我们想感谢曼努埃拉·布达克，Ljiljana Marinkovi和克里斯蒂娜Wullimann的，为优秀的技术援助。我们感谢W.麦克林提供PLP-GFP小鼠。 TM高级研究所（慕尼黑工业大学），德意志研究联合会（DFG Sonderforschungsbereich SFB 596），亚历山大冯洪堡基金会支持，由国家提供资金的机构（的“Bundesministerium献给教化和Forschung” ）ERA-净神经元“iPSoALS”和“2 - 光子成像”中的帧的。 TM和MB支持由集成蛋白质科学中心（慕尼黑）。 PM由研究生院的慕尼黑工业大学（TUM-GS）的支持。

1。三角Sterni外植体（ 图1） 定时：15分钟。 <ol><li>准备手术器械：2双钳，剪刀，春风似剪刀1对，1个组织培养皿中（10厘米）。预鼓泡（至少15分钟）冷却的（4°C）的林格氏溶液，5％CO 2/95％O 2。填充15厘米的冰和组织培养皿中与金属板覆盖。准备解剖显微镜下的清扫范围内，放置在15-cm培养皿中，用冰冷为了冷却剥离时的外植体。 </li><li>致死麻醉小鼠的异氟醚过量（或任何其他批准的方法牺牲）。鼠标喷用70％乙醇，以防止毛皮的外植体的污染。用于SC漂白中，我们使用PLP-GFP小鼠，这可以划线，同时可视化的轴突Thy1肾炎大鼠 -OFP3或大鼠Thy1系-Membow13。 </li><li>对大剪刀，正中切开胸骨上方的皮肤和两个平行的肋条笼形件的下边缘的切口。 </li><li>对使用大剪刀，去除皮肤，打开腹壁，使切口平行的肋骨，脊柱回来的路上。然后解剖胸肌的肌肉插入胸骨切口密切。 </li><li>光阑开剑突软骨下切，沿右肋插入解剖隔膜。 </li><li>的肋笼由剑突过程与镊子一套，开始关闭脊柱切割肋，尽可能地接近其插入。左，右削减收敛高于柄sterni的心脏，对。尽量避免减少大血管（尤其是锁骨下静脉）。剑突上的准备，而用钳子轻轻抬起来实现更好的可视性。 </li><li>做一个横向切的正下方胸骨柄sterni和10-cm培养皿中，将外植体转移到冷却的5％CO 2/95％O 2鼓泡的林格氏溶液。将10-cm培养皿中，在金属板覆盖15厘米的组织培养皿中，用冰冷填充。解剖显微镜下，所有进一步的步骤应该做的。 </li><li>使用小弹簧剪刀除去残存的胸腺，胸膜，膈肌（内部）和胸肌（外图1B-D）。 </li><li>为了适应不同的外植体，以3.5厘米的菜，剖析不插入到胸骨的肋骨。最好的做法是使用小春风似剪刀，切割组织之间的肋骨（ 图1E）。 </li><li>引脚的的三角sterni神经-肌肉植到Sylgard的涂层3.5 cm的组织培养皿中使用minutien引脚（缩短至4毫米; 图1G）。两个引脚应该通过胸骨软骨部分（白色）。通过插入至少两个引脚的肋上的左和右侧的第Ê外植体，旨在为较软软骨份和避免下肋或接近三角肌肉。使用更多的引脚，（我们通常使用8-10）。正如你所牵制的准备，确保肋骨有点蔓延，固定的肌肉稍微伸展的位置。 </li><li> 可选：可视化含有通过加入与Alexa染料（0.8微克/毫升的浓度在5％CO 2/95％O 2 -鼓泡林格氏溶液）耦合到银环蛇毒素的乙酰胆碱受体的突触网站。孵育中：Sylgard的涂布盘为15-20分钟，在室温下的固定的外植体，然后，用5％的CO 2/95％O 2 -鼓泡林格氏溶液洗几次。 </li></ol> 2。漂白SC和可选的时间推移显微镜（ 图2） 定时：30 - 45分钟+可选1 - 5小时为时间间隔。 <ol><li>转让3.5厘米Sylgard的涂层菜，激光共聚焦显微镜配备机智H的氩激光（488nm的波长）和水浸泡的目标（4倍，0.13 NA，20倍，0.5 NA和100​​x，1.0 NA或60倍，0.9 NA）。 </li><li> 可选时间的推移显微镜 ：插入3.5厘米的菜到Sylgard的涂层加热圈，安装灌流系统和温度探头（ 图2A）。确保他们没有接触外植体，边缘的菜或SYLGARD的涂层。样品加热至33-35°C。对于SC漂白没有时间推移，室温下是更好的，随着细胞的显示漂白步骤之间的动态关系。 </li><li>观察了4倍的空中目标模式的三角sterni的肌肉神经支配和找到三角sterni终板带。切换到20倍浸泡锥客观和开始寻找终板带表面的区域内（区域覆盖的肋骨是很好的候选人）。更改目标100倍或60倍的的浸渍锥目标检查个人NMJs。理想是NMJ所涵盖的几个种姓，位于会阴ially（ 图2B）。如果执行时间推移成像漂白后，选择到3 NMJs，是比较密切的合作，以增加产量。 </li><li>采集之前的NMJ光漂白个别SC的（ 图2B）的共焦图像。 （对于全分辨率的成像， 例如在Olympus FV1000显微镜，使用100倍，1.0 NA物镜和2.5放大，这会导致在〜0.1μm的每个像素的奈奎斯特采样有限。） </li><li> “公园”的488 nm激光束集中在细胞核内的SC采用最大功率为5秒（或者可以使用高效的扫描模式，在一个小区域的利益，如“龙卷风扫描”功能的奥林巴斯显微镜上;为我们所用激光共聚焦显微镜，激光被聚焦到的图像共轭平面，因此，在488nm处用客观1.0 NA，&lt;0.5微米的直径）。重新聚焦于细胞和法官漂白结果。如果有必要，请重复漂白工序，直到GFP水平减少d，来接近背景水平。采集漂白后的图像（ 图2C）。这是至关重要的，使确保漂白区域以外的两个单元之间的任何重叠 - 如果出现重叠，漂白的第二小区中是显而易见的。 </li><li>重复上述步骤，直到所有，但一个SC漂白（ 图2D-E）。最后的“漂白的”SC适合共聚焦时间的推移显微镜。单细胞重建图像的减法， 如使用Photoshop软件，划定单一的SC形态和领土（ 图2F）。要知道，减去两个法师都会增加噪声（ 例如通过导入他们连续在Photoshop中的“层”，然后在下拉菜单中的“ 差异化 ”的频道选项卡上方的顶部通道），。这可以规避使用“ 散斑 ”功能之前减法的渠道和种植，显然不是来自任何背景漂白细胞。为了优化对比度，也可以是需要调整的两个通道中的“ 水平 ”窗口的亮度。漂白细胞所得到的图像是叠加在原​​始图像上的突触，在一个独特的色调的伪彩色，透明的，着色的漂白细胞领土中的原始图像（从这样做可用于所有漂白和最后棉纱细胞图2F中的结果）所示的图像。 </li><li> 可选：启动时间的推移显微镜共聚焦后漂白，但一个SCS。在固定的时间间隔（例如每5至10分钟），拍摄图像。使用尽可能少的激光功率。可拍摄最多3个NMJs，在同一届会议。 </li><li>在100x通过拍摄图像无变焦，则该区域的图像，使用20倍和4倍的目标（ 图2G-I）在地图上的时间推移NMJs的。这种“映射”需要找到一个固定的肌肉免疫组化后的NMJs。 </li><li>对于图像处理，转换成个人最大强度的预测，并保存为TIFF格式的图像。将所有Z-投影成叠的时间点在XY和调整， 例如在斐济（包的基础上的开源软件ImageJ的美国国立卫生研究院）使用“stackreg的”插件10。 </li></ol> 3。固定和免疫组化（ 图3） 定时：过夜。 <ol><li>外植体转移到一个50毫升的反应管含有至少15毫升4％PFA通过仔细除去标签。修复后的前胸壁1小时上冰的的三角sterni肌肉。冲洗固定的组织与在PBS中的0.1M甘氨酸（淬灭残留的固定剂，从而减少背景）至少10分钟。样品可以被存储在这一点上，和购买处理。 </li><li>固定植转移到10厘米SYLGARD涂层的组织培养皿中充满了0.01 M PBS。切出一个梯形的形状与一个Si的平行胸骨和其他通过的骨软骨过渡 - 这包含在其表面的三角肌肉。去除低肋的水平（ 即反式胸骨）切割成使得其可以自由访问的三角肌肉的尾端（ 图3A-D）。 </li><li>作为梯形的上部（ 图3E）的销进入插入一个皮下注射针头。使用一个皮下注射针头连接到一个1毫升的注射器和镊子除去三角肌肉的表面上，通过轻轻地保持到尾角的肌肉，和使用针作为微型手术刀。确保削减胸壁的平面平行。被释放后尾部分的肌肉，插入另一个皮下注射针一针下面通过胸壁的肌肉-这固定的编制，并允许使用产钳（ 图3F）对肌肉产生一个温柔的拉。当你在切割结缔组织TISSUE插入“剥离”了从胸部的肌肉。春风似剪刀剪下最后的尾附件。除去脂肪和血管残余，使用产钳。要小心，不要撕裂了的神经。 注意：使用两套独立的工具和餐具现场和固定的组织工作。 </li><li>启动免疫组织化学（使用一个标准的协议）的组织样品转移到封闭溶液，在室温下1小时，在振荡器上。染色的最小可能的体积为200微升，使用96孔板中。 </li><li>应用在封闭溶液，200微升每孔，过夜，4℃下在振荡器上稀释的第一抗体。 </li><li>在0.01M的PBS清洗3次洗涤10分钟。 </li><li>用合适的二级抗体，为1-2小时，在室温下在封闭溶液稀释。 </li><li>在0.01M的PBS清洗3次洗涤10分钟。 </li><li>安装在抗衰落的介质（ 如 Vectashield）和盖玻片。 </li><li>磁性金属板，将磁铁在上面的广场上滑行的样品在4℃下的几个小时或过夜弄平密封指甲油。 </li></ol>

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没有利益冲突的声明。

SC漂白这里介绍的方法很简单，速度快，通用性：（一）它使一个单一的基因-这是一个显着的优势相结合的方法， 例如需要额外的基因与疾病模型，激光共聚焦显微镜的基础上揭示单SC形态和动态。 （ii）该方法具有快速，从解剖，以固定它可以在半天的时间。 （ⅲ）的应用程序的数目是广泛的，因为它可以与其他方法，如细胞消融，成像的细胞器，电生理学或免疫组织化学结合。此外?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司名称（示例） </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> 70％（体积/体积）的乙醇溶液</td><td>罗斯</td><td> T913.1 </td><td>在喷雾瓶</td></tr><tr><td>异氟醚</td><td> Forene，雅培</td><td></td><td>可以使用任何其他核准麻醉致死麻醉</td></tr><tr><td>转基因小鼠（PLP-GFP） </td><td></td><td></td><td>获得援引消息人士</td></tr><tr><td>解剖显微镜冷光照明</td><td>奥林巴斯SZ51 </td><td></td><td>配备了肖特KL 1500 LCD </td></tr><tr><td>镊子＃2 </td><td>精细科学工具</td><td> 11233-20 </td><td></td></tr><tr><td>镊子＃5 </td><td>精细科学工具</td><td> 11295-00 </td><td></td></tr><tr><td>剪刀，大型医疗</td><td>精细科学工具</td><td> 14108-09 </td><td></td></tr><tr><td>剪刀，小角度的春天</td><td>精细科学工具</td><td> 15033-09 </td><td></td></tr><tr><td>列直插式加热器</td><td>华纳仪器</td><td> SF-28 </td><td></td></tr><tr><td> 3.5厘米菜肴的加热圈</td><td>华纳仪器</td><td> 64-0110 DH-35 </td><td></td></tr><tr><td>两路温度控制系统</td><td>华纳仪器</td><td> TC-344B </td><td></td&gt; </tr><tr><td>灌流泵系统</td><td></td><td></td><td>定制的</td></tr><tr><td> 15 cm组织培养皿</td><td>萨尔斯塔特</td><td> 83.1803.003 </td><td>盛有冰和金属板覆盖</td></tr><tr><td> 10 cm组织培养皿</td><td>萨尔斯塔特</td><td> 83.1802.003 </td><td>用含氧林格溶液</td></tr><tr><td> 3.5 cm组织培养皿</td><td>萨尔斯塔特</td><td> 83.1800.003 </td><td>充满Sylgard的聚合物</td></tr><tr><td> Sylgard的聚合物</td><td>道康宁</td><td> Sylgard的184有机硅弹性体套件</td><td></td></tr><tr><td>激光共聚焦显微镜</td><td>奥林巴斯</td><td> FV1000 </td><td>用氩气激光</td></tr><tr><td> 50毫升的反应管</td><td>萨尔斯塔特</td><td> 62.547.254 PP </td><td></td></tr><tr><td>在PBS中的4％PFA </td><td>西格玛</td><td> P6148 </td><td></td></tr><tr><td>套管0.5毫米x 25毫米</td><td> Neolab </td><td> E-1510 </td><td></td></tr><tr><td>注射器1毫升</td><td> Neolab </td><td> E-1496 </td><td></td></tr><tr><td>突触素抗体</td><td> Invitrogen公司</td><td> 18-0130 </td><td>兔</td></tr><tr><td>二级抗体</td><td> Invitrogen公司</td><td> A-11012 </td><td> Alexa的594 </td></tr><tr><td> 24 - 孔板</td><td>萨尔斯塔特</td><td> 83.1836 </td><td></td></tr><tr><td> 0.01 M PBS </td><td>西格玛</td><td> P4417 </td><td></td></tr><tr><td>在PBS中的0.1M甘氨酸</td><td>罗斯</td><td> 3790.1 </td><td></td></tr><tr><td>盖玻片</td><td> Neolab </td><td> E-4132 </td><td></td></tr><tr><td>幻灯片</td><td> Neolab </td><td> E-4121 </td><td></td></tr><tr><td> Vectashield </td><td>矢量实验室</td><td> H-1000 </td><td></td></tr><tr><td> minutien引脚×10 </td><td>精细科学工具</td><td> 26002-20 </td><td>缩短至&lt;4毫米</td></tr><tr><td> α-银环蛇毒素结合网站594 </td><td> Invitrogen公司（Molecular Probes公司） </td><td> B-13423 </td><td></td></tr><tr><td></td><td></td><td></td><td> 封闭液 </td></tr><tr><td> 0。01 M PBS </td><td></td><td></td><td></td></tr><tr><td> 0.2％的Triton-X100 </td><td>罗斯</td><td> 3051.3 </td><td></td></tr><tr><td> 10％正常山羊血清</td><td> Abcam公司</td><td> ab7481 </td><td></td></tr><tr><td> 1％牛血清白蛋白</td><td>西格玛</td><td> A7030 </td><td></td></tr><tr><td></td><td></td><td></td><td> 林格 鼓泡通入95％O 2/5％CO 2的 </td></tr><tr><td> 125毫米的NaCl </td><td>西格玛</td><td> S7653 </td><td></td></tr><tr><td> 2.5毫米氯化钾</td><td>西格玛</td><td> P9333 </td><td></td></tr><tr><td> 1.25毫摩尔的NaH 2 PO 4的 </td><td>西格玛</td><td> S8282 </td><td></td></tr><tr><td> 26毫米碳酸氢钠 </td><td>西格玛</td><td> S6297 </td><td></td></tr><tr><td> 2毫米氯化钙 </td><td>西格玛</td><td> 21114 </td><td></td></tr><tr><td> 1毫摩尔MgCl 2 </td><td>西格玛</td><td> 63020 </td><td></td></tr><tr><td> 20mM葡萄糖</td><td>西格玛</td><td> G7528 </td><td></td></tr><tr><td></td><td></td><td></td><td> 表1中。具体的试剂和设备。 </td></tr></tbody></table>

sequential photo-bleaching to delineate single schwann cells at the neuromuscular junction

顺序光漂白在NMJ标记个别SC的，提供了一种非侵入性的方式。 NMJ是世界上最大的哺乳动物神经系统的突触，并担任指导模式研究突触的结构和功能。电机轴突在鼠标NMJs的终端形成与肌纤维的椒盐卷饼般的接​​触位点。公务员事务局局长电机轴突和终端的护套。在过去的几十年里，转基因小鼠已产生运动神经元和SC可视化，例如大鼠Thy1-XFP和PLP-GFP小鼠，分别。 沿着运动神经元轴突，髓鞘轴索SC的配置在非重叠的节间，分离由节点朗维埃，启用跳跃动作电位传播。相比之下，公务员事务局局长的突触终端是专门的神经胶质细胞，监督和促进神经传导，消化碎片和引导轴突再生。的NMJs盖好了半打非MYELinating端子SC的- ，但是，这些不能被单独用光学显微镜解决，因为它们是在直接膜接触3。 有几种方法可以单独可视化终端种姓。这些都不是完美的，虽然。例如，染料填充，单细胞染料填充微刺穿，需要摧毁了标记的细胞，然后再填充第二个。与随后的时间推移录音3，这是一个不兼容。多光谱“Brainbow”标签的判已经实现，通过使用组合的荧光蛋白的表达4。然而，该技术需要结合几个转基因和限制使用的启动子的表达模式。在未来，“照片切换的”蛋白质在SC的表达可能会对又一替代5。在这里，我们提出了连续的光漂白，漂白单细胞，和自己的形象得到减法。我们相信，这种方法-由于其易用性和多功能性-代表持久除了神经学家的技术调色板，特别是因为它可以在体内使用，转移到其他类型的细胞，解剖部位或物种6。 在下面的协议，我们详细的应用顺序漂白时间的推移和随后的共聚焦显微镜终端公务员事务局局长的三角sterni肌肉植。这本薄薄的，肤浅的，容易解剖神经肌肉准备7,8证明是有益的研究NMJ发展，生理和病理9。最后，我们解释了如何的三角sterni肌肉固定后准备执行相关的高解析度激光共聚焦成像，免疫组化或超微结构检查。

一个准备好成像菜的的三角sterni外植体的一个例子是在图1G所示。此外植体是特别适合用于成像NMJs 体外作为三角的肌肉仅由数层的肌肉纤维。这允许获得高清晰度的图像，从外植体来源于转基因小鼠系，亮点要么判（PLP-GFP 2）和/或轴突（Thy1肾炎大鼠 -XFP 1）。高品质成像的关键因素包括：（i）避免接触三角肌肉外植体的制备过程中，以防止肌肉?...

Watch this Scientific Journal Video about Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction at JoVE.com

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

可视化的单个细胞密集的组织，如终端雪旺氏细胞（SC）的神经肌肉接头（NMJs），是具有挑战性的。 “连续光漂白”可以划定单一的终端南海，例如在的三角sterni肌肉外植体，方便神经肌肉准备，连续的漂白可以结合时间推移成像<em&gt;事后</em&gt; 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 ...

sequential-photo-bleaching-to-delineate-single-schwann-cells-at

Research

JoVE Journal

Neuroscience

10.5K 次观看.  Technische Universität München. 可视化的单个细胞密集的组织，如终端雪旺氏细胞（SC）的神经肌肉接头（NMJs），是具有挑战性的。 “连续光漂白”可以划定单一的终端南海，例如在的三角sterni肌肉外植体，方便神经肌肉准备，连续的漂白可以结合时间推移成像事后 immunostainings。

视频: Sequential Photo-bleaching to Delineate Single Schwann Cells at the 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

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 ...

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 ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

体内基因转移到雪旺细胞在啮齿动物坐骨神经电穿孔

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

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 ...

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 ...

Registration of Calcium Transients in Mouse Neuromuscular Junction with High Temporal Resolution using Confocal Microscopy

Estimation of the presynaptic calcium level is a key task in studying synaptic transmission since calcium entry into the presynaptic cell triggers a cascade of events leading to neurotransmitter release. Moreover, changes in presynaptic calcium levels mediate the activity of many intracellular proteins and play an important role in synaptic plasticity. Studying calcium signaling is also important for finding ways to treat neurodegenerative diseases. The neuromuscular junction is a suitable model for studying synaptic plasticity, as it has only one type of neurotransmitter. This article describes the method for loading a calcium-sensitive dye through the cut nerve bundle into the mice's motor nerve endings. This method allows the estimation of all parameters related to intracellular calcium changes, such as basal calcium level and calcium transient. Since the influx of calcium from the cell exterior into the nerve terminals and its binding/unbinding to the calcium-sensitive dye occur within the range of a few milliseconds, a speedy imaging system is required to record these events. Indeed, high-speed cameras are commonly used for the registration of fast calcium changes, but they have low image resolution parameters. The protocol presented here for recording calcium transient allows extremely good spatial-temporal resolution provided by confocal microscopy.

The protocol describes the method of loading a fluorescent calcium dye through the cut nerve into mouse motor nerve terminals. In addition, a unique method for recording fast calcium transients in the peripheral nerve endings using confocal microscopy is presented.

使用共聚焦显微镜以高时间分辨率记录小鼠神经肌肉接头中的钙瞬变

Estimation of the presynaptic calcium level is a key task in studying synaptic transmission since calcium entry into the presynaptic cell triggers a ...

Visualizing the Morphological Characteristics of Neuromuscular Junction in Rat Medial Gastrocnemius Muscle

The neuromuscular junction (NMJ) is a complex structure serving for the signal communication from the motor neuron to the skeletal muscle and consists of three essential histological components: the pre-synaptic motor axon terminals, post-synaptic nicotinic acetylcholine receptors (AchRs), and peri-synaptic Schwann cells (PSCs). In order to demonstrate the morphological characteristics of NMJ, the rat medial gastrocnemius muscle was selected as the target-tissue and examined by using multiple fluorescent staining with various kinds of biomarkers, including neurofilament 200 (NF200) and vesicular acetylcholine transporter (VAChT) for the motor nerve fibers and their pre-synaptic terminals, alpha-bungarotoxin (&#945;-BTX) for the post-synaptic nicotinic AchRs, and S100 for the PSCs. In this study, staining was performed in two groups: in the first group, samples were stained with NF200, VAChT, and &#945;-BTX, and in the second group, samples were stained with NF200, &#945;-BTX, and S100. It was shown that both protocols can effectively demonstrate the detailed structure of NMJ. Using the confocal microscope, morphological characteristics of the pre-synaptic terminals, post-synaptic receptors, and PSC were seen, and their Z-stacks images were reconstructed in a three-dimensional pattern to further analyze the spatial correlation among the different labeling. From the perspective of methodology, these protocols provide a valuable reference for investigating the morphological characteristics of NMJ under physiological conditions, which may also be suitable to evaluate the pathological alteration of NMJ, such as peripheral nerve injury and regeneration.

The protocol shows a method to examine spatial correlation among the pre-synaptic terminals, post-synaptic receptors, and peri-synaptic Schwann cells in the rat medial gastrocnemius muscle using fluorescent immunohistochemistry with different biomarkers, namely, neurofilament 200, vesicular acetylcholine transporter, alpha-bungarotoxin, and S100.

可视化大鼠内侧腓肠肌神经肌肉接头的形态学特征

The neuromuscular junction (NMJ) is a complex structure serving for the signal communication from the motor neuron to the skeletal muscle and consists of ...