我们感谢他们对这个手稿的有益的意见Nael阿拉米博士和博士南澈金。

1。对免疫的筹备工作： <ol><li>要创建一个解剖面，倒入一个小的组织培养板Sylgard 184硅橡胶基地。确保不来填充板完全使清扫面积低于轮辋。 </li><li>修剪清扫引脚易于操纵增加了约3.5毫米长度，并确保至少有6％的幼虫引脚。 </li><li>您将需要钝钳，让你掌握清扫引脚。 </li></ol> 2。解剖的幼虫： <ol><li>选择流浪三龄幼虫蠕动着小瓶双方还没有开始化蛹。 </li><li>广场上的立体显微镜下解剖面的幼虫。威克走多余的潮湿使用Kimwipe。 </li><li>安排每个幼虫，使背侧朝上。您将能够看到两个气管，白线沿背侧幼虫。中心在中间尽可能多的尝试，因为这标志着背中线。 </li><li>将针，只是上面的嘴钩＃1（图1）。 </li><li>广场针在正上方气孔之间的气管的尾巴＃2。 </li><li>添加少量的PBS幼虫，以防止干燥的角质层。 </li><li>与所有的幼虫，重复上述步骤，确保放置各基因型之间的引脚的空间分隔的基因型。 </li><li>跨越气管（图1）＃2针以上的小横削权。 </li><li>插入你的剪刀剪切和沿背中线之间的气管切开了过去针一路＃1。 </li><li>您将需要在每一侧的角质层非常的顶部和底部的小伤口，使左，右两侧会开放让您针没有角质层拉下来。 </li><li>放置接脚＃“第3，4，5，6，并在每个角落。 </li><li>修正了4％的煤灰样品在室温为20-25分钟</li><li>倒出的煤灰和3-4次，用PBS漂洗，每次调迁。 </li></ol> 3。去除组织： <ol><li>开始删除，因为通常你要保护的区域位于大脑后部组织。 </li><li>而使用steromicroscope下的PBS中的幼虫，先抢后气管结束，并将其拉出，可能会删除一些与脂肪体。重复第二气管。 </li><li>抓住后脂肪体或肠道和拉出来的预习。一旦后组织的大部分已被删除，着眼于消除大脑周围的小组织。如果你只在肌肉，或形态的NMJ感兴趣，可以去除大脑。 </li><li>去除大脑可以更好地肌肉31日，这是一个有用的标记腹段取向的可视化。 </li></ol> 4。为活动区域的免疫： <ol><li>添加阻断剂（5％正常山羊血清的PBS）在解剖盘的解剖PREPS。在室温下1小时座。如果您想封锁和抗体稀释液，不仅使一个解决方案，包括0.1％TritonX - 100你阻止组织通透性的代理。 </li><li>应用的主要抗体阻断0.1％TritonX - 100的试剂稀释。活动区抗体是通过发育研究杂交瘤细胞银行（DSHB，http://dshb.biology.uiowa.edu/）达到。使用引用抗体（如辣根过氧化物酶Cy3标记物）标签NMJ膜和轴突。 </li><li>在4 ° C过夜培养的主要抗体。因此，冰箱干燥不会蒸发的抗体解决方案，创建湿度室，把一些水在使用枪头框的底部。半透明的盖子盖用铝箔，以减少荧光漂白。放置在湿度室的清扫幻灯片，并在4℃孵化器把整个中过夜。 </li><li>第二天早上，洗脸用PBST（0.2％TRITON - X - 100的PBS）的5-10倍，每5-10分钟。 </li><li>孵育的二级抗体（如Invitrogen公司Alexa的山羊抗鼠- 488）在0.1％TritonX - 100阻断试剂在室温覆盖湿度试验箱2-4小时稀释。包括在二次核染色（如DAPI）的，如果你想确定在肌肉或脑细胞核的位置。 </li><li>用PBST（0.02％TRITON - X - 100的PBS），洗净的5-10倍，每5-10分钟。 </li></ol> 5。在幻灯片上安装的幼虫样品： <ol><li>倒出和灯芯最后PBST洗涤。 </li><li>移液器约1毫升80％甘油，以牵制的幼虫毛皮。 </li><li>删除5 6引脚在所有的幼虫。 </li><li>当你准备把幻灯片上的第一基因型，删除最后一个引脚，所有的幼虫，genotyPE。 </li><li>把一个干净的幻灯片上两滴您的安装介质的准备“洗”的幻灯片。这一步很重要的，洗掉的幼虫水解决方案，安装和更清晰的成像。 </li><li>抓住您的钝钳尾一幼虫，幼虫在甘油拖动周围，然后沿夹层菜干边，除去大多数的甘油。放置在第一个安装介质下降的幼虫。 </li><li>同一基因型的幼虫，重复步骤6。 </li><li>一旦所有的幼虫都在第一次安装介质下降，抢到第一幼虫的尾巴，转危为安下降，沿干边的菜，然后删除了大多数中等。然后将安装在第二个中期下降的幼虫。 </li><li>所有具有相同基因型的幼虫重复步骤8。 </li><li>准备好你最后的幻灯片，标签，并在幻灯片上放置一个小的“T”型安装介质的形状。 </li><li>在步骤5.8中，抓住的尾巴的幼虫;转危为安第二安装介质下降，然后沿“洗”幻灯片干燥双方的皮毛。大多数介质脱落后，幼虫皮上安装介质中的最后一张幻灯片。 </li><li>重复所有的幼虫毛皮。 </li><li>确保所有的幼虫毛皮安排需要确保角质层方下来，裸露的肌肉朝上。 </li><li>将幻灯片上的盖玻片覆盖解剖的幼虫，在“T”上线边缘</li><li>盖玻片放在一个小，重量轻（如9伏电池），确保安装介质传播的掩护下滑完全。 </li><li>为了减少可能的荧光猝灭，放置在抽屉或黑暗的空间幻灯片，直到安装介质已治愈。 </li><li>清理安装介质溢出盖玻片一柄。盖玻片或用70％乙醇的幻灯片上，擦去多余的干安装介质。 </li><li>要创建一个最终的密封，指甲油密封。 </li></ol> 6。代表性的成果： 解剖和免疫染色的幼虫例如幻灯片，如图2所示。当幼虫解剖，污渍，并安装得当，用户可以识别A2 - A6段肌肉（图3）幼虫的利益。晃等提供了肌肉位置的详细描述，以及具体的突触boutons（晃和千叶，2001年）的表征。使用高功率的目标用户可以专注于一个单一的突触和采取的Z - Stack的形象。突触6 / 7的一部分的一个代表最高的投影图像（图4）所示。然后用图像处理软件，活跃的区域，可定量评估，尤其是您的突变表型特征。可能在一个特定的突变表型的改变的功能的例子包括的总数量，flourescence强度，活动区域之间和间距。 潜在的隐患： 幼虫神经肌肉交界处的免疫评价是一个有价值的的系统，可以到突触生物学提供了宝贵的见解，虽然有潜在的缺陷，可以限制这一制度的效用。例如，它常常是可取的图像在同一样品的两个或两个以上的主要抗体染色模式。选择适当的主要抗体，适当的荧光基团，和相应的阴性对照的是取得成功的关键。 如果成像两个抗原在实验中，可以肯定，使每一个感兴趣的蛋白质，可以唯一一个荧光标记的第二抗体确定不同物种的主要抗体。一个推荐的方式，以减少背景是使用阻断剂含有从中派生的二级抗体（如用正常山羊血清作为阻断剂时，使用羊抗兔或抗鼠二抗）的物种的正常血清。 规划的小学和中学的抗体在实验中，您将使用后，你还必须考虑包括在您的染色的控制设置，以确保您看到的荧光信号是真实的，不只是背景。如果可能的话，包括阴性对照样品，不包含感兴趣的蛋白质。中的外源基因的过度表达的研究，这是很容易做到，包括野生型动物，但在某些情况下可以使用一个你感兴趣的蛋白质的纯合子突变的完成。另一个重要的负面控制，用于测量您的荧光信号的背景水平，是不包括主使用的二级抗体。成像时产生的PREPS，重要的是理解的荧光信号，你看到的所有可能不是真正的的，可能从哟背景UR二次抗体或内源性组织背景。考虑背景荧光，荧光强度之间的变化化验时，您可能需要将您的图像的正常化。 荧光的选择也必须小心。每个荧光基团和能力的唯一检测每个信号的激发和发射光谱的代价是必要的，以避免渠道流失，通过的。如果有任何的不确定性，一个简单的方法来确定是否出血的程度，通过免疫染色与荧光标记的单一抗体的时间和检查，每个通道的图像，你会在你最后的实验中使用。这应该发现，如果通过流血与您的显微镜过滤器设置发生。 <img src="/files/ftp_upload/2676/2676fig1.jpg" alt="图1" /> 图1。角质层的切割和安置的针脚1-6。流程图<a href="http://jove.com/files/ftp_upload/2676/2676fig1_large.jpg">点击这里查看大图</a> <img src="/files/ftp_upload/2676/2676fig2.jpg" alt="图2" /> 图2。安置的最后一张幻灯片上的幼虫。确保，幼虫PREPS暴露肌肉侧和角质层一面向下。在每个样本之间至少留出幼虫体宽度。 <img src="/files/ftp_upload/2676/2676fig3.jpg" alt="图3" /> 图3。成像时，找到利益的肌肉和段。常见的神经肌肉接头的特点支配肌肉的6 / 7，肌肉13 12，肌肉或肌肉4。在腹中线的对面，有一个镜像显示半段的肌肉结构。作为一个标记，以确定哪个段是成像肌肉31。相同的突触图片和段其余样品，收集许多突触％的基因型，可用于定量使用的图像。 <img src="/files/ftp_upload/2676/2676fig4.jpg" alt="图4" /> 图4。NMJ支配肌肉的6 / 7的一部分的一个有代表性的最大投影图像显示。 NC82（Bruchpilot）染色显示为绿色。 HRP的前突触染色显示为红色。注意活动区域punctae图像处理软件，可以进一步量化。

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神经元突触终端领域是至关重要的，是适当的沟通后，突触前细胞之间的桥梁。调查疾病模型的神经元的健康，是一种强有力的方式分析，免疫荧光蛋白质的突触终端。这里介绍的免疫荧光法，使研究者检查许多幼虫的同时，限制群体之间的环境差异。 果蝇三龄幼虫的中央神经系统有许多优点，包括谷氨酸的突触，突触末端，交通方便，重现性和遗传操作的权力映射和重复。特别在果?...

<table border="1"><tbody><tr><td>试剂名称</td><td>公司</td><td>目录编号</td><td>评论</td></tr><tr><td> Sylgard 184有机硅弹性体基地</td><td>道康宁</td><td> 68037-59-2 </td><td>混合后让气泡上升缓慢肩或允许坐30分钟或更慢了。 </td></tr><tr><td>不锈钢Minutien引脚</td><td>精细科学的工具</td><td> 26002-10 </td><td>修剪到约。 3 - 4mm的长度与普通剪刀</td></tr><tr><td>椎板钳（抓销钝） </td><td>精细科学工具</td><td> 11223-20 </td><td>使用钝钳抓销</td></tr><tr><td>解剖钳</td><td>世界精密仪器</td><td> 501985 </td><td></td></tr><tr><td>超炫Vannas剪刀，8厘米长</td><td>世界精密仪器</td><td> 501778 </td><td></td></tr><tr><td>鼠抗BRP抗体</td><td> DSHB </td><td> NC82 </td><td>使用1:50稀释</td></tr><tr><td> Cy3标记Affinipure山羊抗辣根过氧化物酶</td><td>杰克逊Immunoresearch </td><td> 123-165-021 </td><td>用1:200稀释</td></tr><tr><td>的Alexa Fluor 488山羊抗鼠IgG </td><td> Invitrogen公司</td><td> A11001 </td><td>使用约。 1:200稀释</td></tr></tbody></table>

dissection and imaging of active zones in the drosophila neuromuscular junction

果蝇幼虫神经肌肉接头（NMJ）突触的结构和功能研究的一个很好的模型。果蝇是缓解强大的遗传操作和幼虫的神经系统已被证明不仅在学习的正常功能特别有用，但也扰动伴随一些神经系统疾病（Lloyd和泰勒，2010）。在果蝇中发现的许多关键的突触分子还发现，在哺乳动物中，最喜欢在哺乳动物中枢神经系统兴奋性突触，果蝇NMJ是谷氨酸和演示活动依赖的重塑（Koh等 ， 2000）。此外， 果蝇的神经细胞可以单独确定，因为他们的神经支配模式都是千篇一律和重复，使我们能够研究确定的突触终端，如运动神经元和体壁肌纤维之间，他们支配（Keshishian和Kim ，2004）。进化上保守的突触组件以及与遗传和体质操纵缓解的存在， 使得果蝇调查的内在机制，突触功能的（Budnik 1996）的理想模型。 在突触终端的活动区域特别感兴趣，因为这些神经递质释放的网站。 NC82是一种单克隆抗体，能够识别的果蝇蛋白Bruchpilot（BRP），CAST1/ERC家庭成员的活动区（Wagh 等人 ，2006年），是一个重要组成部分。 BRP是直接形成活动区的T -酒吧，并负责有效聚类的Ca 2 +通道下方的T -酒吧密度（富凯等人，2009年） 。 BRP突变体减少的Ca 2 +通道密度，郁闷诱发囊泡释放，并改变短期的可塑性（ 基特尔等，2006） 。活跃区的改建已发现在果蝇的疾病模型。例如，使用的NC82抗体的免疫荧光显示，活跃的区域密度下降在肌萎缩性脊髓侧索硬化症和皮特霍普金斯综合征（Ratnaparkhi等，2008; Zweier等，2009）。模型。因此，活跃的区域，或其他疾病的果蝇幼虫模型，突触蛋白的评价可能会提供一个宝贵的初步线索，一个突触缺陷的存在。 准备整个安装解剖果蝇幼虫的免疫分析NMJ需要一些技巧，但可以稍加练习，大多数科学家所完成。是提供多个幼虫在同一解剖盘解剖和免疫染色方法，限制各基因型之间的环境差异，并提供足够的动物重现性和统计分析的信心。

Watch this Scientific Journal Video about Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction at JoVE.com

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

神经肌肉接头（NMJ）<em&gt;果蝇</em&gt;是一个重要的模型系统，为研究正常的突触功能以及某些神经系统疾病中发现的突触功能的扰动。我们提出了一个解剖的协议<em&gt;果蝇</em&gt;幼虫电机系统和免疫活性区蛋白内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 ...

dissection-imaging-active-zones-drosophila-neuromuscular

Research

JoVE Journal

Neuroscience

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

15.3K 次观看.  St. Jude Children’s Research Hospital. 神经肌肉接头（NMJ）果蝇是一个重要的模型系统，为研究正常的突触功能以及某些神经系统疾病中发现的突触功能的扰动。我们提出了一个解剖的协议果蝇幼虫电机系统和免疫活性区蛋白内NMJ。

视频: Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

Single Drosophila Ommatidium Dissection and Imaging

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative diseases because of its powerful genetics1. The fly eye in particular has been the organ of choice for neurodegeneration research, being the most accessible and life-dispensable part of the Drosophila nervous system. However the major caveat of intact eyes is the difficulty, because of the intense autofluorescence of the pigment, in imaging intracellular events, such as autophagy dynamics2, which are paramount to understanding of neurodegeneration.
We have recently used the dissection and culture of single ommatidia3 that has been essential for our understanding of autophagic dysfunctions in a fly model of Dentatorubro-Pallidoluysian Atrophy (DRPLA)3, 4.
We now report a comprehensive description of this technique (Fig. 1), adapted from electrophysiological studies5, which is likely to expand dramatically the possibility of fly models for neurodegeneration. This method can be adapted to image live subcellular events and to monitor effective drug administration onto photoreceptor cells (Fig. 2). If used in combination with mosaic techniques6-8, the responses of genetically different cells can be assayed in parallel (Fig. 2).

Single Drosophila Ommatidium Dissection and Imaging

The limiting factor in the use of the adult Drosophila eye to study neurodegeneration and cell biology is the difficult imaging of intracellular processes. We describe the dissection of single ommatidia to generate a bona-fide primary neuronal cell culture, which can be subject to drug treatment and advanced imaging.

单果蝇 Ommatidium解剖和影像

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative ...

科研

神经科学

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 ...

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 ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

一个简单的一步解剖协议成人全安装准备<em&gt;果蝇</em&gt;脑

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

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 ...

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 ...

在活动区域​​的解剖和影像果蝇神经肌肉交界处

在活动区域的解剖和影像果蝇神经肌肉交界处