我们承认维也纳果蝇资源中心和布卢明顿果蝇股票中心（NIH P40OD018537）用于提供果蝇株。我们感谢来自显微成像中心杰克·弗兰森在成像专家支持。这项研究是由VIDI和荷兰科学研究组织（NWO）TOP补助（917-96-346，912-12-109），由德国智障网络支持由两个DCN / Radboud大学医学中心博士奖学金教育与研究部（BMBF）德国联邦财政部的NGFN +程序，并通过欧盟FP7大规模集成网络Gencodys（HEALTH-241995），以AS资助。该资助者在研究设计，数据收集和分析，决定发表或准备手稿没有作用。

1.需求之前图像处理<ol><li>执行第三龄幼虫徘徊（L3）的果蝇翻书制剂，如前所述19。 </li><li>共immunolabel使用两种标记的组合果蝇 NMJ端子:DLG-1或的Hrp连同BRP与" 果蝇 NMJ形态特征"的分析，和嘉庆或CSP一起BRP与" 果蝇 NMJ布顿形态特征" 20分析。 注:来自相同物种的抗体可以通过预先标记一个与抗体缀合试剂盒如泽农Alexa的标记试剂盒17相结合。 </li><li>图像NMJ终端使用的选择， 例如，荧光（有或没有ApoTome）或共焦显微镜的显微镜。 <ol><li>获取NMJ终端的2通道的图像栈。 <ol><li>以这样的方式调整显微镜设置，信道1获取与DLG-1（或者HRP，嘉庆，CSP）和信道2免疫标记与BRP免疫标记的NMJ终端的NMJ终端。 </li><li>任选地，分析（与单一抗体免疫标记的突触）一个通道的图像与所述宏。图像NMJs与DLG-1或唯一的Hrp用于免疫标记以" 果蝇 NMJ形态特征"，或嘉庆或CSP为" 果蝇 NMJ布顿形态测量"分析。 注意:这是不可能的分析，只有抗BRP免疫染色突触。 </li></ol></li><li>导出所获得的图像作为单独的" .tiff文件。倒置之前运行的宏如果所指示的未获取的信道的顺序。 </li></ol></li></ol> 2.软件需求和安装<ol><li>从以下网站" 果蝇 NMJ形态特征"和" 果蝇 NMJ布顿形态特征":下载宏https://figshare.com/s/ec634918c027f62f7f2a <sup类= "外部参照"&gt; 21。 </li><li>将光标移动到文件夹"宏更新1"，然后单击出现的选项"视图"。会出现该文件夹的内容列表。该文件夹包含宏" 果蝇 NMJ形态特征"和" 果蝇 NMJ布顿形态特征"。 注:这两个宏与斐济版本1.4，这也是在同一个文件夹中提供兼容。宏可能无法在新版本上运行。请使用所提供的1.4版本。这是没有问题的，开始这个版本，甚至计算机上可用较新版本的斐济。 </li><li>点击"全部下载"。该文件夹的内容将被下载到计算机的.zip文件。解压下载的文件。 </li><li> 果蝇 _NMJ_Morphometrics.ijm和果蝇 _NMJ_Bouton Morphometrics.ijm文件复制到Fiji.app/plugins/目录。重新启动程序时，宏将出现在插件的底部下拉式菜单。 </li></ol> 3.运行子宏"转换为栈"创建NMJ图像的Z-预测和Hyperstacks <ol><li>通过工具栏中选择插件在下拉菜单中启动图形界面，然后选择" 果蝇 NMJ形态特征"。 </li><li>定义宏的图形界面的"唯一文件字符串"设置。 注:显微镜软件使用的标识签名存储堆栈作为单独的" .tiff文件时，组织飞机和渠道。所输入的唯一文件字符串设置需要指定由软件分配到第一通道的第一平面上的签名（重要:最低平面和通道数量需要注明）。 </li><li>只选择子宏"转换为堆"，然后点击"确定"，然后选择其中的图片所在的文件夹。如果选择了几个子文件夹的主目录下，所有的个人" .TIFF中的文件主目录和子匹配的唯一文件字符串标准将被处理。 <ol><li>如果z-栈仅包含一个信道，选择框"通道只有1"。 </li></ol></li><li>请注意，每个NMJ图像两个新文件，在默认情况下称为stack_image_name和flatstack_image_name会出现。只存储这些堆栈和flatstack作进一步的分析。该.TIFF文件系列可在此时被删除，最大限度地减少所需的存储容量，并避免可能的误差来源。 </li></ol> 4.运行子宏"定义ROI"划定利益的NMJ终端<ol><li>开始" 果蝇 NMJ形态特征"的图形界面。 </li><li>仅选择复选框"定义ROI"，然后按"确定"按钮并选择题为flatstack_name存储图像，然后按"选择"主目录。子宏"定义ROI"通过所有子文件夹自动搜索所选择的主目录内。 </li><li>作为第一投影打开时，选择工具栏上的"写意选择"工具。使用鼠标绘制一个专门包含感兴趣完整的NMJ终端的选择，并在窗口中的"定义终端"，单击"确定"。宏将与下一个投影进行。 </li><li>描绘未来的投资回报率和重复，直到所有ROI的定义。的ROI的图像文件，命名为"roi_image_name"，将被存储在相同的目录中的每个处理的图像的先前生成的堆栈和投影图像。此子宏的输出是在白色的ROI在黑色背景上的二进制图像。 </li></ol> 5.运行子宏"分析"来量化NMJ终端特点<ol><li>转到工具栏中，选择"插件"，并使用: " 果蝇 _NMJ_Morphometrics"分析用抗DLG-1或抗HRP（信道1）免疫标记的突触时一起用抗BRP（通道2），或" 果蝇 _NMJ_Bouton_Morphometrics"分析用抗嘉庆或抗CSP与BRP（信道1）一起免疫标记突触时（信道2）。 <ol><li>当使用一个通道图像栈要分析（对于"Drosophila_ NMJ_Morphometrics"，或嘉庆或CSP为" 果蝇 _NMJ__Bouton_Morphometrics"结构性通道DLG-1或HRP），选择框"通道只有1"。 </li></ol></li><li>调整对应于图像中的刻度进行分析。如果图像中的一个像素对应于2.5微米，表明在微米= 2.5量表像素= 1，尺度距离。在情况下，两个设置在0时，NMJ面积，周长，长度和最长长度分支将在像素的个数来表示。 </li></ol><ol start="3"><li>如果需要，调整宏的默认分析设置。执行调整只有在子宏"分析"（成绩不理想的这一部分，而DETE在第6 rmined更好的设置）已经运行。 </li><li>选中复选框"分析"和"等待"，然后按"确定"。 <ol><li> 2个通道图像运行子宏"分析"时，选择"等待"复选框。否则，在活动区域​​计数错误可能是由于有限的计算机能力。 </li></ol></li><li>作为一个新的窗口，"选择目录"打开，选择图像所在，然后按"选择"的目录。宏将分析存储在主目录下的所有图片，如果适用，后续的文件夹（使用从执行前一子宏的三个文件:stack_image_name，flatstack_image_name和roi_image_name）。宏处理每个图像单独地和连续。这可以（取决于计算机容量）采取每图像堆栈几分钟。 </li><li>运行宏后，请注意，新的命名为res_image_name每个图像文件进行分析突触存储将是CREA泰德在亲本文件夹中。定量测量将被存储为"RESULTS.TXT"文件。 </li><li>检查所有结果图像来检测和排除与分割错误的图片。可能分割错误在表3中所述，建议如何调整设置，以规避这些沿。与这样的分割误差结果图像被设置为在图4中的例子。 注意:当运行在所述用户界面中观察到的默认设置宏，有大约95％的精确度，当宏评估相比手动评估17。 </li></ol> 6.调整宏设置到图像<ol start="1"><li>当图像的超过5％的显示分割错误，探索不同的算法来定义/选择的图像中的最合适的宏设置。 </li><li> 调整滚动球半径值 注:滚动球的半径函数减去图像的背景。与荧光显微镜获得的图像时，此功能是至关重要的和/或当图像具有高背景噪声。背景减法将帮助宏的自动阈值的步骤，以产生NMJ终端的足够分割。 <ol><li>选择三个NMJ Z-突起（由子宏"转换为堆栈"而生成flatstack_image_name图像）是表示该图像的数据集。 </li><li>在工具栏中，选择图片|颜色|拆分通道。两个图像将被创建，一个表示信道1和其它信道2。 </li><li>只保留属于通道1门打开，对应于DLG-1，HRP，嘉庆或CSP免疫标记，并通过关闭图像丢弃BRP通道图像的图像。 </li><li>在下拉菜单中选择工具栏，然后在"过程"，"扣除背景..."运行过滤器"扣除背景"。 </li><li>点击在弹出窗口中的预览复选框并调整滚球半径增加突触和背景之间的对比度在面板（ 图5A"）的值。 注意:请参阅图5中的示例。在Figure5A，突触的部分表示同一灰度级为背景，而在图5A"的‘风水球半径’设置为500产生突触和背景之间的强烈的对比。 </li><li>当定义为滚球半径为适当的值，运行所选的Z-突起具有相同的滚珠半径值"减去背景"算法，并将其保存（在任何目录）。 注:对于8位或RGB图像的滚动球半径值应至少为不是背景的一部分的图像中的最大对象的半径一样大。对于16位和32位图像的半径应INVersely成比例的像素值范围22。 </li></ol></li><li> 确定将使用不同的自动阈值 <ol><li>打开保存在前面的步骤（6.2.6）的Z-预测和选择图片|调整| AutoThreshold |尝试所有。 </li><li>作为二进制阈值化的结果图像将与所有不同的自动阈值算法的出现，确定用于图像的最适合的算法。 <ol><li>当以后运行宏，在宏设置更改相应的阈值。 </li><li>使用更严格的阈值，如"RenyiEntrophy"或"时刻"一样的NMJ轮廓门槛，如"礼"更宽松的阈值来确定NMJ骨架，"黄"，以确定活动区域。当图像是非常尖锐的几乎没有背景，用"黄"为" NMJ轮廓门槛，否则突触部分可能的图像分割后会丢失。 </li><li>参见图5B中的示例。与由绿框突出自动阈值获得的突触适当的分割。非合适的阈值的一些例子是由红色框（检查在高放大倍率突触）高亮显示。在后者中，突触的任一部分丢失或背景的部分被包括在内。见参考文献23获取更多信息。 </li></ol></li></ol></li></ol><ol start="4"><li> 确定所述小颗粒的最大尺寸 注:该函数将排除了比在"小颗粒设置"从分析所定义的值的情况下由NMJ轮廓阈值和骨架阈值来检测所有的颗粒。该值以像素为单位定义的。该功能用作噪声过滤器，是非常有用的，当非均匀背景高速率（如晶体/粉尘）存在于所获得的图像。 <ol><li>打开保存在步骤6.2.6 Z-突起。并设置规模检测通过分析像素数|设定的比例。应用下列设置:距离以像素= 1，已知的距离= 1，像素纵横比= 1，长度=像素和按下"OK"的单位。点击工具栏上的"椭圆选择"工具。 </li><li>使用鼠标绘制一个选区紧紧围绕单一颗粒存在于免疫染色，但不属于NMJ。按Ctrl + M用于Windows用户或CMD + M Mac用户。一个结果窗口将打开，表明像素数中选择的颗粒的面积。 </li><li>与存在于图像，以确定最大的污染颗粒/伪影区域多个工件重复上述步骤数次。这将是以后在运行宏时在设置中设置的值。当运行宏设置的"小颗粒尺寸"为最小粒径观察脓25％的裕度。 </li><li>参见图5D的例子。最大的晶体检测ED具有112个像素的区域。 "小颗粒大小"的设置，与宏处理该图像时，应设置TO125 - 150。 </li></ol></li></ol><ol start="5"><li> 确定最小布顿大小 注:此功能将排除比从分析定义的值由NMJ轮廓的阈值检测到的所有终扣。该值以像素为单位定义的。 <ol><li>按照同样的步骤，如第6.4节描述的那样，但是在这种情况下，绘制周围存在于NMJ终端的最小终扣的选择。选择对应的实测最小布顿最小面积。这是后来上运行宏时在最小布顿大小设置设定值。 </li></ol></li></ol><ol start="6"><li> 定义"查找最大噪声容限"值 <ol><li>使用6.2.1节中选择的Z-hyperstacks。 </li><li>在工具栏中，选择图片|颜色|拆分渠道CREATE 2堆叠（用于信道1和信道2），并保持该图像对应于所述信道BRP开放。通过关闭它丢弃其他信道的图像。 </li><li>转到在弹出菜单中的插件选项卡，选择流程|最大（3D），以及maximum_image_name出现时（这可能需要长达几分钟）后，关闭原图像栈。 </li><li>选择最大... IMAGE_NAME（所获得的图像栈），然后选择插件|流程|最小（3D），关闭最大... IMAGE_NAME堆栈。 </li><li>在工具栏中，选择Process |查找最大值...。一个新窗口"查找最大值..."将打开。单击勾选"预览点的选择......"，并与默认的宏设置50千里马点将图像为小十字中应标明填充"噪声容限"复选框。 <ol><li>加大对"噪声容限"值，如果观察过量注解的活动区域， 即十字架上没有的活动区域的顶部是在着眼于所选择的堆栈平面，或在背景中检测到的错误的活动区域。 <ol><li>在另一方面，如果观察不完全注释的活动区域， 即活动区域中不被认可的标记重点，减少"噪声容限"值。不断尝试执行此过程，直到十字适当聚焦标注活动区域不同的值。填写"查找最大噪声容限"这个值。 </li><li>参见图5C中的例子。检测到太多的活动区域。在图5C中"，仅聚焦活动区域被检测到。 </li></ol></li></ol></li><li>运行子宏"分析"在步骤5.1中选择的代表图像，与在所有前面的步骤中定义的设置。 </li></ol></li><li> 调整BRP-斑点下部和上部阈值 <ol><li>请注意，新文件将运行第m后出现根据ACRO到步骤6.6，称为2_active_zone_stack_image_name。在这种图像叠加由"查找最大值"功能检测到通过在每个平面中的白点指示的活动区域。 </li><li>通过拖放到工具栏，然后选择图片打开此文件|堆栈| Zproject |投影型=萨姆切片。所述2_active_zone_stack_image_name的投影将被获得。 </li><li>选择图片|调整|阈。一个新窗口"门槛"将打开。滑动上部杆选择在何处，所有期望的病灶/ BRP阳性斑点在红色可视化的阈值。 注意:如果阈值设置得太低，过量的活性区将被计数。如果设置得太高，在活动区域​​的一小部分将被错过。 <ol><li>参见图5E为一个例子。当阈值设置为400，最有源区域（符号化为1个像素灶）的不包括在所述分割的，因为它们没有在红色（ 图5突出E）。当阈值被设定为值50的所有活动区域以红色突出显示（ 图5E"）。 </li></ol></li><li>定义该值作为最小阈值。离开"上的斑块门槛"上的最大值。 </li><li>重新运行子宏"分析"与本节中的所有前面的步骤中定义的设置的代表图像。审慎评估所产生的图像文件，并确保该细分做得好。如果这不是这种情况，根据所述分割误差的性质（ 图4， 表3）重新调整设置。 </li></ol></li></ol>

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作者有没有利益冲突披露。

" 果蝇 NMJ形态特征"和" 果蝇 NMJ布顿形态特征"是感兴趣的评估突触形态研究人员的强大工具。 NMJ参数的手动评估是费力;据估计，宏会节省一个有经验研究者〜15分钟/ NMJ花费在手动图像分割。一到两打每个条件或基因型评估的突触，这种迅速总结了相当大量的节省了时间，甚至在小规模研究。当执行大屏幕，采用高通量分析，相比手动评估和量化的增益，可以是巨大的。除了增加的吞吐量，宏容易地提供目标数据;他们排除，否则需要蒙蔽实验以及当多个研究人员参与分析发生人际差异的个人偏见。最后，宏提供了一个敏感和准确的的NMJ功能alysis，从而导致比戏剧性NMJ缺陷相当微妙且迄今仍然在研究人员的眼睛赏识突触监管机构的鉴定。有关验证程序和在宏使用的算法的详细信息在出版物Nijhof 等中找到。 17。 宏的功能已经被验证在肌肉4.以适当地测量果蝇 NMJs的形态特征。随后，它证实了宏也适于分析在其它肌肉突触该生物体。它是可能的宏也可以被用于测量NMJ的形态学参数与其他物种中，包括其它果蝇和进一步昆虫类似的结构。即使NMJs在进化中非常遥远， 如小鼠NMJs，展现出非常相似的结构构象29。宏尚未在NMJ准备测试来自其他物种，但我们鼓励潜在用户测试宏用于上述目的。 这是非常重要的是，用户将探讨不同的自动阈值和算法来确定/选择图像最合适的宏设置。通过这些设置，当宏评估相比手动评估的大约95％的精确度得以实现。调整宏设置才能正常段的图像的100％，并且是非常费力或甚至不可能的过程。因此，如果它们的数量在5％以下，建议无法正常分割图像的排斥。显然，如果该图像的质量为低时，宏将产生不满意的图像分割的比例较高。低质量的图像同样会影响到人工评估，因此无法联系到宏的性能。尽管如此，宏是相当强大的，因为他们设计的图像S ON高含量显微镜（自动化荧光显微镜，其允许大量样品的成像）17产生。 一个关键的一点是，用户在视觉上检查由宏产生的所有结果的图像。这将允许检测并排除与不令人满意的分割画面。在这个协议中的部分6中，用户被引导如何在运行子宏时"分析"调整为正确的图像分割的设置。为了迅速与宏的要求以及如何调整宏设置了一个名为"Examples_adjusting宏设置"文件夹熟悉包含在宏库https://figshare.com/s/ec634918c027f62f7f2a。十三子文件夹，每个在不同的平台显微镜获得的（高含量/共焦/荧光显微镜）的例子的图像和不同的免疫染色中，提供了。甲PDF题为"范例指南"被包含在相同的其中提供对于每个实施例所需要的设置，与一个文本文档提供了预期的效果和结果图像一起夹。 宏被设计为.TIFF分开要处理的文件保存的图像，但是一些用户可能会以不同的格式保存其影像。下面的网站https://figshare.com/s/ec634918c027f62f7f2a 21包含一个名为" 果蝇 NMJ"，其中三个示例文件（示例1 - 3）文件夹及文件"的例子指南"以详细说明如何将图像导入宏如果不是因为.TIFF分隔的文件存储也可以在同一个文件夹中找到。 总之，" 果蝇 NMJ形态特征"和" 果蝇 NMJ布顿形态特征"的宏量化十个不同的NMJ特性:NMJ面积，周长NMJ，终扣的数目，NMJ布顿区，NMJ长度，最长NMJ枝长，岛上的数NDS，分枝数，分枝点的数量和活动区域的数量。这提供了可评估只有一个或几个突触功能30，31，到目前为止可用的工具有很大的优势。多参数的定量分析，承担新的发现， 例如，巨大的潜力，以确定控制一个高达突触生物学的许多方面新的监管机构。它还提供所要求的分辨率确定coregulate完全相同的或重叠的NMJ特征，因此有可能在常见的分子途径来操作的基因。最后，它会打开的可能性，调查不受干扰的条件下17和基因确保这种协调的形态相关突触参数彼此多么的不同相关。 总之，该协议说明了如何使用两个宏" 果蝇 NMJ形态特征"和" 果蝇 NMJ布顿形态特征"，其在高通量的方式执行的10个形态NMJ特征客观和敏感定量。

认知障碍如智力障碍，孤独症谱系障碍和精神分裂症的特征通常在于异常突触功能1，2，3。突触的形态和功能是紧密相互交织的;形态缺陷会导致突触故障和，相反地，异常突触传递会影响突触成熟和形态4，5，6。 许多模式生物的人为了更好地理解突触生物学和突触的变化如何影响健康和疾病的7，8，9脑功能阐明了使用。 果蝇 NMJ是被广泛研究的和成熟的体内模型用于谷氨酸SYnapse生物10，11。在过去几十年里，这种模式已被用于生理和基因研究的重点，以及大规模的遗传筛选，目的是检测NMJs之间的形态差异。特别是，向前的遗传筛选已经鉴定了许多重要调节剂和机制突触发育底层和功能12，13，14，15，16。然而，大多数这些屏幕的依赖NMJ终端形态的视觉评估和突触异常的定性检测或很少形态特征半定量评分。因此，相当微妙的突触形态异常是不明显的人眼很容易错过。为了能够全面检测定量差异，NMJ必须由感兴趣的形态参数的定量系统进行精确评估。人工测量NMJ特征是费力的，尤其是当有感兴趣的几个NMJ特征和/或执行大规模的遗传筛选时。为了支持多参数，高通量的形态分析，并实现客观量化，两个宏" 果蝇 NMJ形态特征"和" 果蝇 NMJ布顿形态特征"被开发17。这两个宏在开源的图像分析软件斐济18运行，并且可以量化共焦和非共图像。 "果蝇 NMJ形态特征"的措施与突触后标记圆盘大-1（DLG-1）或突触前辣根过氧化物酶（HRP）进行免疫染色NMJ终端，共标记与活性区域标志bruchpilot（BRP）。它量化9个形态parameTER值（下面进一步描述）:NMJ区域，NMJ周长，终扣的数目，NMJ长度，NMJ最长分支长度，岛的数，分支数，分支点和在突触终端活性区的数目的数目（ 图1） 。虽然用于确定终扣的数目的算法存在于该宏，它不符合为精度17的标准。正确评估终扣的数目，有必要使用" 果蝇 NMJ布顿形态测量"宏，它是专门设计来定量使用抗突触结合蛋白（嘉庆）或抗半胱氨酸字符串蛋白（CSP）进行免疫染色NMJ制剂终扣，和联合免疫标记与BRP。的"果蝇 NMJ布顿形态测量"宏量化参数如下:活性ZO的终扣的数目，NMJ布顿面积，NMJ长度，NMJ最长分支长度，岛数，分支数，分支点的数量和数目NES（ 图2）。 宏包括3个子宏:（I）"转换为堆栈"标识所有可用的图像文件并创建Z-hyperstacks和两个通道的最大强度投影。作为输出，这个宏会产生每突触称为"stack_image_name"和"flatstack_image_name"两个新文件。 II）"定义ROI"将打开所有最大的投影图像"flatstack_image_name"连续，并用手动定义感兴趣区域（ROI），其中感兴趣的特定突触终端存在的区域中的请求呈现它们。这被实现以允许连接至相邻的肌肉和/或其它类型的突触终端（如1秒），可以存在于图像11突触排斥。 （III）"分析"用全自动化分析的图像的所有区域中的ROI的边界内。如此步骤的结果，用户将获得两个新文件:"RESULTS.TXT"，其中所有的数值测量将被注释，并且其中由宏产生的底层图像分割将被示出的"res_image_name.tif"。的NMJ轮廓，所述NMJ骨架，BRP-正极活性区的数目:在图像分析三种结构从每个突触终端的。所述NMJ轮廓被用于确定所述NMJ区域和其周边和随后的分水岭分离提供终扣的数目。从骨架，五个NMJ特征推导出:总NMJ长度，最长的连续路径连接任何两个端点的长度的总和（最长分支长度），每NMJ未连接的隔室的数目（以下称为"岛" ），支链的数量，和分支点（一个支化点连接三个或更多个分支）的数目。有源区的数目在BRP通道通过计数确定BRP-阳性斑点。带注释的NMJ轮廓（黄线）时，NMJ骨架（蓝线），和BRP-正极活性区域（由白色灶表示）的数目被显示在结果图像和参数的测量结果进行处理，以一个（。 TXT）输出文件（ 图3）。 果蝇 NMJ形态特征"和‘ 果蝇 NMJ布顿形态特征’进行了首次描述，并通过Nijhof 等人 17广泛验证。这手稿着重于方法使用宏来分析NMJ形态‘ 果蝇 NMJ形态特征’和‘ 果蝇 NMJ布顿形态特征’。尽管如此，NMJ解剖和免疫染色需要之前宏辅助分析被执行。这是关键步骤，并且用于免疫组织化学标记物的组合，需要以适合于宏分析。简要地提及这些步骤本身此协议的ction 1和指导用户在详细描述的协议来执行这些程序的引用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Immunostainings</td>
			<td></td>
			<td></td>
			<td>Dilution</td>
		</tr>
		<tr>
			<td>Mouse anti-discs large 1</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>AFFN-DLG1-4D6</td>
			<td>1/25 (conjungated using the Zenon Alexa Fluor 528 Labeling Kit)</td>
		</tr>
		<tr>
			<td>Rabbit anti-horseradish peroxidase</td>
			<td>Jackson IR</td>
			<td>323-005-021</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Rabbit anti-Synaptotagmin</td>
			<td>Gift from Hugo Bellen</td>
			<td></td>
			<td>Jan-00</td>
		</tr>
		<tr>
			<td>Mouse anti-Cysteine string protein</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>DCSP-1(ab49)</td>
			<td>1/10 (conjungated using the Zenon Alexa Fluor 528 Labeling Kit)&#160;</td>
		</tr>
		<tr>
			<td>Mouse anti-Bruchpilot</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>nc82</td>
			<td>Jan-50</td>
		</tr>
		<tr>
			<td>Goat anti-mouse Alexa Fluor 488</td>
			<td>Life technologies</td>
			<td>A11029</td>
			<td>1/200</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit Alexa Fluor 568</td>
			<td>Life technologies</td>
			<td>A11011</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Zenon Alexa Fluor 568 Mouse IgG1 Labeling Kit</td>
			<td>ThermoFisher</td>
			<td>Z25006</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>ThermoFisher</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Material</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">Confocal microscope or fluorescence microscope</td>
			<td>Leica SP5</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Axio imager</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>Mac or Pc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Material</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FIJI</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

two algorithms for high-throughput and multi-parametric quantification of drosophila neuromuscular junction morphology

突触形态是紧密相关的突触效能，并且在许多情况下，形态突触缺陷最终导致突触故障。 果蝇幼虫神经肌肉接头（NMJ），对谷氨酸突触一套行之有效的模式，已被广泛研究了几十年。导致NMJ形态缺陷的突变的鉴定表明，调节突触发育和功能基因库。许多这些在集中于定性的方法来检测果蝇 NMJ的形态异常的大规模研究，确定。定性分析的一个缺点是，很多细微的玩家奉献NMJ形态可能被忽视了。而需要定量分析检测微妙的形态差异，这样的分析还没有普遍执行，因为他们是勤劳的。该方案描述了详细的两个图像分析算法" 果蝇 </em&gt; NMJ形态特征"和‘ 果蝇 NMJ布顿形态特征’，可作为斐济兼容的宏，在果蝇 NMJ的定量，准确和客观的形态分析，这种方法被开发的分析与常用的标记免疫标记NMJ终端DLG-1和BRP此外，其广泛应用到其它标记物如HRP，CSP和嘉庆呈现在这个协议中的宏能够评估9个形态NMJ功能:。NMJ区域，NMJ周长，终扣的数目，NMJ长度，NMJ最长分支长度，岛数，分支数，分支点和在NMJ终端活性区的数目的数目。

文本结果文件将出现在主目录。它总结了每个图像中的所有测量参数。结果链接到文件名称和参数在表1和2中所示的顺序随后总结。 Res_image_name是三图像栈。第一图像突出的轮廓，并根据信道1由宏确定的NMJ终端的骨架（免疫标记DLG-1，HRP，嘉庆，或CSP）。第二图像是第一图像的副本，并另外示出了在信道2作为示意灶中检测到的标识BRP阳性斑点。第三图像一起提供所述第二信道的最大突起与识别BRP阳性病灶。 所述NMJ轮廓阈值在黄色宏输出结果图像中表示。 NMJ区，丕林岛ETER和终扣的数目从该阈值推导出来。 所述NMJ骨架阈值在宏输出结果图像中的蓝色表示。 NMJ长度，最长枝长，分枝数，分枝点和岛屿从这个阈值推断。 所述NMJ有源区阈值未被宏输出结果图像中表示。该阈值判定，其中BRP阳性病灶潜在地由宏可能遇到的区域。它的目的是创建一个NMJ面积比由NMJ轮廓门槛定义的一个稍微大一点。当选择了太多限制阈值时，位于突触的余量BRP阳性病灶可被排除。当阈值是太许可，背景噪声可被算作BRP-阳性斑点（ 图1 - 2）。 为了validaTE的" 果蝇 NMJ形态测量"宏观的性能，那些已经描述的存在于不同的参数NMJ突触缺陷3个诱变条件进行了测试。每个缺陷是通过由宏（NMJ轮廓，骨架或活性区，分别为17）进行不同的图像分割程序进行检测。针对由诱导RNAi的兴趣，并进行解剖和L3幼虫NMJ免疫染色三种基因后，宏被运行。然后将获得的形态学NMJ测量使用t-检验成对地比较（RNAi的相对于其控制）。在这三种情况下，被突变，影响是与先前报道的形态缺陷的协议参数控制之间存在统计学差异。这证实了宏的确能够在果蝇 NMJ充分识别先前描述的缺陷。 锚蛋白 2（ANK2，CG42734）突变体是已知的，以显示突触形态缺陷，包括稠合的终扣和较小NMJs。观察到对于ANK2突变体24，25，这些缺陷和ANK2击倒苍蝇26。泛神经元Ank2- RNAi敲除蝇NMJ端子（ 瓦特; UAS-的Dicer-2 / UAS-ANK2的RNAi KK107238; ELAV-GAL4 / +）显示出更小的显著NMJ面积（平均值= 339.25微米是2; t检验p = 2.18 ×10 -8）和周长（平均值= 238.24微米，t检验p = 1.82×10 -3），相比于遗传背景控制数据集（ 瓦特; UAS-的Dicer-2 / UAS-KK60100; ELAV-GAL4 / + ）（平均值= 451.95微米2和运行" 果蝇 NMJ形态测量"（ 图6A和图4B）之后分别是指= 288.62微米，）。 GTP酶Rab3（CG7576）所需的适当bruchpilot分布和RUP突变体具有作用区27的显著减少数目呈现。观察到活性区的数目甲显著减少由泛神经元Rab3击倒蝇（ 瓦特的NMJ端子" 果蝇 NMJ形态测量"宏观测量BRP阳性病灶时; UAS-的Dicer-2 / UAS-RNAi的KK100787; ELAV -Gal4）。在Rab3 -RNAi每NMJ终端活性区域的平均数量为138对比在控制数据集（/ +）T检验p检测290 = 4.43×10 -29）（ 图6A＆4C）。 HIGHWIRE（HIW，CG32592）是NMJ生长的重要调节剂;在HIW基因突变铅过度生长并延长了NMJ端子28的支化。测量泛神经元HIW -RNAi击倒线（ 瓦特的NMJ端子;ûAS-切酶- 2 / UAS-RNAi的GD36085; ELAV-GAL4 / +）与" 果蝇 NMJ形态特征"，在骨架衍生的参数中观察到显著差异:长度（平均= 147.36微米;对照平均值= 122.07微米;吨-test p = 7.31×10 -7），最长长度分支（平均值= 122.19微米;对照平均值= 105.65微米，t检验p = 4.62×10 -4）的分支（平均值= 7.69的数;控制平均= 5.74; t检验p = 2.52×10 -2）和分支点（平均值= 2.73的数;控制平均= 1.79，t检验p = 3.31×10 -2）。所有这些参数显著增加（120 - 180％）相比，遗传背景的对照（ 瓦特; UAS-的Dicer-2 / UAS-GD60000; ELAV-GAL4 / +）（ 图6A＆4D）。 <img alt="图1" src="/files/ftp_upload/55395/55395fig1.jpg" /> 图1: 果蝇 _NMJ_Morphometrics措施Drosophi的9个参数LA NMJ。左边是DLG-1和BRP免疫标记NMJ终端，成像在与ApoTome荧光显微镜。在右边是运行" 果蝇 NMJ形态特征"后，结果图像。参数面积，周长和终扣是由所指示的宏注释的黄色轮廓表示。参数长度，最长枝条长度（LBL），分支，分支点，岛屿是由宏观标注蓝色的轮廓呈现。 BRP-免疫标记灶（活性区）由宏如在结果图像的白点表示。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55395/55395fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55395/55395fig2.jpg" /> 图2: 果蝇 NMJ布顿形态特征的测量果蝇 NMJ的8个参数。在左边是SYT-1和BRP免疫标记NMJ终端，成像在与ApoTome荧光显微镜。在右边是运行" 果蝇 NMJ布顿形态特征"后，结果图像。参数终扣和布顿面积由宏观标注黄色的轮廓表示。参数长度，最长枝条长度（LBL），分支，分支点，岛屿是由宏观标注蓝色的轮廓呈现。 BRP-免疫标记灶（活性区）由宏如在结果图像的白点表示。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55395/55395fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55395/55395fig3.jpg" /> 图3:流程图代表果蝇 NMJ形态特征和果蝇 NMJ布顿形态测量宏。第一子宏"转换ŧO栈"创成像NMJs的凸起和hyperstacks，第二子宏‘定义ROI’需要手动输入定义感兴趣NMJ终端的位置，子宏3，‘分析’，测量所有NMJ参数。文本包含定量值和结果图像文件描绘参数划分的文件被创建，以帮助的微距性能的用户的评价，当图像在不同条件下获得的，宏观的设置必须进行测试和调整，以确保准确的分析。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55395/55395fig3large.jpg" target="_blank">请点击这里以查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55395/55395fig4.jpg" /> 图4:不适当宏分割结果的例子。运行" 果蝇 NMJ Morphometr后，结果图像集成电路"或‘ 果蝇 NMJ布顿形态特征’。突触终端的部分不包括在所述黄色轮廓（A）。背景技术的部分包括在由黄色轮廓（B）的突触终端。蓝骨架线延伸超出突触终端。（C - D）检测到太多的活动区域（E - E"）（一些活动区域保持不被分析ģ - G）未被发现"。有源区被突触（F）不正确布顿分割外检测（仅适用运行果蝇 NMJ布顿形态测量时），终扣被错过（H）或太多的终扣由分割（I）检测。这是被包括在分割的背景的一部分的粒子这样的晶体或灰尘（J） 。在表3中提供的信息如何更改设置，以避免这些错误</st荣&gt;。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55395/55395fig4large.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/55395/55395fig5.jpg" /> 图5:宏设置的调整及对图像分割后果的例子。 （A）中减去一个DLG-1免疫标记突触，成像在与ApoTome荧光显微镜的背景预览，当"风水球半径"被设定为20（A）或500（A"）。运行图像后获得的（B）输出的图像|调整|自动阈值|尝试所有的图像示出了由16个不同的自动阈值的算法获得的图像分割。 （C）在50（C）和500（C"）的设置的"噪声容差"，当"查找最大值"的预览; AC由分割检测略去区由一个小十字标记。 "小颗粒"出现在与抗-HRP免疫标记突触，成像在一个共焦显微镜的图像背景的（D）的测量。从2_active_zone_stack_ima-ge_name得到（E） "萨姆切片"投影。阈值设定为400（E），并在50（E"）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55395/55395fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/55395/55395fig6.jpg" /> 图6:在运行上DLG-1和BRP" 果蝇 NMJ形态测量"宏之后宏观评价和定量对肌肉4.（A）结果的图像NMJs的免疫标记NMJ端子。参数面积，周长和布顿s的由宏注释的黄色轮廓表示。参数长度，最长枝条长度（LBL），分支，分支点，岛屿是由宏观标注蓝色的轮廓呈现。 BRP-免疫标记灶（活性区）由宏如在结果图像的白点表示。中的比例尺为20μm以下。 （B）Ankyrin2 RNAi敲除表现出更小的NMJ面积和周长相比遗传背景对照。 （C）Rab3敲低导致NMJs具有较低数目相比遗传背景对照BRP阳性活性区。 （D）HIGHWIRE敲低导致更长的时间，较高的最长分枝长度，多种支化并与每相比遗传背景控制NMJs NMJ端子更多分支点。误差棒表示SEM，** P &lt;0.01，双尾t检验。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55395/55395fig6large.jpg" target="_blank">请点击他</a>再查看该图的放大版本。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 参数 </td><td> NMJ结构 </td><td> 说明 </td></tr><tr><td>面积（平方微米） </td><td> NMJ大纲</td><td>完整的标记NMJ的面积</td></tr><tr><td>周长（μm）的</td><td> NMJ大纲</td><td>属于该地区的周边</td></tr><tr><td> #Boutons </td><td> NMJ大纲</td><td>在NMJ突触终扣（"在一根绳子上的珍珠"）的数</td></tr><tr><td>长度（μm）的</td><td>骨架</td><td>完整NMJ终端的总长度</td></tr><tr><td>最长分枝长度（μm）的</td><td>骨架</td><td>最长的连续路径的长度的连接NMJ的任何两个端点之</td></tr><tr><td> ＃科ES </td><td>骨架</td><td>分支机构总数</td></tr><tr><td> #Branching点</td><td>骨架</td><td>分支点的数量（几个分支可以从一个分支点得出） </td></tr><tr><td> ＃岛屿</td><td>骨架</td><td>非连接DLG1阳性突触隔室（或任何其它染色）数量</td></tr><tr><td> #Active区</td><td> BRP-阳性斑点</td><td>活动区域的基础上，BRP染色数</td></tr></tbody></table> 表1:NMJ参数由" 果蝇 NMJ形态测量"进行测量。由" 果蝇 NMJ形态测量"宏观测量的参数NMJ将出现在所获得的文本文件的列表，按照本表所描述的顺序。此表由Nijhof 等转载。 17 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 参数 </td><td> NMJ结构 </td><td> 说明 </td></tr><tr><td>终扣</td><td> NMJ大纲</td><td>在NMJ突触终扣（"在一根绳子上的珍珠"）的数</td></tr><tr><td>布顿区</td><td> NMJ大纲</td><td>所有终扣的总面积</td></tr><tr><td>长度（μm）的</td><td>骨架</td><td>完整NMJ终端的总长度</td></tr><tr><td>最长分枝长度（μm）的</td><td>骨架</td><td>最长的连续路径的连接NMJ的任何两个端点的长度的总和</td></tr><tr><td> #Branches </td><td>骨架</td><td>分支机构总数</td></tr><tr><td> #Branching点</td><td>骨架</td><td>钍支化点电子数（几个分支可以从一个支化点导出） </td></tr><tr><td> ＃岛屿</td><td>骨架</td><td>非连接DLG1阳性突触隔室（或任何其它染色）数量</td></tr><tr><td> #Active区</td><td> BRP-阳性斑点</td><td>活动区域的基础上，BRP染色数</td></tr></tbody></table> 表2:NMJ参数由" 果蝇 NMJ布顿形态测量"进行测量。由" 果蝇 _Bouton_NMJ_Morphometrics"宏观测量的参数NMJ将出现在所获得的文本文件的列表，按照本表所描述的顺序。此表由Nijhof 等转载。 17 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 分割 </td><td&gt; 观察到的错误 </td><td> 例 </td><td colspan="2"> 所需的调整 </td></tr><tr><td> NMJ面积和周长（由黄色轮廓在结果图像表示） </td><td>可能是没有包含在黄色轮廓或背景的部件突触终端的部分被包含在黄色概述的突触终端。 </td><td> 图5A-B </td><td>调整"滚动球的半径"值。见6.1。 </td><td>调整"NMJ轮廓门槛"。见6.2。 </td></tr><tr><td> NMJ长度相关的参数（通过将结果图像中的蓝色骨架线表示） </td><td>蓝色干线或者延伸超过或不是沿着整个突触终端存在。 </td><td> 图5C-D </td><td>调整"滚动球的半径"值。见6.1。 </td><td>调整"NMJ轮廓门槛"。见6.2。 </td> &lt;/ TR&gt; <tr><td> BRP阳性斑点（由点在结果图像表示） </td><td>检测到太多的活动区域。 </td><td> 图5E-E" </td><td>降低"查找最大噪声容限的价值。参见6.5节。 </td><td></td></tr><tr><td> BRP阳性斑点（由点在结果图像表示） </td><td>活动区域被通过分析错过。 </td><td> 图5G-G" </td><td>增加了"查找最大噪声容限的价值。参见6.5节。 </td><td>减少"BRP-斑点下限阈值"。见6.6。 </td></tr><tr><td> BRP阳性斑点（由点在结果图像表示） </td><td>有源区的伪影突触终端的外部检测。 </td><td> 图5F </td><td>调整"活动区门槛"第6.2节。 </td><td>增加"BRP-斑点下限阈值"。见6.6。 </td></tr><tr><td>小颗粒</td><td>颗粒，例如一个晶体或粉尘是背景的一部分显示为被包括在分割。 </td><td> 图5J </td><td>选择框"中取出小颗粒"。参见6.3节。 </td><td>确定小颗粒最大尺寸。参见6.3节。 </td></tr><tr><td>布顿分割</td><td>不正确布顿分割（只适用于果蝇NMJ布顿形态特征;不要使用果蝇NMJ形态特征为布顿分割）。 </td><td> 图5H-I </td><td>调整"NMJ轮廓门槛"。见6.1。 </td><td>确定的最低布顿大小"。参见6.4节。 </td></tr></tbody></table> 表3:故障排除指南误差在图像分割不同种类可以由宏来制造。此表描述了不同种类的由宏生成的图像分割误差。这些可以很容易地在结果图像来检测。每个错误类型的实例示于图4。在该表中的"调整部分"，即需要调整的设置被突出显示，并且用户被称为部分6的临界子步骤，它说明如何调整这些设置。

Watch this Scientific Journal Video about Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology at JoVE.com

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

两个图像分析算法，" 果蝇 NMJ形态特征"和" 果蝇 NMJ布顿形态特征" 被创建 ，以自动量化果蝇神经肌肉接头（NMJ）九个形态特征。

两种算法的高通量和多参数定量<em&gt;果蝇</em&gt;神经肌肉接头形态

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The ...

two-algorithms-for-high-throughput-multi-parametric-quantification

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

9.6K 次观看.  Radboud University Medical Center. 两个图像分析算法，" 果蝇 NMJ形态特征"和" 果蝇 NMJ布顿形态特征" 被创建 ，以自动量化果蝇神经肌肉接头（NMJ）九个形态特征。 

视频: Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

在活动区域​​的解剖和影像果蝇神经肌肉交界处

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

科研

神经科学

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

在发展中国家小鼠视网膜体内电穿孔

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

连续光漂白划定单雪旺细胞在神经肌肉接头处

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

全山神经肌肉交界分析的 横向解剖阿布多米尼斯 肌肉

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

检测<em&gt;原位</em&gt;蛋白质 - 蛋白质复合物的<em&gt;果蝇</em&gt;幼虫神经肌肉接头使用接近结扎分析

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

FIM Imaging and FIMtrack: Two New Tools Allowing High-throughput and Cost Effective Locomotion Analysis

The analysis of neuronal network function requires a reliable measurement of behavioral traits. Since the behavior of freely moving animals is variable to a certain degree, many animals have to be analyzed, to obtain statistically significant data. This in turn requires a computer assisted automated quantification of locomotion patterns. To obtain high contrast images of almost translucent and small moving objects, a novel imaging technique based on frustrated total internal reflection called FIM was developed. In this setup, animals are only illuminated with infrared light at the very specific position of contact with the underlying crawling surface. This methodology results in very high contrast images. Subsequently, these high contrast images are processed using established contour tracking algorithms. Based on this, we developed the FIMTrack software, which serves to extract a number of features needed to quantitatively describe a large variety of locomotion characteristics. During the development of this software package, we focused our efforts on an open source architecture allowing the easy addition of further modules. The program operates platform independent and is accompanied by an intuitive GUI guiding the user through data analysis. All locomotion parameter values are given in form of csv files allowing further data analyses. In addition, a Results Viewer integrated into the tracking software provides the opportunity to interactively review and adjust the output, as might be needed during stimulus integration. The power of FIM and FIMTrack is demonstrated by studying the locomotion of Drosophila larvae.

FIM is a novel, cost effective imaging system designed to track small moving objects such as C. elegans, planaria or Drosophila larvae. The accompanying FIMTrack program is designed to deliver fast and efficient data analysis. Together, these tools allow high-throughput analysis of behavioral traits.

FIM成像和FIMtrack:两个新的工具允许高吞吐量和成本效益分析步态

The analysis of neuronal network function requires a reliable measurement of behavioral traits. Since the behavior of freely moving animals is variable to ...

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the &#34;greater of two goods.&#34; However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

高通量检测，以检查个体产蛋首<em&gt;果蝇</em

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

为什么量化事项:表型的表征在<em&gt;果蝇</em&gt;幼虫神经肌肉接头

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

测量神经肌肉接头功能

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

Speed

Subtitles

两种算法的高通量和多参数定量