We are grateful to Dr. Andrea Chai for her insightful comments on the manuscript. This work was supported by the Wellcome Trust (grant number: Pennetta8920) and by the Motor Neuron Disease Association (grant number: Pennetta6231).

1.实验准备注意:夹层动脉瘤和在第2免疫组织化学程序和3是根据参考文献3-6进行的，但具有修改。 <ol><li>制备1×磷酸盐缓冲盐水（PBS）和含有0.1％的Triton-X 100的PBS（PBT）。让他们在冰上。 </li><li>制备布安的固定液（15苦味酸:10甲醛:1冰醋酸）。使这种试剂新鲜。 </li><li>选择干净的不锈钢minutien销和细镊子。 </li><li>准备含5cm的培养皿的Sylgard盘夹层板。 </li></ol> 2.第三龄幼虫NMJs剖析<ol><li>从这里选择小瓶或用细刷瓶流浪三龄幼虫，并将它们放到含有4℃PBS洗涤残留的食物客场2厘米的培养皿。 </li><li>放置在夹层板的SYLGARD表面的最上面的一个幼虫并确保它是POSITIoned其背侧，使得两个纵向气管导管是在顶部可见。 </li><li>使用镊子保持销，销的幼虫向下在其前端，右下嘴钩。舒展幼虫出尽可能多地和引脚及其后端下来。 </li><li>添加足够的PBS盐水到达板的壁和完全沉浸幼虫。 </li><li>重复从步骤2.1的方法，以2.3的相同基因型的其他幼虫。单5厘米培养皿夹层板可以轻松容纳多达8个幼虫。 </li><li>使用显微解剖剪，微微抬起背侧角质层并在销附近的后端使一小的水平切口。 </li><li>插入剪刀入切口，切开幼虫一路沿着气管的两个纵向束之间的中线的前端。确保中线削减足够的肤浅，只是通过角质层，避免通过肌肉切腹侧。 </li><li>在每一端，切断左侧和右两侧两个槽口。 </li><li>通过把两个引脚上的前切口两侧打开圆角。重复与后端相同。当将针，一定要掰开体壁。 </li><li>清理出使用镊子和PBS液内脏。离开中枢神经系统完好无损。轻轻地用针角落伸展幼虫直到完全伸展，但要确保肌肉不会在此过程中撕裂。 </li><li>重复同样的解剖程序在同一夹层板其他幼虫。 </li><li>用PBS盐水洗涤三次以除去所有的内部器官。 </li><li>与布安固定液更换PBS，在室温下放置10分钟。 </li><li>与PBT洗几次。 </li><li>小心取出针和各项准备工作，目前已相当严格，转移到了免疫染色1.5 ml的微离心管中。</li></ol>用特异性抗体肌肉和肌细胞核果蝇 NMJs 3.免疫组织化学染色<ol><li>快速漂洗PBT幼虫鱼片。 </li><li>通过用在PBT中的10％正常山羊血清（NGS）孵育下恒定搅拌2小时阻断制剂。 </li><li>孵育在含有NGS的5％，和兔抗核纤层蛋白抗体（以1:1的浓度:500）的PBT一套解剖NMJs的并用豚鼠抗DVAP抗体（以1:1的浓度:500）2小时在室温下，或在4ºC过夜。而抗拉明染色检测肌细胞核的轮廓抗DVAP抗体染色的横纹肌。 </li><li>洗一次迅速PBT除去过量的抗体。通过改变PBT缓冲每15分钟洗在PBT中2小时。 </li><li>孵育样品在含有PBT的5％NGS和荧光标记的二抗以1:在室温下500稀释2小时。相同的样品C一个受到与在同一时间的多个抗体染色，如果具有不同的发色团共轭次级抗体用于。 </li><li>除去次级抗体和通过改变PBT缓冲每15分钟在PBT中洗涤2小时。 </li><li>染色内部的肌细胞核洗样品三次，用PBS和步骤如下: <ol><li>在PBS 1000和孵育在搅拌条件下20分钟:添加核标记TO-PRO-3以1:1的稀释度。该标记物在该研究中使用，但任何其他市售的核标记物可以用作良好。 </li><li>安装前应清洗在PBS快三倍。 </li></ol></li></ol> 4.在幻灯片上安装样本<ol><li>从1.5毫升微量离心管钳挑样品起来，躺在下来在处理幻灯片。 </li><li>通过使用显微解剖剪，切头和圆角的尾部，并保持其内部表面上。 </li><li>日准备e。通过以约1厘米的相互距离在一个干净的幻灯片的每一侧纤维素带的三个板条缠绕安装滑动。一旦盖玻片被定位在两个条的顶部，间隙将生成，这将避免样品的平坦化。这是至关重要的，如果结构的三维体积渲染要制成。 </li><li>把约20微升所述安装平台的一小滴在三纤维素带条之间的安装滑道的中间。 </li><li>推广用干净的镊子安装介质后，拖动解剖幼虫到安装滑入安装介质，保持内表面的。尝试装入他们的四个或五个行。 </li><li>轻轻地砸在安装滑道顶部盖玻片，并确保没有气泡产生。密封透明指甲油的幻灯片。让试样干燥成像前至少10分钟。 </li></ol> 5.设置共聚焦成像注:电子倒置显微镜:在本研究中呈现的图像是使用集成到钛尼康A1R共聚焦单元拍摄。然而，用最少的在488纳米，561纳米和642纳米和一个3通道的检测系统的波长区域的3-激光单元的任何共聚焦显微镜是适合于该目的。 <ol><li>接通激光器，探测器单元，汞灯泡，舞台控制器，显微镜和电脑。启动控制软件和安全在舞台上持有人的幻灯片。 </li><li>为了使成像速度，选择并通过使用20X物镜标记的样品上感兴趣区域（ROI）中的所有区域。 </li><li>仔细摆动物镜转换器的60X高倍物镜（60X计划阿婆VC / NA 1.4 OIL）。 </li><li>放置浸油下降到物镜，然后从XYZ概述窗口在计算机上的标记的ROI中的一个。 </li><li>通过使用下面的光学设置启动成像:选择第一分色镜:48分之4058/561/640。选择488nm的激光发射滤波器在信道1，561 nm激光器在信道2和642纳米的激光五十零分之五百九十五发射滤波器与信道3个长通滤波器650纳米525/50纳米。 </li><li>启动图像采集之前，请使用以下扫描设置:选择电扫描器;扫描方向:一种方式;扫描速度:每秒0.5帧。 </li><li>选择频道系列，以避免渠道流失通。 </li><li>调整针孔大小为1通风装置。扫描和调整激光功率，检测增益并适当地偏移对每个信道，以避免像素饱和度和背景水平。 </li><li>通过使用体素尺寸0.2×0.2×0.5微米3为所有感兴趣区和滑动制剂获得的z栈。如果图像将经受卷积然后设置体素尺寸，以0.06×0.06×0.15微米3。 </li><li>保存.nd2文件格式或的.ics格式的图像。 </li></ol> 6.计算之内横纹核之间的距离60，肌肉由最近邻分析方法<ol><li>对于这种分析，使用共焦图像显示与DVAP抗体可视化的肌肉和与核纤层蛋白的抗体和核标记来突出细胞核染色的幼虫体壁肌肉。 </li><li>为了估计原子核之间的最近距离，使用图像分析软件（ 如了Imaris）的MeasurementPro模块内测量点。用于图像分析其它类似的软件应用程序可以被用于相同的目的。 </li><li>打开共聚焦图像。要启动，软件图标双击。拖动和共聚焦Z堆栈图像拖放到竞技场。 </li><li>在图像上双击在超越视图中自动打开它们，菜单工具栏图标下<img alt="图片1" src="/files/ftp_upload/53821/53821img1.jpg" /> 。该超越观主要有三个工作区面板:查看区域，对象列表和对象属性区。 </li><li>创建一个卷呈现Ť通过点击菜单图标3D视图重稀土通道图像<img alt="图片2" src="/files/ftp_upload/53821/53821img2.jpg" /> 。 </li><li>点击添加新的测量点图标<img alt="图片3" src="/files/ftp_upload/53821/53821img3.jpg" />来自 对象工具栏并按照出现在对象属性区创建向导。 </li><li>从创建向导首先选择编辑选项卡，然后特定通道。选择核标记或核纤层蛋白通道突出核。 </li><li>通过按下键盘上的ESC选项卡中设置的指针选择模式。 </li><li>调整3D光标框的大小与鼠标滚轮以包含在图像中的给定核。通过举办同一核Shift键并点击鼠标左键添加一个测量点。 </li><li>通过重复前面的步骤中添加上的相同肌肉附近的核的第二点。一条线是在两个点之间的距离测量之间的自动绘制<html>被显示在两个细胞核。这两个核之间的距离，现在被记录为对象属性区域标签下的统计&gt;详细&gt;距离数据的统计变量。 </li><li>从重复步骤6.6步骤6.10围绕给定的所有核细胞核。 </li><li>从统计&gt;详细&gt;距离数据显示所有收集到的测量点，选择最短的距离。 </li><li>通过单击导出统计上的标签显示到文件<img alt="图片4" src="/files/ftp_upload/53821/53821img4.jpg" />对象属性区下提供的数据将被保存在一个电子表格。 </li><li>重复同样的程序为其它周围细胞核和为每基因型肌纤维的选定数量。 </li><li>使用导出到电子表格文件中的数据，计算使用下列公式肌肉的所有M个的平均最短距离（D AVE）: OAD / 53821 / 53821eq1.jpg"/&gt; 二是对于给定的核其中i从0变化到N，N是每块肌肉分析核数目相邻核的最短距离。从j值的总和= 0到j = m表示分析肌肉的M个。 </li><li>另外，使用 斑点创建向导和斑点到斑点最近距离来估计到最近的相邻核的平均距离。为此，需要一个Matlab扩展模块。 <ol><li>关于竞技场和3D容量图像一个特定的图像，双击将显示在查看区域。点击对象创建图标，并添加新景点<img alt="图像5" src="/files/ftp_upload/53821/53821img5.jpg" />从对象工具栏。 </li><li>从对象属性区创建向导，点击选择跳过自动创建，编辑手动。 </li><li>选择的核纤层蛋白或核标记物通道，以显示细胞核。 </li><li>按住Shift键并单击LEF对图像中的特定肌肉的所有核吨鼠标按钮。每个核的点就会出现。 </li><li>选择斑点斑点在对象属性区域的工具选项卡中列出最近的距离。出现的结果模式和Matlab的窗口下选择景点统计。 </li><li>根据统计选项卡，选择Detailed，然后具体数值。点击Distmin并为每核的最小距离的值将出现。 </li><li>将数据导出到电子表格文件，并计算出每肌肉这些距离的平均值。重复该过程对于一个给定的基因型的所有肌肉。 </li></ol></li></ol> 7.确定果蝇幼虫的体壁肌肉内的细胞核形态<ol><li>对于这种分析，请使用核纤层蛋白和核标记以可视化的细胞核染色体壁肌肉的共聚焦图像。 </li><li>来评价肌细胞核，测量球形的形状（定义为的河畔的比率与同体积的给定核一个球体，以核的表面积）或椭圆（的面部区域扁圆/扁长椭球和球状体）进行了区分。 </li><li>如前所述打开图像。点击对象工具栏图标添加新面<img alt="图像6" src="/files/ftp_upload/53821/53821img6.jpg" /> 。 </li><li>在出现的对象属性区创建向导中，选择核标记染色来源渠道，以显示细胞核。 </li><li>设置选项绝对强度为阈值。确保大部分核的表示由阈值曲线上改变的值的平滑和不超载渲染。同时，使用相同的曲线避免孔或不完整的掩模的存在的任何核。 </li><li>使用 过滤工具排除在表面呈现的任何噪声。在新创建的表面层，斯普利特的编辑选项卡或合并是错误的重原子核表面ndered。 </li><li>导出到电子表格文件表面呈现核是统计标签下的椭圆球形和价值观。 </li><li>或者，使用ImageJ的或斐济软件来测量核的圆度，其中圆形度（C）被定义为C <img alt="公式（2）" src="/files/ftp_upload/53821/53821eq2.jpg" /> 。一项的值代表一个完美的圆，而接近零值表示一个逐渐拉长的形状。 </li><li>创建菜单图像&gt;栈&gt;以Z项目的Z堆栈的最大强度的预测。设置投影类型最大强度。 </li><li>拆分渠道和选择核标志的通道。 </li><li>从主菜单中，选择图像&gt;调整&gt;阈值。 </li><li>段的核通过调整强度阈值。如果附近的核被分割为单个单元，点击处理&gt;二进制&gt;分水岭工具断开细胞核。 </li><li>从主菜单中，选择Edit&gt;取值选&gt;创建的选择。 </li><li>通过点击菜单上的分析&gt;工具&gt; ROI管理器添加所有选定的投资回报的投资回报率经理。在ROI管理器窗口中单击添加。在同一个窗口中，选择更多&gt;分割。 </li><li>选择分析&gt;设置测量形状描述。在ROI管理器窗口中点击标签 测量。这将显示所有选择的核的圆的值的列表。 </li><li>此外，为了测量核体积，使用如在小节7.3-7.7中描述的核标记物染色通道遵循表面创建向导。 </li></ol>在果蝇幼虫体壁肌肉内的核选择8. 3D体渲染到评估特定蛋白质的核内本地化<ol><li>利用共聚焦图像与申报核标志物，并与特定的核纤层蛋白和DVAP抗体染色体壁肌肉。 </li><li>通过启动软件和SEL打开图像待分析等的特定核。这可以通过选择主菜单项编辑&gt;作物3D来完成。 </li><li>使用如第7.3-7.7描述拉明频道关注表面创建向导。 </li><li>一旦表面被创建，在对象属性区域点击编辑，然后选择屏蔽所有的细胞核内的信号隔离开来。这将创建一个新的窗口。 </li><li>从选择通道下拉菜单中选择DVAP信号。通过点击选项设置体素外表面零选择细胞核内的DVAP免疫反应信号。一个新的掩码通道被创建，并在选择显示器调整窗口中。 </li><li>以可视化的细胞核内信号的存在通过点击图标添加新的裁剪平面创建一个平面轮廓<img alt="图像7" src="/files/ftp_upload/53821/53821img7.jpg" />从对象工具栏。 </li><li>交互式调整剪裁的角度平面和其可视化的核内的信号的分布位置。 </li></ol>

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The authors have nothing to disclose.

在过去，中和实验组之间的形态差异很少考虑。然而，定量方法的应用现在已经成为在形态和解剖形式的数学描述的比较研究规范进行计算。采用定量的评估特定的细胞过程的遗传操作的影响进行分析，拿在提高我们检测形态变化能力，改善与这些变化中描述的准确性的承诺。此外，量化数据的统计分析使我们能够评估表型之间观察到的差异是否显著。 在横纹肌，细胞核表现出明显的圆形结构，并沿肌纤维均匀地分布。尽管建立和保持的大小，形状和原子核的结构的分子机制尚不清楚，这些核特征可能吨Ø在控制肌肉功能的基础性作用。事实上，几个肌病是由突变基因的肌肉内调节原子核的形态和位置造成的。形状和小区内的核的分配的功能的重要性并不限于肌肉。越来越多的证据表明，核的缺陷也与神经变性疾病如帕金森氏病18,24相关联。此外，我们开始明白，形态，大小和其他细胞器的细胞内分布，包括内质网和线粒体可能有功能的后果。例如，在线粒体形态的改变与神经性疾病如视神经萎缩型-1（OPA1）和腓骨肌萎缩2A型神经病25相关联。 协助阐明潜在这些重要过程的分子机制的过程中，我们建议高分辨率共焦结合与成像软件和形态数据分析，以定量地评价遗传操作如何肌纤维内影响的形状，大小，与原子核的位置。电源和果蝇遗传学的多功能性加上在果蝇幼虫的神经肌肉系统的高度定型性质使幼虫NMJ的实验模型特别适合这种类型的分析。在幼虫NMJs，表型分析可以在单个突触分辨率允许其中多个NMJs可以在同一飞行中进行研究，甚至同一识别NMJ可以不同基因型3,4的苍蝇之间进行比较精确的形态测定分析来进行。 在果蝇幼虫NMJ核位置，形状和大小的表型特征通过与突出的肌肉内的肌肉和细胞核抗体进行解剖NMJs的免疫染色开始。在协议THI概述造纸，肌细胞核用针对核纤层蛋白，核膜的标志多克隆抗体染色，用核标记突出特定DVAP弄脏整个肌肉核内部和抗体。在这些实验中使用的核纤层蛋白抗体麻烦由Paul费舍尔19-22提供，但可以使用的抗拉明抗体的替代来源。另外，许多特异于核膜其他抗体是市售的。最后，核标记物，如DAPI和碘化丙锭，也可同时肌肉可以通过用抗肌动蛋白或抗微管蛋白抗体染色可视化。如果比那些在本实验过程中使用的其他抗体使用的，免疫染色协议将需要额外的步骤，其中的固定条件和工作浓度为新的抗体将需要优化。在这个协议中，特别是当体积渲染需要分析一个关键的步骤，是第Ë安装在幻灯片上的样品。在这种情况下，以包括载玻片和盖玻片使得试样没有得到挤压之间的间隔是重要的。绕在盖玻片的两侧滑动缠绕纤维素带的三个带代表制作隔板的简单方法。 虽然被用于2D图像ImageJ的，大部分的3D多通道图像在本文分析介绍，通过使用，因为其内部的可用性了Imaris完成。然而，任何其他类似的商业软件包可用于这些应用。 有几个开源（例如，ImageJ的，CellProfiler，Vaa3D，冰爽，KNIME等）和商业软件平台，可用于共聚焦图像的分析。 ImageJ的26日 ，由美国国立卫生研究院或其更多加强版，被称为斐济27免费软件，具有可用于全球成像一个省交通大量进口过滤器，宏和插件TY。大多数这些插件都集中在加工的片逐片方式的信息。也有可用于多通道的3D图像的可视化和分析的插件。然而，他们通常被设计为一个特定的任务，用户可根据需要这些插件扩展或适应自己的需要。在另一方面，商业平台的目标相对缺乏经验的用户，而且往往集中在易于使用，具有令人难以置信的高速图像处理任务覆盖面广。 在这个协议中所概述的定量表型分析的实验程序一起，可以协助在阐明的分子机制控制细胞器的形态和细胞内的分布。但是，这种方法具有一个特定终点分析这些过程的明显的限制。控制的形态和细胞器的分配过程中可能是非常动态的，并不仅不同细胞TY之间变化PES而且取决于发育或生理状态在同一小区内。这一分析的进一步实施将通过时间推移成像，允许在细胞器的形态和位置的变化随着时间的推移进行监控来表示。

Qualitative analysis restricts the focus of most experimental studies to the examination of genetic manipulations leading to large phenotypic effects or to phenotypes that are not amenable to quantification, e.g., absence/presence. Quantification of phenotypes is not usually performed and variations present within members of a phenotypic group are not taken into consideration. Additionally, without mathematical descriptions of morphologies, it may be difficult to determine whether fine scale phenotypic changes are the result of genetically induced alterations or whether observed changes are simply due to random fluctuations. 
We propose that for an accurate and unbiased analysis of phenotypic defects due to gene disruption, quantitative methodologies should accompany more traditional qualitative approaches. Quantitative evaluation of phenotypes is particularly beneficial for structures such as synapses that present a high degree of variability due to their intrinsic morphological and functional plasticity. We have selected examples of quantitative analyses applied to areas of investigation with which we are most familiar, namely the Drosophila larval neuromuscular junctions (NMJs). However, concepts and principles apply equally well to other experimental systems. 
The larval NMJ is an excellent model system to study synaptic development and function because of the highly stereotyped nature of its structure. Each hemi-segment of the larval neuromuscular system contains 32 identifiable motor neurons forming synaptic contacts with their postsynaptic target muscle. Every hemi-segment also contains a fixed number of muscles visible as multinucleated fibers attached to the internal surface of the cuticle1. Another advantage of using the larval neuromuscular system is the power and versatility of Drosophila genetics that easily allows the generation of a number of mutant alleles and the possibility to modify gene expression in a time- and tissue-restricted manner. Finally, 75% of the human genes causing a disease have an evolutionarily conserved orthologue in Drosophila2. Indeed, entire genetic pathways are conserved between flies and humans. Because of this, the Drosophila larval neuromuscular system is a very popular experimental model to elucidate the molecular mechanisms underlying a number of human diseases including amyotrophic lateral sclerosis (ALS)1. 
Here we show that the availability of several reliable immuno-histochemical markers, combined with bio-imaging techniques and accurate morphometric analyses can describe anatomical traits that are likely to play an important functional role3,4,5. Among the cellular processes that are amenable to quantitative analyses, we focus on changes in shape, size and position of intracellular structures such as the nuclei. All these are processes that we know very little about.
The challenge for molecular geneticists in the coming decades will be to extend our current knowledge by analyzing the effect of genetic mutations that produce very subtle phenotypic defects. Quantitative methodologies that allow researchers to meticulously explore the effects of genetic mutations can provide a more comprehensive understanding of how genotypes relate to phenotypes, especially for poorly understood cellular processes.

<table><tbody><tr><td>Micro-Forceps 0.3 mm x 0.25 mm</td><td>Fine Science Tools&amp;nbsp;</td><td>11030-12</td><td></td></tr><tr><td>Sylgard dissection plates&amp;nbsp;</td><td>SIGMA-ALDRICH</td><td>76103</td><td>Mix the pre-weighed elastomer base with curing agent. Poor the mixture into a 5 cm Petri dish. Let cure it at 60 &amp;deg;C for at least 24 hr</td></tr><tr><td>Stainless/Steel Minutien Pins 0.1 mm diameter</td><td>Fine Science Tools&amp;nbsp;</td><td>26002-10</td><td></td></tr><tr><td>Microdissection scissors (ultra-fine)&amp;nbsp;</td><td>Fine Science Tools&amp;nbsp;</td><td>15200-00</td><td></td></tr><tr><td>1x PBS (Phosphate Buffered Saline). Composition: 3 mM NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 7 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 130 mM NaCl, pH 7</td><td></td><td></td><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; (SIGMA-S8282), Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; (SIGMA-S7907), NaCl (SIGMA-S7653)</td></tr><tr><td>Bouin&#039;s Solution. Composition: Picric Acid, Formaldehyde, Acetic Acid (15:10:1)</td><td></td><td></td><td>Picric Acid (SIGMA 197378), Formaldehyde (F8775), Acetic Acid (SIGMA-1005706)</td></tr><tr><td>1x PBT (Phosphate Buffered Saline with Triton). 1x PBS + 0.1% Triton&amp;nbsp;</td><td></td><td></td><td>Triton-X100. SIGMA-T8787</td></tr><tr><td>Normal Goat Serum&amp;nbsp;</td><td>SIGMA</td><td>G9023</td><td></td></tr><tr><td>Guinea Pig anti-DVAP antibody</td><td></td><td></td><td>Provided by Dr. Giuseppa Pennetta (University of Edinburgh, UK). Use at 1:200 dilution in 5% NGS</td></tr><tr><td>Rabbit anti-HRP (Horseradish peroxidase)</td><td>Jackson ImmunoResearch</td><td>123-065-021</td><td>Use at 1:500 dilution in PBT containing 5% NGS</td></tr><tr><td>Rabbit anti-Lamin antibody</td><td></td><td></td><td>Provided by Dr. Paul Fisher (State Univeristy of New York at Stony Brook). Use at 1:500 dilution in PBT containing 5% NGS&amp;nbsp;</td></tr><tr><td>TO-PRO-3</td><td>Molecular Probes</td><td>T3605</td><td></td></tr><tr><td>Goat anti-rabbit antibody, Alexa Fluor488, conjugated</td><td>Jackson ImmunoResearch</td><td>bs-295G-A555-BSS</td><td>Use at 1:500 dilution in PBT containing 5% NGS</td></tr><tr><td>Goat anti-guinea pig IgG antibody, Cy3, conjugated</td><td>Jackson ImmunoResearch</td><td>bs-0358G-Cy3-BSS</td><td>Use at 1:500 dilution in PBT containing 5% NGS</td></tr><tr><td>Vectashield mounting medium for fluorescence</td><td>Vector laboratories</td><td>H-1000</td><td></td></tr></tbody></table>

why quantification matters: characterization of phenotypes at the drosophila larval neuromuscular junction

形态上的大多数研究依赖于如何解剖性状是由特定的基因和遗传途径的破坏影响的定性描述。定量描述很少进行的，虽然基因操作产生一系列表型的影响和变化即使在对照组中的个体观察。越来越多的证据表明，形态，大小和细胞器的位置，起到细胞功能和生存以前怀才不遇，但根本性的作用。在这里，我们在果蝇幼虫神经肌肉接头（NMJ）进行表型的定量分析提供一步一步的指示。我们使用多种可靠的免疫组化标记与生物成像技术和形态分析，研究特定的细胞过程的基因突变的影响相结合。特别是，我们专注于表型的定量分析影响的形态，大小，n的位置uclei内​​果蝇幼虫的横纹肌。 果蝇幼虫NMJ是一个有价值的实验模型来研究的结构和神经肌肉系统的功能，无论是在健康和疾病的分子机制。然而，我们在这里描述的方法可扩展到其他系统也是如此。

ALS是专门影响运动神经元导致横纹肌7的渐进和致命性瘫痪退行性疾病。在人类VAMP相关蛋白B（hVAPB）错义突变导致一系列运动神经元疾病，包括ALS 8型8-12。在hVAPB基因的错义突变（V234I）已在人类13 ALS的典型一例最近已确定。评估其致病潜力，我们产生表达hVAPB 果蝇直向同源物DVAP携带致病突变（DVAP-V260I）的转基因苍蝇。这种转基因的表达被靶向使用UAS / GAL4系统和肌肉特异性驱动BG57-Gal4的14,15的肌肉。 DVAP-V260I转基因表达的影响进行比较和对比于其他两种转基因（DVAP-WT1和DVAP-WT2），其表达不同水平的野生型DVAP蛋白16的。莫尔ê具体地，在DVAP免疫反应的增加比为DVAP-WT2线控制2.2倍高，而相同的信号16的DVAP-V260I和DVAP-WT1展览可比和更低的水平。 核的改变已经与衰老和几种神经变性疾病，包括帕金森氏病17,18相关联。为了评估我们的ALS8蝇模型是否表现出核架构，位置和大小的变化，我们的适当基因型横纹肌内染色的核具有核标记物和抗核纤层蛋白抗体19-22，其中可视化核膜。以突出的肌肉，特定DVAP抗体也被加入到同一样品（ 图1）。共焦图像收集和使用图像分析软件进行详细形态分析。在控制的肌肉，发现核沿着肌肉均匀分布纤维而在DVAP-V260I和DVAP-WT表达肌肉，细胞核表现出以重新分布密切相关的簇（ 图1）的倾向。 我们进行了最近邻分析来执行核的沿着每一基因型的肌纤维的分布的定量评价。最近邻分析，通过测量给定核的中心，并且每个其他的周边核的中心之间的距离的第一识别用于每个核的最接近的邻居。然后重复该过程沿着肌肉纤维隔核。最后，一​​个特定的肌肉内原子核之间的最短距离，通过平均每个核和其最近的邻居的最短距离来计算。 （ 图2A - C）。与对照组相比，肌肉表达任DVAP-V260I转基因或任何转基因过表达的野生型蛋白的中，存在于细胞核之间的平均最短距离的显着降低，并且作为结果，核似乎在簇密切相关。所述的ALS引起等位基因DVAP-V260I效果比，与野生型蛋白质的过表达有关的更严重，即使最强DVAP-WT2转基因时（ 图1和图2D）。 要么DVAP-V260I或DVAP-WT转基因的过表达也显示出核架构造成与一个细长结构（ 图1）变形核严重恶化。这个结构畸变是通过使用其中圆形度由式C所限定的的ImageJ软件定量<img alt="公式3" src="/files/ftp_upload/53821/53821eq3.jpg" /> ，衡量的宽度与C = 1代表一个完美的圆和C = 0无限拉长的多边形每个核的长度的比值。在对照醇细胞核表现出明显的圆形状，C等于1，而在转基因突变体形状的改变与圆的因此而丧失，导致从该值（ 图1和图3）一个显著偏差。 我们还发现，在表达相同转基因的肌肉，细胞核与对照相比显示一个标记放大核体积，虽然ALS引起等位基因的似乎是诱导这种表型相比，DVAP-WT转基因（ 图4）更有效。 几乎所有的神经变性疾病是由含有致病蛋白质聚集体的细胞内积累为特征。我们做了三维重建和核的体积渲染和我们发现，在肌肉表达突变体转基因或过表达野生型蛋白质，DVAP免疫反应形成簇第二，其中一些也被本地化为核（ 图5）。相反，在控制NMJs，DVAP免疫反应是微弱地分散在整个肌肉纤维，并从核16排除。 <img alt="图1" src="/files/ftp_upload/53821/53821fig1.jpg" /> 图1:横纹肌表达任DVAP-WT或DVAP-260I转基因内肌细胞核的共聚焦图像 （A）的BG57-Gal4的/ +对照，（B）的BG57; DVAP-V260I;（C）BG57; DVAP-WT1和（D）BG57;表达所指示的转基因DVAP-WT2肌肉染色与特异于DVAP（红色信号），核纤层蛋白（绿色信号），并用一个核特异性标记物来可视化的细胞核（蓝色信号）的抗体。比例尺= 30微米<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53821/53821fig1large.jpg" target="_blank">请点击这里查看</a>更大的版本这个数字。 <img alt="图2" src="/files/ftp_upload/53821/53821fig2.jpg" /> 图2:最近邻居分析来确定核和其单最近的邻国之间的平均距离 （B）代表性的结果显示肌肉过度表达DVAP-WT2基因改变核的定位相比，在（A）的控制。在指定基因型的肌肉平均核距离用下式（C）中，估计和数据（D）中被报告。幼虫NMJs沾满特异性DVAP（红色信号），核纤层蛋白（绿色）的抗体，并用核标记（蓝色信号）。星号表示统计显着性。 *** P &lt;0.001，** P &lt;0.01。对这个实验的统计分析，并使用报告低于一个单向ANOVA测试的所有实验以及Tukey的MULTIP勒对比试验涂布作为事后测试时发现的基因型之间的差异是由ANOVA检验显著。误差棒表示SEM。比例尺= 30微米<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53821/53821fig2large.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/53821/53821fig3.jpg" /> 图 3: 照片显示，在核体积的计算步骤代表 （A）的代表图像显示使用表面创建向导分割核。在图像的边界细胞核被忽略了。 （B）中示出了周边DVAP染色后核DVAP信号的图像已经通过使用在核标记信道创建的表面屏蔽。 （C）表面层提供的附加 ​​参数信息包括核体积和球形。不同基因型的核体积（D）的数据。星号表示统计显着性。解剖NMJs用抗DVAP抗体（红色信号），抗核纤层蛋白抗体（绿色信号）和核标记（蓝色信号）染色。 *** P &lt;0.001，** P &lt;0.01。误差棒表示SEM。比例尺= 30微米<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53821/53821fig3large.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/53821/53821fig4.jpg" /> 图4: 图像显示在核形状由ImageJ的估计代表性的步骤。 &#160;&#160;使用ImageJ肌肉内估计核圆图像的最大强度投影进行了分析。 （ 一 ）密度投影的一个代表性的例子作为该协议的步骤7.9三个通道图像。 （B）中示出，其中信道被分割的协议和核标记信道的步骤7.10的图像被选择。 示出在ImageJ的施加强度阈值，以段细胞核和ROI经理插件后，所有感兴趣的核可以选择和它们的形状，通过形状描述符测定（℃）的代表性图像（步骤7.11-7.15）。不同基因型的圆度（D）量化。上幼虫NMJs，红色信号指示DVAP染色而绿色概述核并对应于拉明染色。每个核的内部被标记为蓝色由于与核标记在染色。星号表示统计显着性。 *** P &lt;0.001，** P &lt;0.01。误差棒表示SEM。比例尺= 30微米<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53821/53821fig4large.jpg" target="_blank">请点击她的</a>E要查看此图的放大版本。 <img alt="图5" src="/files/ftp_upload/53821/53821fig5.jpg" /> 图5:图像显示在创建肌细胞核的体积渲染的具体步骤 。 （A）显示3D图像的强度混合视图与DVAP蛋白（红色），核纤层蛋白（绿色）和DNA标记（蓝色）染色的肌肉。 （B）表示通过使用拉明信道段的原子核产生的表面层的图像。 （C）表示，其中表面层已被用来掩盖所选择核外DVAP信号的核图像。以黄色突出显示的是已被添加到图像的剪取平面。其视和位置角可以交互调节到可视信号的细胞核内的分布。 （D）的一种图像报告了核表面层产生USI的剖视图NG的核纤层蛋白通道与非屏蔽DVAP和核标记信号合并。 （E和F）相同的细胞核的附加 ​​切片体积渲染。比例尺= 10微米<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53821/53821fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction at JoVE.com

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 ...

why-quantification-matters-characterization-phenotypes-at-drosophila

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

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

8.1K 次观看.  University of Edinburgh. 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. 

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

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

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

果蝇幼虫NMJ夹层

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

生物学

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

果蝇幼虫NMJ免疫

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 - A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination1. Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP&#8217;s) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP&#8217;s and minature excitatory junction potentials (mEJP&#8217;s) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution2 (HL3) that contains 5 mM Mg2+ and 1.5 mM Ca2+. Changes in the amplitude of evoked EJP&#8217;s can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.

Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle.

电生理方法记录从果蝇幼虫NMJ突触潜力

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

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

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The Drosophila larval neuromuscular junction (NMJ), a well-established model for glutamatergic synapses, has been extensively studied for decades. Identification of mutations causing NMJ morphological defects revealed a repertoire of genes that regulate synapse development and function. Many of these were identified in large-scale studies that focused on qualitative approaches to detect morphological abnormalities of the Drosophila NMJ. A drawback of qualitative analyses is that many subtle players contributing to NMJ morphology likely remain unnoticed. Whereas quantitative analyses are required to detect the subtler morphological differences, such analyses are not yet commonly performed because they are laborious. This protocol describes in detail two image analysis algorithms "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics", available as Fiji-compatible macros, for quantitative, accurate and objective morphometric analysis of the Drosophila NMJ. This methodology is developed to analyze NMJ terminals immunolabeled with the commonly used markers Dlg-1 and Brp. Additionally, its wider application to other markers such as Hrp, Csp and Syt is presented in this protocol. The macros are able to assess nine morphological NMJ features: NMJ area, NMJ perimeter, number of boutons, NMJ length, NMJ longest branch length, number of islands, number of branches, number of branching points and number of active zones in the NMJ terminal.

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

Two image analysis algorithms, "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics" were created, to automatically quantify nine morphological features of the Drosophila neuromuscular junction (NMJ).

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 ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

从果蝇幼虫神经肌肉交界处的突触电流的焦 Macropatch 记录

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

Drosophila melanogaster represents a genetically tractable model to study neuronal structure and function, and subsequent changes in disease states. The well characterized larval neuromuscular junction is often used for such studies. However, rapid larval development followed by muscle histolysis and nervous system remodeling during metamorphosis makes this model problematic for the study of slow age-dependent degenerative changes like those occurring in amyotrophic lateral sclerosis. Alternatively, adult flies live for 90 days and the adult leg can be used to study motor neuron changes over the course of adult lifespan using in vivo fluorescent imaging through the cuticle. Here, we describe a leg dissection technique coupled with immunocytochemistry, which allows for the study of molecular changes at the neuromuscular junction of identified adult leg motor neurons. These techniques can be coupled with a myriad of antibodies labeling both pre- and post-synaptic structures. Together these procedures allow a more complete characterization of slow age-dependent changes in adult flies and can be applied across multiple motor neuron disease models.

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

We describe a dissection technique that preserves the architecture of the neuromuscular junction and enables a detailed immunocytochemical study of motor neurons in the adult Drosophila leg.

果蝇成腿的解剖和免疫组织化学，以检测已识别运动神经元的神经肌肉接头处的变化

Drosophila melanogaster represents a genetically tractable model to study neuronal structure and function, and subsequent changes in disease states. The ...

Characterization of Neuromuscular Junctions in Mice by Combined Confocal and Super-Resolution Microscopy

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the transmission of molecules from the nervous system to voluntary muscles, leading to contraction. They are affected in many human diseases, including inherited neuromuscular disorders such as Duchenne muscular dystrophy (DMD), congenital myasthenic syndromes (CMS), spinal muscular atrophy (SMA), and amyotrophic lateral sclerosis (ALS). Therefore, monitoring the morphology of neuromuscular junctions and their alterations in disease mouse models represents a valuable tool for pathological studies and preclinical assessment of therapeutic approaches. Here, methods for labeling and analyzing the three-dimensional (3D) morphology of the pre- and postsynaptic parts of motor endplates from murine teased muscle fibers are described. The procedures to prepare samples and measure NMJ volume, area, tortuosity and axon terminal morphology/occupancy by confocal imaging, and the distance between postsynaptic junctional folds and acetylcholine receptor (AChR) stripe width by super-resolution stimulated emission depletion (STED) microscopy are detailed. Alterations in these NMJ parameters are illustrated in mutant mice affected by SMA and CMS.

This protocol describes a method for morphometric analysis of neuromuscular junctions by combined confocal and STED microscopy that is used to quantify pathological changes in mouse models of SMA and ColQ-related CMS.

通过联合共聚焦和超分辨率显微镜表征小鼠的神经肌肉接头

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the ...

观察 果蝇 神经肌肉接头和肌肉细胞中的微管网络

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of &#945;-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster,&#160;and then examining the microtubule networks in the neuromuscular junction and muscle cells, we&#160;accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila&#160;melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

作者聚焦:了解果蝇神经肌肉接头中的微管网络 

Here, we present a detailed protocol to visualize the microtubule networks in neuromuscular junctions and muscle cells. Combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule-dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

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在活动区域的解剖和影像果蝇神经肌肉交界处

果蝇幼虫NMJ夹层

果蝇幼虫NMJ免疫

电生理方法记录从果蝇幼虫NMJ突触潜力

两种算法的高通量和多参数定量

从果蝇幼虫神经肌肉交界处的突触电流的焦 Macropatch 记录

果蝇成腿的解剖和免疫组织化学，以检测已识别运动神经元的神经肌肉接头处的变化

通过联合共聚焦和超分辨率显微镜表征小鼠的神经肌肉接头

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络

为什么量化事项:表型的表征在

摘要

探索更多视频

此视频中的章节

在活动区域​​的解剖和影像果蝇神经肌肉交界处

果蝇幼虫NMJ夹层

果蝇幼虫NMJ免疫

电生理方法记录从果蝇幼虫NMJ突触潜力

两种算法的高通量和多参数定量

从果蝇幼虫神经肌肉交界处的突触电流的焦 Macropatch 记录

果蝇成腿的解剖和免疫组织化学，以检测已识别运动神经元的神经肌肉接头处的变化

通过联合共聚焦和超分辨率显微镜表征小鼠的神经肌肉接头

使用 果蝇 幼虫神经肌肉接头和肌肉细胞可视化微管网络

使用 果蝇 幼虫神经肌肉接头和肌肉细胞可视化微管网络

在活动区域的解剖和影像果蝇神经肌肉交界处

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络