法计算软件。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54359/Video3.mov" target="_blank">请点击此处观看该视频。 （右键点击下载）。</a> <img alt="视频4" src="/files/ftp_upload/54359/54359video4.jpg" /> 视频4:不曲率依靠游泳办法计算软件地图。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54359/Video4.mov" target="_blank">请点击此处观看该视频。 （右键点击下载）。</a>

Watch this Scientific Journal Video about Automated Analysis of C. elegans Swim Behavior Using CeleST Software at JoVE.com

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

对于在液体线虫游泳行为的基于计算机的分析一个有效和简单的方法进行说明。该方法需要很少或几乎没有投资线虫实验室。所使用的硬件是标准的，并且该计算机软件的行为分析（CELEST）是一个开放源码之一。

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 ...

automated-analysis-of-c-elegans-swim-behavior-using-celest-software

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

12.3K 次观看.  Rutgers University. 对于在液体线虫游泳行为的基于计算机的分析一个有效和简单的方法进行说明。该方法需要很少或几乎没有投资线虫实验室。所使用的硬件是标准的，并且该计算机软件的行为分析（CELEST）是一个开放源码之一。 

视频: Automated Analysis of C. elegans Swim Behavior Using CeleST Software

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 &#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 ...

VisioTracker, an Innovative Automated Approach to Oculomotor Analysis

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, robust, and simple to manipulate. A suitable model has been found in the optokinetic response (OKR), a reflexive behavior present in all vertebrates due to its high selection value. The OKR involves slow stimulus-following movements of eyes alternated with rapid resetting saccades. The measurement of this behavior is easily carried out in zebrafish larvae, due to its early and stable onset (fully developed after 96 hours post fertilization (hpf)), and benefitting from the thorough knowledge about zebrafish genetics, for decades one of the favored model organisms in this field. Meanwhile the analysis of similar mechanisms in adult fish has gained importance, particularly for pharmacological and toxicological applications.
Here we describe VisioTracker, a fully automated, high-throughput system for quantitative analysis of visual performance. The system is based on research carried out in the group of Prof. Stephan Neuhauss and was re-designed by TSE Systems. It consists of an immobilizing device for small fish monitored by a high-quality video camera equipped with a high-resolution zoom lens. The fish container is surrounded by a drum screen, upon which computer-generated stimulus patterns can be projected. Eye movements are recorded and automatically analyzed by the VisioTracker software package in real time.
Data analysis enables immediate recognition of parameters such as slow and fast phase duration, movement cycle frequency, slow-phase gain, visual acuity, and contrast sensitivity.
Typical results allow for example the rapid identification of visual system mutants that show no apparent alteration in wild type morphology, or the determination of quantitative effects of pharmacological or toxic and mutagenic agents on visual system performance.

The VisioTracker is an automated system for the quantitative analysis of visual performance of larval and small adult fish based on the recording of eye movements. It features full control over visual stimulus properties and real-time analysis, enabling high-throughput research in fields such as visual system development and function, pharmacology, neural circuit studies and sensorimotor integration.

VisioTracker，创新的自动化到动眼神经分析方法

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, ...

The Mouse Forced Swim Test

The forced swim test is a rodent behavioral test used for evaluation of antidepressant drugs, antidepressant efficacy of new compounds, and experimental manipulations that are aimed at rendering or preventing depressive-like states. Mice are placed in an inescapable transparent tank that is filled with water and their escape related mobility behavior is measured. The forced swim test is straightforward to conduct reliably and it requires minimal specialized equipment. Successful implementation of the forced swim test requires adherence to certain procedural details and minimization of unwarranted stress to the mice. In the protocol description and the accompanying video, we explain how to conduct the mouse version of this test with emphasis on potential pitfalls that may be detrimental to interpretation of results and how to avoid them. Additionally, we explain how the behaviors manifested in the test are assessed.

The forced swim test is validated as an experimental approach to assess potential antidepressant efficacy in rodents. Experimental animals are placed in a tank of water and escape-related mobility behavior is quantified. The common procedures for the mouse version of this test are described.

小鼠强迫游泳实验

The forced swim test is a rodent behavioral test used for evaluation of antidepressant drugs, antidepressant efficacy of new compounds, and experimental ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

突触荧光自动定量<em&gt; C。线虫</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

C. elegans Tracking and Behavioral Measurement

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

C. elegans Tracking and Behavioral Measurement

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

线虫跟踪和行为测量

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

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

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

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

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

长期的嗅觉适应性的分子读数<em&gt; C.线虫</em

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

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 ...

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