一些菌株由美国国立卫生研究基金会研究基金项目办公室（P40 OD010440）资助的秀根索氏杆菌遗传中心（CGC）提供。这项工作得到霍华德休斯医学研究所的支持，PWS是研究者。

行为室装配注意:行为室由近似立方体形状的框架组成，该框架由挤压铝条制成，覆盖着不透明的织物罩，透明的丙烯酸底部和相机支架。形成行为室的挤压铝条之间的接头都是垂直的，并使用"L"形的角支架（1英寸宽，1英寸支脚）固定，用一个角支架腿将螺钉和滑入式T型螺母。每个接头用一个或两个角落支架固定，如下所述。在房间上的织物覆盖物用相同的螺钉和滑入式T形螺母连接到铝框架杆上，但是通过织物角落处的垫圈附接。螺丝已正确插入滑入式T型螺母的埋头端，而不是突出的唇缘。将螺丝/ T形螺母紧固件组件安装到铝棒上时，将T型螺母滑入通道在杆的适当面上，螺钉的头部伸出槽。 <ol><li> 准备面料盖。 <ol><li>使用剪刀切割五块不透明的聚酯织物，以覆盖室的顶部和四侧。 <ol><li>切割一个方形14 x 14英寸2张织物的顶部的房间。切割四个长方形11 x 14英寸2张织物的两侧面。 </li></ol></li><li>在织物片的角落安装垫圈。 <ol><li>使用铅笔和直尺标记所有五个聚酯织物片的每个边缘0.5英寸的参考线。 </li><li>对于四个矩形片材，使用索环钳子插入中心的线圈，其中铅笔线在每个角落相交（即每张4个垫圈）。 </li><li>对于方形片材，沿着每个边缘插入两个垫圈，每个护环以铅笔线为中心，并定位在距离每个边缘末端1.5英寸四条铅笔线（即共有8个扣眼，每个角落附近两个）。 </li></ol></li><li>将螺丝插入每个索环孔，并松开地将T形螺母连接到每个。 </li></ol></li><li> 制造相机安装杆。 <ol><li>使用带锯或等效物为每个相机切割一个2英寸长的铝挤压条，确保条的切割端垂直于条的长度，以便稍后装配。 </li><li>使用具有0.25英寸直径钻头的钻床，通过每个相机安装杆的中心腹板钻一个孔，从一端0.75英寸，使得孔垂直于顶面和底面并穿过杆的中心线。可选地，使用具有0.25英寸钻头的手持式电钻钻孔，但确保孔垂直于杆的顶面和底面。 </li></ol></li><li> 准备所有的角落支架。 <ol><li>将螺丝插入孔中每个角支架腿，从内侧角插入螺丝。将T型螺母松动地拧到每个螺丝上。 </li></ol></li><li> 准备相机安装组件（ 图1B ，第2层）。 注意:相机安装组件是支持三个相机皮套的"H"形组件。 <ol><li>将摄像机安装杆连接到摄像机安装组件的中心杆。 <ol><li>布置在步骤1.2中制造的三个相机安装杆，钻孔垂直定向。将两个角支架滑入每个安装杆的距离钻孔最远端的两侧。 </li><li>将支架的支架放置在与支架末端齐平的角支架上，并拧紧螺丝固定到位。这形成"T"形的"T"底部的钻孔。 </li><li>在工作表面上放置1英尺铝挤压件。滑动一个相机安装杆的T形螺母mbly（从上一步）到1英尺的酒吧的一边。沿着1英尺长的长度将安装座中心，并拧紧螺丝固定到位。将另外两个安装杆滑到1英尺的对面。 </li><li>将两个条子从1英尺的杆的中间等距离放置，其内角部支架间隔约2.5英寸。将角落支架滑入1英尺的酒吧的两侧，在酒吧的每一端两个。将支架的支架定位在支架的两端与杆的两端齐平并拧紧螺丝固定到位。 </li></ol></li><li>将端棒连接到中心杆。 <ol><li>从上一步形成"H"形状，将两个1英尺铝挤压件从组件的每一端滑动到T形螺母上。将中心杆上的每个端杆中心并拧紧四个螺钉。 </li><li>将四个角落支架滑入上一步骤中添加的两个端栏的顶部，每个端部一个的每个酒吧。将支架的支架与支柱的两端齐平并拧紧螺丝固定到位。 </li></ol></li><li>将相机支架和皮套连接到相机安装组件。 <ol><li>对于三个显微镜相机架中的每一个，通过松开指旋螺钉并从支架杆上滑下来取出相机皮套。 </li><li>对于每个相机安装杆（现在固定到"H"的中心杆），将一个垫圈放在圆头螺丝上，将螺丝拧到相机安装杆的钻孔中，并将另一个垫圈放在螺丝上。将相机支架杆的螺纹孔螺纹拧到螺丝上，紧固在相机安装杆上。 </li><li>对其他两个相机安装杆重复步骤14.3.2。 </li><li>将相机皮套滑到每个相机支架上，将更宽的开口朝向相机安装杆和螺丝。将皮套临时固定到相机支架上od与皮套的指旋螺丝。 注意:最后的操作位置将在第2节中设置。 </li></ol></li></ol></li><li> 组装行为室框架（ 图1A ）。 注意:为了方便施工，将框架上下颠倒，从工作台面上的房间框架的"顶部"（ 图1B ，第1层）开始，朝向框架的"脚"向上工作（ 图1B ，层3）。 <ol><li>为了组装行为室的顶层，将四个1英尺挤出的铝条连接到正方形聚酯片（ 图1B ，第1层）。 <ol><li>沿着织物片的每个边缘将一个杆滑动到两个T形螺母上，沿着织物宽度定位杆。拧紧紧固件以将织物片固定到每个条上。 </li><li>将正方形聚酯薄片铺在工作面上在四个附加的铝条在顶部。将八个角支架滑入四个铝条的顶面，每个条的每一端一个。将支架放在杆上，将支架的垂直支柱与杆的末端齐平。拧紧T型螺母固定。 </li></ol></li><li>反过来，在每个角落，直立一个额外的1英尺铝挤压，将挤压物滑过两个角支架腿上的T形螺母。 </li><li>通过拧紧紧固件将直立杆固定到水平杆上，从而连接两个相邻的水平杆。 注意:完成后，八个挤出（四个水平和四个垂直）将在桌子上形成一个方形环，四个柱向上（ 图1B ，第一层）。 </li><li>将"H"型相机安装组件的松散T形螺帽从第1.4节向下滑入上一步中的框架立柱上。让"H＆＃34;一直滑落到框架每个角落的角落托架的顶部。通过拧紧四个角支架螺丝将相机安装组件固定到位。 </li><li>使用最后的四个1脚挤压铝棒（ 图1B ，第3层）组装腔室的底层。 <ol><li>将一个角落支架滑入每个1英尺杆的每个端部，将角撑架放置在支架的两端与杆的两端齐平并拧紧螺丝固定到位。 </li><li>在立柱与立柱之间松动的T形螺母之间滑动杆。定位杆，使其角撑架的顶部边缘在立柱的开口端下方1英寸处，并通过拧紧角落托架螺钉固定到位。 </li></ol></li></ol></li><li>将四个矩形聚酯薄片的T形螺母向下滑入直立挤压棒上的通道中，以覆盖坡口的两侧r并拧紧紧固件以固定到位。 </li><li>选择一侧是进入腔室内部的门，并拆下腔室底层的两个螺钉和T形螺母。 注意:当框架直立时，此盖的底部将在底部松动，并可以提起以进入室内部。 </li><li>在每个角落的顶端放置一个塑料盖，作为完成框架的脚。将框架右侧朝上。每侧在框架底层内插入两个面板支架，并扭曲以将其固定到位（ 图1B ，第3层）。在面板支架的顶部放置12 x 12英寸2片透明丙烯酸，为测定板提供了一个阶段（ 图1B ，第3层）。 </li></ol>相机和舞台模板定位<ol><li>将显微镜摄像机视频采集软件下载并安装到计算机上从制造商的网站。 </li><li>将显微镜相机滑入三个相机套中的每一个，相机光学指向丙烯酸阶段和背光。将相机连接到计算机的USB端口。将红外LED背光面板放置在框架下面，以舞台为中心。插入背光面板打开。 </li><li>在透明度工作表上打印提供的解决方案和平板对齐模板（请参阅补充文件 ）。 注意:在溶液放置模板上，板周长圆直径为3.8厘米，目标圆圈的内部区域直径为0.9厘米。 </li><li>沿着网格线切割，以获得围绕圆形模板的矩形透明件。 </li><li>启动视频采集软件，然后单击窗口顶部的编号缩略图，查看摄像机的信息。 </li><li> 调整显微镜相机对准和放大。 </strong&gt; <ol><li>将平板对准模板放置在显微镜相机镜头下（ 图2B ）。 </li><li>通过沿相机支架杆垂直滑动相机皮套来更改工作距离，并通过旋转相机的放大倍数拨盘调整相机的倍率，直到模板的数字标记在相机视野的边缘清晰可见。 </li><li>将板对准模板粘贴到丙烯酸阶段。 </li><li>在显微镜照相机下将样品盘放在板对准模板上，进一步细化放大倍数和工作距离，直到达到蠕虫的清晰图像，同时仍将数字标记保持在视场内。 </li><li>对其他两台显微镜相机重复步骤2.6.1-2.6.4。 </li></ol></li></ol> 3.线虫生长与同步<ol><li>实验前四天，种子二十四个60毫米线虫生长培养基（NGM）琼脂平板与50μLOP50 大肠杆菌在Luria-Bertani（LB）肉汤（见第5部分的配方）。 注意:每个60 mm的板将为一个测定板提供足够的蠕虫。建议每次治疗或基因型获得6次重复。 </li><li>离开播种板直立，盖上盖子，室温过夜。 </li><li>第二天，将15个喂养好的妊娠雌雄同株转移到每个种子板上，并允许蠕虫产卵6小时。这将产生每盘360克鸡蛋。 </li><li> 6小时后，从种子板上取出蠕虫，让后代在20℃下长大成人三天。 </li></ol>化学感应优选测定<ol><li> 在实验前一天，准备35mm NGM琼脂测定板。 <ol><li>准备250 mL的NGM琼脂（参见第5部分的配方）。 </li><li>在平坦的su上安排二十八个空的35毫米的五个或更少的板rface。向每个测定板上加入5mL NGM琼脂。将试验板用盖子直立干燥，室温过夜。 注:根据经验，为偶然发生的每种处理或基因型准备一个额外的测定板进行分析。 </li></ol></li><li>在实验当天，打开背光，启动电脑，并启动显微镜摄像机视频采集软件。 </li><li> 将显微镜相机连接到计算机并调整录像设置。 <ol><li>单击软件窗口顶部的编号相机缩略图可查看相机的直播。 </li><li>拉下相机进纸左上角的"视频格式"菜单，然后选择"MJPG 1280 x 1024"。拉下"视频格式"菜单旁边的"文件夹"菜单，然后选择一个保存视频文件的文件夹。 </li><li>点击右上角的"自动曝光"图标相机进纸并关闭自动曝光。点击"自动曝光"图标右侧的"LED"图标，关闭内置的显微镜相机LED。点击相邻的"设置"图标:选择"单色"，最大化"对比度"，并调整"亮度"，直到蠕虫出现与背景不同。 </li></ol></li><li> 获取校准视频。 <ol><li>将解决方案放置模板对齐在平台对齐模板的顶部（ 图2A ）。点击相机进纸左侧的"时间过长的视频"录制图标，输入&#39;5&#39;持续时间，&#39;1&#39;为间隔，以1帧/秒记录5秒的视频。点击"开始"开始录像。 </li></ol></li><li>对于其他两台显微镜相机重复步骤4.3和4.4。 </li><li>从室中取出溶液 - 放置模板。 </li><li><strong&gt;用洗涤缓冲液洗涤蠕虫三次以除去食物。 <ol><li>使用5 mL玻璃移液管将1.2 mL的NGM缓冲液加入到同步蠕虫的平板上，轻轻搅拌板，将蠕虫置于缓冲液中。从板上取出缓冲液，将缓冲液中的蠕虫分配到干净的微量离心管中。 注意:蠕虫对其培养琼脂盐浓度的偏差敏感14 。为了尽可能减少蠕虫放置后琼脂盐浓度的变化，建议使用NGM缓冲液进行洗涤过程（参见第5部分的配方）。不要使用塑料移液器从板上除去蠕虫，因为蠕虫会粘在两侧。准备等效数量的管作为同时运行的测定板的数量。 </li><li>让三根管子放置1-2分钟，让蠕虫沉降到底部。使用塑料移液器吸头从管中移除尽可能多的洗涤缓冲液，而不会干扰聚集体的沉淀在管底部的蠕虫。用1 mL干净的NGM缓冲液重新填充管子。 </li><li>重复步骤4.7.2一次。 </li></ol></li><li> 将10mM CuCl 2和M13缓冲溶液加入测定板（参见第5节中的配方）。 <ol><li>在解剖显微镜的舞台上贴上一个溶液放置模板。 </li><li>在平板对准模板的任一侧放置一条双面胶带。确保磁带与平板周边环重叠，但不会在模板中心遮挡感兴趣的记录区域。 </li><li>将测定板的周长与板对准模板上的圆对齐，然后向下按压直到模板粘附到板的底部。将测定板模板号标记与解剖显微镜上的溶液 - 放置模板标记对齐。 </li><li>使用P2移液器和塑料尖端将1μLM13缓冲液放在象限1和4（ 2溶液分别放置在象限2和3（ 图2A ）中。 </li><li>对其他两个测定板重复步骤4.8.2-4.8.4。 </li></ol></li><li>一旦溶液滴已被完全吸收到琼脂中，则使用玻璃巴斯德吸管将洗涤的蠕虫从每个微量离心管转移到相应的测定板上。 </li><li>在圆形处理区域上方和下方的区域放置一滴蠕虫。折叠组织四次，并使用钝边吸收所有测定板上的过量洗涤缓冲液。 </li><li>通过对齐模板角落处的数字，立即将三个测定板在显微镜照相机上放置在行为室中。等待2-3分钟以使蠕虫适应测定板。 注意:在下一步开始视频录制之前，请确保行为室门板关闭。 </li><li&gt;点击相机进纸左侧的"时间过长的视频"录制图标，并输入"10"分钟持续时间，"1"为间隔，以每秒1帧的速度录制10分钟的视频。点击"开始"开始录像。记录和保存视频后，从室中取出测定板。 </li><li>对下一轮测定板重复步骤4.7-4.12。获得每次治疗或基因型至少六次重复或两轮记录。 </li></ol> 5.试剂准备<ol><li> 准备NGM琼脂和缓冲液。 <ol><li>在1升玻璃瓶中，加入1.5克氯化钠，8.5克琼脂和1.25克蛋白胨。向瓶中加入500 mL蒸馏水和搅拌棒。高压灭菌瓶内容物。 </li><li>将瓶子放在搅拌盘上并打开搅拌功能。 </li><li>使用无菌技术按顺序加入:0.5mL 5mg / mL胆固醇，0.5mL 1M钙氯化物，0.5mL 1M硫酸镁和12.5mL 1M磷酸钾。 </li><li>重复步骤5.1.1至5.1.2制作NGM缓冲液，但在步骤5.1.1中不含琼脂和蛋白胨。 </li></ol></li><li> 准备LB肉汤 <ol><li>在1升玻璃瓶中加入5克胰蛋白胨，2.5克酵母提取物，2.5克氯化钠，500毫升蒸馏水和0.5毫升1N氢氧化钠。高压灭菌瓶内容物。 </li></ol></li><li> 准备M13缓冲液。 <ol><li>在1升玻璃瓶中，加入1.817克Tris，2.922克氯化钠，0.373克氯化钾和500毫升蒸馏水。用内容物高压灭菌。 </li></ol></li><li> 制备氯化铜（CuCl 2 ）溶液。 <ol><li>称量0.134克CuCl 2 。向15 mL塑料管中的10 mL M13缓冲液中加入溶质，制成100 mM CuCl 2储液。倒置塑料管，直到所有的溶质都弄脏LVED。 </li><li>使用注射器和0.2μm膜过滤器将100 mM CuCl 2溶液净化成干净的15 mL塑料管。 </li><li>为了制备10mM CuCl 2溶液，向微量离心管中加入900μLM13缓冲液和100μL100mM CuCl 2储备溶液。倒置微量离心管混匀。 </li></ol></li></ol>视频分析<ol><li>下载Python 3（https://www.python.org/downloads/）和OpenCV软件库（http://opencv.org/downloads.html）。 </li><li>下载三个Python脚本进行分析（https://github.com/WormLabCaltech/ResidentWorm）。 注意:在继续之前，请确保所有使用的视频文件都是mp4格式。 </li><li>要查看功能和参数说明，请在控制台中键入"help（enter function name）"，然后按Enter键。例如，键入"help（calibration）"，然后按Enter键。 </li><li> 执行校准功能具有用于定义感兴趣的视频区域（ROI）的输入参数。 <ol><li>输入第4节中为变量&#39;mp4filename&#39;获取的校准视频的文件名。输入文件名时包括引号。 </li><li>从"Q * x"和"Q * y"变量的值600和360开始。 *输入象限号1到4.从"rad"变量的值310开始。调整变量值，直到ROI掩模与模板上的象限轮廓对齐。 </li><li>记录执行此功能后将在控制台中打印的每个ROI中的像素总数。在preference_index函数中为"ROI_size"参数输入此值。 </li></ol></li><li>执行threshold_constant函数来确定适当的阈值常数。调整"常数"值，直到可以清楚地看到蠕虫（白色），背景中的盐和胡椒噪音最小（黑色）。 </li><li> 执行preference_index函数生成csv数据表，视频的偏好索引以及随附的蠕虫占用情况。 <ol><li>使用从步骤6.4和6.5确定的&#39;Q * x&#39;，&#39;Q * y&#39;，&#39;rad&#39;，&#39;ROI_size&#39;和&#39;常数&#39;作为preference_index函数的输入参数。 注意:对于每个视频帧，preference_index函数计算每个ROI中的蠕虫像素数，并计算出ROI像素总数中蠕虫像素的百分比。该函数然后使用以下公式为每个视频帧生成偏好索引值: <img alt="方程" src="/files/ftp_upload/55963/55963eq1v2.jpg" /> 注意:一旦程序分析了视频中的所有帧，就会为视频生成平均偏好索引值。具有视频首选项索引的蠕虫占用情况打印在顶部以及包含的csv数据表一旦程序运行完毕，蠕虫像素，蠕虫占有率百分比和每个视频帧的偏好索引将被保存到指定的文件夹中。 </li></ol></li></ol>

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作者没有什么可以披露的。

该方案的关键步骤是确保测定板在不同的实验日内具有一致的干燥度。不同的干燥度将导致溶液进入琼脂的不同扩散速率，从而导致行为结果的变化。因此，测试板应在实验前的下午始终保持新鲜。为了便于比较处理，也应调整每次测定的蠕虫数量。作为参考，如果上述蠕虫同步协议遵循17 ，则野生型蠕虫平均产生每次测定产生&gt; 360个蠕虫的4-10个卵/ h。如果某些突变菌株产卵有缺陷，选择更多的妊娠成虫虫卵来达到目标​​数量的后代。协议的另一个重要步骤是在洗涤过程和蠕虫放置期间轻轻地处理蠕虫。蠕虫对机械刺激敏感，引起应激反应<html>的逆转和产卵抑制18 。而且，在进行视频分析之前，应该注意准确定义ROI并确定特定照明条件的最佳阈值常数值。还建议如果自上次实验运行以来经过了漫长的一段时间，则重复校准和阈值处理。 这种方法的局限性在于它不适用于测定小蠕虫群体。然而，如果进行适当的控制来确定食物的存在对感觉行为的影响，那么利用食物来限制蠕虫的空间探索位置，如在保留测定中也是可能的。此外，该方法不是用于研究刺激梯度导航由于紧密接近和少量的控制和实验溶液滴使用。 e_content"&gt;在未来，编程软件，使多虫跟踪和单蠕虫特征提取可以集成到该系统19，20。记录的单个蠕虫行为微妙行为参数，如反转速度和身体弯曲的幅度将提供更详细地描述了在基于群体的检测方法中个体蠕虫的化学感应行为，还可以通过使用具有适当规格尺寸的注射器针孔通过ROI钻孔来修改测定以研究习惯，并用琼脂灌注孔与实验化合物或对照缓冲液，这将确保化合物在习性研究期间在更长的记录时间内更加一致的表面浓度。该方法的另一个潜在应用是对不同线虫种进行比较行为研究。另外，行为室可以通过多种方式修改以研究对多模态刺激的行为反应。对于光生应用，高强度LED阵列可以安装在相机底座旁边，以便在分析过程中选择性地激活感兴趣的神经元。加热元件，冷却系统和温度传感器也可以添加到设置中，以研究温度对感官行为的影响。此外，气味递送系统可以安装在室内以调查感器和化学感应模式之间的相互作用。

觅食动物必须整合来自多种感官模式的输入，并选择适当的行为策略，以便成功地导航他们的环境。了解外部感觉输入如何接收和转导到神经信息中以指导动作选择是神经生物学领域的核心目标。遗传易感线虫秀丽隐杆线虫是一种有吸引力的模型生物体，用于研究感官生物学和多模态整合的神经机制。尽管线虫仅具有302的神经元，它可以检测和各种环境刺激包括可溶性化合物，挥发性芳香剂，和环境温度1，2，3，4，5，6，7的区分。该线虫C.线虫在很大程度上依赖其化学感应器来本地化食物来源，并提醒自己潜在的威胁。因此，设计用于筛选野生型和突变体线虫对化学刺激的反应的行为测定在解剖线虫的显着感官能力的基因，细胞和神经机制中起着至关重要的作用。 为了测定对可溶性化合物的反应，已经描述了三种类型的测定 - 滴剂测试，趋化性测定和保留测定。在跌落试验中，该化合物的一小滴被放置在移动蜗杆和蜗轮的决定逆转或向前移动，一旦液体到达前感觉装置被拿下4的尾部。滴液测试需要很少的实验准备，并且当蠕虫的样品尺寸小时，如在激光操作的蠕虫的情况下是有用的。但是，只有一个蠕虫可以一次测定，并且实验者必须在整个测定期间存在，滴液测试可能是耗时的。跌落测试也容易受到样品中每个蠕虫之间滴落递减的变化的影响，这可能影响测定的总体结果。跌落测试的另一个限制是，它只能用于测定蠕虫对厌恶化合物的反应，因为不可能区分化合物与蠕虫向前运动的有吸引力或中性作用。 对于可溶性化合物的趋化性试验通常涉及将在琼脂平板分成四个象限，以混合成两个相对的象限的琼脂并混合到其他两个象限8,9对照溶液实验溶液。在测定开始时，将一滴含有蠕虫的缓冲液放置在板的中心，并将蠕虫的数量放入<html>每个象限在不同的时间点得分。与滴定试验相比，趋化性测定提供了更大的统计学功效，因为在每个测定中测试了大量蠕虫。然而，该方法的一个限制是趋化性测定板的制备需要大量的实验化合物。如果需要具有有限产率的复杂纯化方法来获得感兴趣的化合物，如在白加罗糖信号分子10的情况下，这将使得难以进行大规模行为筛选。此外，在整个测定过程中手动计数蠕虫易受错误影响，并且计数过程中板的扰动可能会影响结果。 与上述两种方法不同，保留测定利用机器视觉，其在评分过程中最小化误差并降低实验者在测定过程中的干扰<html> &gt; 11。计算机化分析的蠕虫行为的视频记录也可能潜在地显示微妙的行为动态，只有在几个离散的时间点进行评分时才会错过。在保留测定中，在小圆形细菌食物贴片的相对侧上加入两个溶液斑点，随后在食物贴片中间放置少量蠕虫。然后将该蠕虫的行为进行视频记录，分析，并且基于每个解决方案区域中的蠕虫像素的总数来计算偏好索引值。尽管存在有吸引力的食品贴片能够在每个测定中使用更少的蠕虫群体，但是食物以前已被证明可以使回避行为对可溶性驱避剂12敏感。此外，蠕虫对短波长光表现出光亮响应，并且在行为记录设置中使用发出白光的显微镜光源可能影响行为s ="xref"&gt; 13。 本文讨论的方法的目的是使用基于群体的测定来记录和分析秀丽隐杆线虫对可溶性化合物的偏好。为此，目前的方法整合并改进了前面讨论过的三种方法的各个方面。它可以测试大量的蠕虫，并且只需要在每个测定中使用少量的实验溶液。此外，该测定在具有红外LED背光的定制封闭行为室内进行，以最小化短波长光对行为的影响。每个室也可以配备多台显微镜相机，这样可以提高实验产量而不会影响工作台面积。最后，视频分析软件输出每个视频的偏好索引值，以及随附的蠕虫占用情况，以随时间推测人口行为动态。房间设置和assay方案可以进一步修改以研究多模态行为响应，例如气味剂或温度对化学感觉行为的影响。 本文介绍了行为室和测定方案的构建。它还证明了该方法在测定野生型蠕虫和化学感染缺陷突变体对已知可溶性驱避剂铜离子的反应的实用性4 。最后，详细介绍使用提供的软件进行视频分析的过程。

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</table>

automated analysis of a nematode population-based chemosensory preference assay

线虫， 秀丽隐杆线虫的紧凑型神经系统只有302个神经元，构成了各种各样的行为。为了便于基于这些行为的神经回路的解剖，需要开发健壮和可重复的行为测定。以前的线虫行为研究已经使用"滴定测试"，"趋化性测定"和"保留测定"的变化来研究秀丽隐杆线虫对可溶性化合物的反应。本文中描述的方法旨在结合上述三种测定的互补优势。简而言之，每个测定板中间的一个小圆圈被分成四个象限，其中交替放置对照和实验溶液。在添加蠕虫之后，将测定板装载到行为室中，其中显微镜照相机记录蠕虫与被处理区域的相遇。然后执行自动视频分析a并生成每个视频的偏好索引（PI）值。该方法的视频采集和自动分析功能最大限度地减少了实验者的参与和任何相关错误。此外，每个测定使用微量的实验化合物，并且行为室的多摄像机设置增加了实验吞吐量。该方法对于进行遗传突变体和新化合物的行为筛选是特别有用的。然而，这种方法不适合于研究刺激梯度导航由于控制和实验解决方案区域的紧密接近。当只有少量蠕虫可用时，也不应该使用它。虽然适用于仅测定其当前形式的可溶性化合物的反应，但是该方法可以容易地修饰以适应多峰感官相互作用和光遗传学研究。该方法也可适用于测定其他线虫物种的化学感应反应。 </p&gt;

图3A显示了针对不同基因型和治疗配对获得的偏好指数值。偏好指数值1表示对实验ROI中的溶液的强吸引力，而偏好指数值-1表示强排斥。当将M13缓冲溶液置于对照和实验ROI两者中时，获得了0.02的偏好指数，表明没有对ROI的空间偏差。 N2蠕虫强烈地避免了铜离子，导致偏好指数为-0.67，这证实了以前的发现，铜离子是强驱虫剂（ 补充电影1 ） 4 。 osm-3突变体缺乏适当形成感觉纤毛的远端部分，显示出明显减少的铜离子回避（PI = -0.19） 15 。 ocr-2突变体，<html>许多ASH介导的伤害感受反应（包括铜避免）中的有效性也显示出显着减少的避免，甚至对铜离子的一些温和吸引（PI = 0.19）（ 补充电影2 ） 16 。 图3B显示了代表性的蠕虫占据图，其表示在每个视频中随着时间的推移，控制中蠕虫的密度与实验的ROIs密切相关。绘图区域的颜色越暗，ROI中的蠕虫像素数越多。用M13缓冲液对照处理的N2蠕虫的占用情况表明，在整个测定过程中，两个ROI中蠕虫的数量保持不变。然而，用铜离子处理的N2蠕虫的占用情况表明，蠕虫在整个测定中强烈而一致地避免了实验性ROI。 gimg"src ="/ files / ftp_upload / 55963 / 55963fig1.jpg"/&gt; 图1:行为室设计的照片和原理图表示。 （ A ）行为室设置的前视图（左）和腔室框架的对应示意图（右）。不透明的聚酯薄片包围除了底面以外的所有面上的腔室，这允许来自红外背光面板的光通过。数字1,2和3对应于（B）中的挤出层。 （ B ）包括行为室框架的挤出层的顶视示意图。这包括顶层（1），中间相机安装组件层（2）和底层层（3）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55963/55963fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55963/55963fig2.jpg"/&gt; 图2:解决方案放置和板对齐模板。 （ A ）溶液放置模板有四个象限和数字对齐标记。将控制溶液放置在左上和右下象限中，同时将实验溶液置于右上和右下象限中。 （ B ）板对准模板仅具有数字对准标记，以在视频记录和分析期间最小化视野中的蠕虫的阻塞。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55963/55963fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/55963/55963fig3.jpg" /> 图3:使用此测定收集的示例数据。 （ A ）野生型N2和突变体线虫的偏好指数（PI）值</e<html> m&gt;响应于M13缓冲液和10mM CuCl 2 。当将M13对照溶液放置在两个ROI中时，N2蠕虫在对照和实验ROI之间没有显示出偏好，但是当放置铜离子（PI = 0.02和-0.67）时，强烈地避免了实验ROI。与N2相比， osm-3（p802）突变体和ocr-2（ak47）突变体显示出明显减少的铜离子回避（PI = -0.1和0.2）。每个基因型治疗配对的N = 6个测定，每次测定的&gt; 360个蠕虫，误差线表示±1个SEM，*** p &lt;0.001，单因素方差分析，然后进行事后Tukey诚实显着差异（HSD）检验。 （ B ）在两个ROI（顶部）和N2蠕虫中，具有M13缓冲区的N2蠕虫在控制ROI中的代表性蠕虫占有率图和实验ROI（底部）中的铜离子。每个占用图下方的颜色标尺表示蠕虫像素的数量。pload / 55963 / 55963fig3large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 补充电影1:N2蠕虫对10 mM氯化铜的行为反应。蠕虫被M13缓冲器（左上和右下）的控制象限吸引，但强烈地避免了含有溶解在M13缓冲液中的铜离子的象限（右上和左下）。视频以每秒1帧的速度记录，并加快了15倍。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55963/Supplementary_Video_1.mp4">请点击这里下载此文件。</a> 补充电影2: ocr-2（ak47） 蠕虫对10mM氯化铜的 行为反应 。蠕虫在M13缓冲器（左上和右下）和象限的控制象限中大致相等程度地漫游s含有溶解在M13缓冲液中的铜离子（右上和左下）。在记录开始时，突变体对含有铜离子的象限略显偏爱。视频以每秒1帧的速度记录，并加快了15倍。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55963/Supplementary_video_2.mp4">请点击这里下载此文件。</a> 补充文件:解决方案放置和板对齐模板。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55963/JoVE_template.pptx">请点击这里下载此文件。</a>

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Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

我们提出一种行为记录设置和协议，能够自动分析线虫， 秀丽隐杆线虫在基于群体的测定中对可溶性化合物的偏好。本文介绍了行为室的构造，行为测定方案和视频分析软件的使用。

基于线虫人群的化学感应优选测定的自动分析

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection ...

automated-analysis-nematode-population-based-chemosensory-preference

Nanyang Technological University

Research

JoVE Journal

Behavior

7.4K 次观看.  California Institute of Technology. 我们提出一种行为记录设置和协议，能够自动分析线虫， 秀丽隐杆线虫在基于群体的测定中对可溶性化合物的偏好。本文介绍了行为室的构造，行为测定方案和视频分析软件的使用。 

视频: Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

Fat Preference: A Novel Model of Eating Behavior in Rats

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to this multifactorial disorder is the consumption of a high fat diet, a behavior that has been shown to increase both caloric intake and body fat content. However, the elements regulating preference for high fat food over other foods remain understudied.
To overcome this deficit, a model to quickly and easily test changes in the preference for dietary fat was developed. The Fat Preference model presents rats with a series of choices between foods with differing fat content. Like humans, rats have a natural bias toward consuming high fat food, making the rat model ideal for translational studies. Changes in preference can be ascribed to the effect of either genetic differences or pharmacological interventions. This model allows for the exploration of determinates of fat preference and screening pharmacotherapeutic agents that influence acquisition of obesity.

Dietary fat content influences both energy intake and body fat composition in mammals. By examining rats&#8217; preference for high fat food in a series of choice experiments, it is possible to test genetic differences and pharmacological interventions on their preference for high fat food.

脂肪偏好:饮食行为的一种新模型大鼠

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to ...

科研

行为学

Experimental Assessment of Mouse Sociability Using an Automated Image Processing Approach

Mouse is the preferred model organism for testing drugs designed to increase sociability. We present a method to quantify mouse sociability in which the test mouse is placed in a standardized apparatus and relevant behaviors are assessed in three different sessions (called session I, II, and III).
The apparatus has three compartments (see Figure 1), the left and right compartments contain an inverted cup which can house a mouse (called &#34;stimulus mouse&#34;).
In session I, the test mouse is placed in the cage and its mobility is characterized by the number of transitions made between compartments. In session II, a stimulus mouse is placed under one of the inverted cups and the sociability of the test mouse is quantified by the amounts of time it spends near the cup containing the enclosed stimulus mouse vs. the empty inverted cup. In session III, the inverted cups are removed and both mice interact freely. The sociability of the test mouse in session III is quantified by the number of social approaches it makes toward the stimulus mouse and by the number of times it avoids a social approach by the stimulus mouse.
The automated evaluation of the movie detects the nose of the test mouse, which allows the determination of all described sociability measures in session I and II (in session III, approaches are identified automatically but classified manually). To find the nose, the image of an empty cage is digitally subtracted from each frame of the movie and the resulting image is binarized to identify the mouse pixels. The mouse tail is automatically removed and the two most distant points of the remaining mouse are determined; these are close to nose and base of tail. By analyzing the motion of the mouse and using continuity arguments, the nose is identified.
<img alt="Figure 1" src="/files/ftp_upload/52508/52508fig1.jpg" /> 
Figure 1. Assessment of Sociability During 3 sessions. Session I (top): Acclimation of test mouse to the cage. Session II (middle): Test mouse moving freely in the cage while the stimulus mouse is enclosed in an inverted cup. Session III (bottom): Both test mouse and stimulus mouse are allowed to move freely and interact with each other.

This protocol describes a method to quantify mouse sociability. Mice are videotaped as they move and interact in a special cage. Movie processing allows for the automated quantification of sociability with excellent accuracy and reliability.

使用自动图像处理方法鼠标社交性的实验评估

Mouse is the preferred model organism for testing drugs designed to increase sociability. We present a method to quantify mouse sociability in which the ...

Assessment of Cocaine-induced Behavioral Sensitization and Conditioned Place Preference in Mice

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to drugs of abuse make lasting alterations in the brain function of vulnerable individuals, and these persistent alterations likely serve to maintain the maladaptive drug seeking and taking behaviors characteristic of addiction/dependence2. In rodents, reward experience and contextual associations are frequently measured using the conditioned place preference assay, or CPP, wherein preference for a previously drug-paired context is measured. Behavioral sensitization, on the other hand, is an increase in a drug-induced behavior that develops progressively over repeated exposures. Since sensitized behaviors can often be measured after several months of drug abstinence, depending on the dose and length of initial exposure, they are considered observable correlates of lasting drug-induced plasticity. Researchers have found these assays useful in determining the neurobiological substrates mediating aspects of addiction as well as assessing the potential of different interventions in disrupting these behaviors. This manuscript describes basic, effective protocols for mouse CPP and locomotor behavioral sensitization to cocaine.

This protocol is intended to enable researchers to conduct experiments designed to test these aspects of addiction using the conditioned place preference and locomotor behavioral sensitization assays.

可卡因诱导行为敏和小鼠位置偏爱评估

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to ...

Protocol for Data Collection and Analysis Applied to Automated Facial Expression Analysis Technology and Temporal Analysis for Sensory Evaluation

We demonstrate a method for capturing emotional response to beverages and liquefied foods in a sensory evaluation laboratory using automated facial expression analysis (AFEA) software. Additionally, we demonstrate a method for extracting relevant emotional data output and plotting the emotional response of a population over a specified time frame. By time pairing each participant's treatment response to a control stimulus (baseline), the overall emotional response over time and across multiple participants can be quantified. AFEA is a prospective analytical tool for assessing unbiased response to food and beverages. At present, most research has mainly focused on beverages. Methodologies and analyses have not yet been standardized for the application of AFEA to beverages and foods; however, a consistent standard methodology is needed. Optimizing video capture procedures and resulting video quality aids in a successful collection of emotional response to foods. Furthermore, the methodology of data analysis is novel for extracting the pertinent data relevant to the emotional response. The combinations of video capture optimization and data analysis will aid in standardizing the protocol for automated facial expression analysis and interpretation of emotional response data.

A protocol for capturing and statistically analyzing emotional response of a population to beverages and liquefied foods in a sensory evaluation laboratory using automated facial expression analysis software is described.

协议进行数据采集和分析应用到自动人脸表情分析技术和时序分析感官评价

We demonstrate a method for capturing emotional response to beverages and liquefied foods in a sensory evaluation laboratory using automated facial ...

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration

Neurodegenerative diseases are frequently associated with a progressive loss of movement ability, reduced life span, and age-dependent neurodegeneration. To understand the mechanism of these cellular events, and their causal relationships with each other, Drosophila melanogaster, with its sophisticated genetic tools and diverse behavioral features, are used as disease models for assessing neurodegenerative phenotypes. Here we describe a high-throughput method to analyze Drosophila adult negative geotaxis behavior, as an indication for possible motor defects associated with neurodegeneration. An automated machine is designed and developed to drive fly synchronization using an initial electric impulse, later allowing the recording of negative geotaxis behavior over a course of secs to mins. Images from the digitally recorded video are then processed with the self-designed RflyDetection software for statistical data manipulation. Different from the manually controlled negative geotaxis assay based on single fly, this precise, fast, and high-throughput protocol allows data acquisition from more than hundreds of flies simultaneously, providing an efficient approach to advance our understanding in the underlying mechanism of locomotor deficits associated with neurodegeneration.

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration

This step-by-step protocol analyzes Drosophila negative geotaxis behavior using an automated multi-cylinder system that hosts hundreds of flies and synchronizes their action by an electric motor. Upon synchronization, fly negative geotaxis behavior is assayed, digitally recorded, and analyzed using the self-designed RflyDetection software.

一种自动快速迭代阴性 Geotaxis 分析方法在神经的果蝇模型中的成人攀爬行为

Neurodegenerative diseases are frequently associated with a progressive loss of movement ability, reduced life span, and age-dependent neurodegeneration. ...

Reinstatement of Drug-seeking in Mice Using the Conditioned Place Preference Paradigm

The present protocol describes the Conditioned Place Preference (CPP) as a model of relapse in drug addiction. In this model, animals are first trained to acquire a conditioned place preference in a drug-paired compartment, and after the post-conditioning test, they perform several sessions to extinguish the established preference. The CPP permits the evaluation of the conditioned rewarding effects of drugs related to environmental cues. Then, the extinguished CPP can be robustly reinstated by the non-contingent administration of a priming dose of the drug, and by exposure to stressful stimuli. Both methods will be explained here. When the animal reinitiates the behavioral response, a reinstatement of the conditioned reward is considered to have taken place.
The main advantages of this protocol are that it is non-invasive, inexpensive, and simple with good validity criteria. In addition, it allows the study of different environmental manipulations, such as stress or diet, which can modulate relapse into drug seeking behaviors. However, one limitation is that if the researcher aims to explore the motivation and primary reinforcing effects of the drug, it should be complemented with self-administration procedures, as they involve operant responses of animals.

This protocol describes the Conditioned Place Preference (CPP) as a model of relapse. This procedure permits the measurement of relapse in laboratory animals, considering the impact of drug-associated environmental cues as craving and relapse in abstaining addicts is currently the focus of drug-abuse treatment programs.

用条件位置偏好范式恢复小鼠药物寻毒作用

The present protocol describes the Conditioned Place Preference (CPP) as a model of relapse in drug addiction. In this model, animals are first trained to ...

Automated, Long-term Behavioral Assay for Cognitive Functions in Multiple Genetic Models of Alzheimer's Disease, Using IntelliCage

Multiple factors&#8212;such as aging and genes&#8212;are frequently associated with cognitive decline. Genetically modified mouse models of cognitive decline, such as Alzheimer&#39;s disease (AD), have become a promising tool to elucidate the underlying mechanisms and promote the therapeutic advances. An important step is the validation and characterization of expected behavioral abnormality in the models, in the case of AD, cognitive decline. The long-term behavioral investigations of laboratory animals to study the effect of aging demand substantial efforts from researchers. The IntelliCage system is a high-throughput and cost-effective test battery for mice that eliminates the need for daily human handling. Here, we describe how the system is utilized in the long-term phenotyping of a genetic Alzheimer&#39;s disease model, specifically focusing on the cognitive functions. The experiment employs repeated battery of tests that assess spatial learning and executive functions. This cost-effective age-dependent phenotyping allows us to identify the transient and/or permanent effects of genes on various cognitive aspects.

This paper describes a protocol for cognitive assessments for genetic models of the Alzheimer&#39;s disease using the IntelliCage system, which is a high throughput automated behavioral monitoring system with operant conditioning.

基于 IntelliCage 的阿尔茨海默病多基因模型认知功能的自动、长期行为检测

Multiple factors&#8212;such as aging and genes&#8212;are frequently associated with cognitive decline. Genetically modified mouse models of cognitive ...

A Conditioned Place Preference Protocol for Measuring Incubation of Craving in Rats

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed incubation of drug craving. Here, we describe a morphine conditioned place preference (CPP) protocol for measuring the incubation of craving in rats. In this protocol, a CPP paradigm mainly employing somatosensory cues is used to establish a long-term reward memory of morphine. A three-chamber CPP box that differs in the texture of the chamber floor is constructed. First, the animals are tested for their baseline preference to the two side chambers for three consecutive days. Then, they are injected intraperitoneally with morphine/saline and put into their non-preferred/preferred chamber for 45 min. After 6 days of conditioning, their preference to the side chambers is tested for 15 min at different time points after the last conditioning session. With this paradigm, the reward memory of morphine could last for at least 18 days. To test whether the above-mentioned protocol can model increased craving, the number of entrances into the two side chambers are counted during the abstinence period. The results show that the entrances increased, suggesting that the CPP paradigm could mimic the incubation of craving. Future studies can employ this model to study neural mechanisms underlying long-term memory and incubation of craving.

Here, a morphine conditioned place preference (CPP) protocol is described to measure incubation of craving in rats.

一种用于大鼠渴望培养的条件场所偏好协议

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed ...

Paw-Print Analysis of Contrast-Enhanced Recordings (PrAnCER): A Low-Cost, Open-Access Automated Gait Analysis System for Assessing Motor Deficits

Gait analysis is used to quantify changes in motor function in many rodent models of disease. Despite the importance of assessing gait and motor function in many areas of research, the available commercial options have several limitations such as high cost and lack of accessible, open code. To address these issues, we developed PrAnCER, Paw-Print Analysis of Contrast-Enhanced Recordings, for automated quantification of gait. The contrast-enhanced recordings are produced by using a translucent floor that obscures objects not in contact with the surface, effectively isolating the rat&#8217;s paw prints as it walks. Using these videos, our simple software program reliably measures a variety of spatiotemporal gait parameters. To demonstrate that PrAnCER can accurately detect changes in motor function, we employed a haloperidol model of Parkinson&#8217;s disease (PD). We tested rats at two doses of haloperidol: high dose (0.30 mg/kg) and low dose (0.15 mg/kg). Haloperidol significantly increased stance duration and hind paw contact area in the low dose condition, as might be expected in a PD model. In the high dose condition, we found a similar increase in contact area but also an unexpected increase in stride length. With further research, we found that this increased stride length is consistent with the bracing-escape phenomenon commonly observed at higher doses of haloperidol. Thus, PrAnCER was able to detect both expected and unexpected changes in rodent gait patterns. Additionally, we confirmed that PrAnCER is consistent and accurate when compared with manual scoring of gait parameters.

Paw-Print Analysis of Contrast-Enhanced Recordings PrAnCER: A Low-Cost, Open-Access Automated Gait Analysis System for Assessing Motor Deficits

We describe a novel gait analysis system, Paw-Print Analysis of Contrast-Enhanced Recordings (PrAnCER), an open-access automated system for the quantification of gait characteristics in rats that utilizes a novel semitransparent floor to automatically quantify gait. This system was validated using the haloperidol model of Parkinson&#8217;s Disease.

对比度增强记录 (PrAnCER) 的爪打印分析:用于评估电机缺陷的低成本、开放访问自动步态分析系统

Gait analysis is used to quantify changes in motor function in many rodent models of disease. Despite the importance of assessing gait and motor function ...

A System for Tracking the Dynamics of Social Preference Behavior in Small Rodents

Exploring the neurobiological mechanisms of social behavior requires behavioral tests that can be applied to animal models in an unbiased and observer-independent manner. Since the beginning of the millennium, the three-chamber test has been widely used as a standard paradigm to evaluate sociability (social preference) and social novelty preference in small rodents. However, this test suffers from multiple limitations, including its dependence on spatial navigation and negligence of behavioral dynamics. Presented and validated here is a novel experimental system that offers an alternative to the three-chamber test, while also solving some of its caveats. The system requires a simple and affordable experimental apparatus and publicly available open-source analysis system, which automatically measures and analyzes multiple behavioral parameters at individual and population levels. It allows detailed analysis of the behavioral dynamics of small rodents during any social discrimination test. We demonstrate the efficiency of the system in analyzing the dynamics of social behavior during the social preference and social novelty preference tests as performed by adult male mice and rats. Moreover, we validate the ability of the system to reveal modified dynamics of social behavior in rodents following manipulations such as whisker trimming. Thus, the system allows for rigorous investigation of social behavior and dynamics in small rodent models and supports more accurate comparisons between strains, conditions, and treatments.

Described here is a novel automated experimental system that offers an alternative to the three-chamber test and also solves several caveats. This system supplies multiple behavioral parameters that enable rigorous analysis of small rodent behavioral dynamics during the social preference and social novelty preference tests.