这个项目是由CIHR和NSERC。我们要感谢创造表达DsRed的标记SSR（北京）和不列颠哥伦比亚大学生物成像设施的飞株倒钩Jusiak。

第1部分：组织准备<ol><li>我们的目标是组织编制的神经系统是完整的蝇蛆，但体壁肌肉的内表面接触到一个人工血淋巴，可定位接近一个良好的光学显微镜盖玻片。换句话说，里面出幼虫准备。 由于传统的组织筹备工作涉及切割，固定和体壁的肌肉伸展，有时消除神经系统的一部分，我们需要一种不同的方法。 我们希望内而外的动物，因为我们想获得以上时间在突触的结构特点和结构变化的一个很好看，我们希望保持神经系统完好无损。我们也希望，以避免伸展的组织，并激活牵张感受器，这将使肌肉抽搐，破坏我们的形象。 </li> <li>对于初学者来说，现阶段的动物。喂第三幼虫和流浪第三幼虫大，易于解剖，所以我们将演示W3的幼虫。这种动物是有短粗的表型，是广泛的，所以容易埃弗特。 我们只用幼虫，我们看到积极地爬行，包括W3的幼虫。 </li><li>蛆表面清洁的双蒸馏水的培养皿中的一个非常柔软的油漆刷。清洁幼虫有较好的光学和清洁减少细菌。 </li><li> HL - 6约3毫升冰冷的小培养皿，人工血淋巴转移动物。放在冰盘，直至停止不动的动物，并放宽（约5分钟）。 </li><li>持有一方面和在其他弹簧剪刀很精尖的镊子。我用我的惯用手的剪刀。设为一个小孔，在体壁用剪刀equlibrate横跨体壁的压力。 </li><li>倒在盘底部镊子轻轻地握住动物和切断后两部分。解剖了内脏和体腔脂肪体可能会移动。这个组织还切去。 </li><li>保持幼虫对菜用细镊子底部。按住＃0昆虫针（用钝尖），推动对幼虫的口器。推动通过体腔口部位，像你转动内而外的袜子。 </li><li>出组织内将类似于图：下面。超精细的解剖工具，解剖离体壁脂肪体和trachioles。试一下真的很难不拉的trachioles，或断开的神经系统。翻录trachioles RIP在体壁肌中的漏洞。 删除尽可能多的脂肪体，你可以。这两种结构的破坏您的组织的光学质量。您可能想跳过前这样做准备的咖啡。 </li> <li>当你完成的肌肉将半透明，不透明的或白色的。如果你把HL - 6没有谷氨酸的组织准备，在室温下，它可能会合同的节奏，因为在中枢神经系统的运动模式发生器工作。 避免用体壁肌肉明显收缩，或不定期承包PREPS。 容易出完整的体壁内往往折沿背，腹中线的一半，所以准备给左或右的“半动物的”可视化。 </li><li> 1.9安装在体积小商会，如华纳室，或与桥接盖玻片安排显微镜玻片上的组织。请参阅安装组织的建议的第3部分。 </li></ol>第2部分：对辣根过氧化物酶的主要的抗体与荧光标记的神经元布顿标签。 <ol><li>在培养皿中放入HL - 6 50荧光标记对辣根过氧化物酶的主要反身微升。沉浸在染浴内而外的体壁的准备。您可能会看到大约5分钟后，神经元终端标签，但孵育10-20分钟，一个完整的，明亮的标签，。 </li><li>在室温下，HL - 6冲洗10-30秒染料关闭。非绑定的染料冲洗快，所以冲洗周期需要进取。 </li><li>不同染料浓度和培养时间需要。 </li></ol>第3部分：Perisynaptic右旋糖酐flurophore共轭空间标签<ol><li>稀释荧光标记的葡聚糖染料稀释在HL - 6。 A的浓度，以及工作对我们的目的是： </li><li>如果您不是对染料的访问到您的外空间的时机，把一个“桥接”盖玻片（见第4部分）20微升染料下降和存款准备在染浴。 </li></ol>第4部分：安装可视化（共聚焦显微镜）组织。 如果您没有需要灌注你的准备（简称意见），或者你想保持洗澡HL - 6的小体积，使用双桥玻片法如果你想灌注你的准备，尽量使用灌注室。我们用一个莫dified商会从华纳仪器。 为后续的详细信息。 安装双桥的幻灯片上的准备。 <ol><li>使用一个非常干净的显微镜幻灯片。两个强力胶平方米，18毫米（1.5）盖玻片上的幻灯片。盖玻片边缘之间留下一个2毫米的差距。 </li><li>让胶水完全干燥，否则它会在你准备的水介质，形成一个奇怪的电影。 </li><li>出盖玻片边缘之间的幼虫内的位置。您可能需要一个对角线上的位置的准备，如果幼虫大您的图像采集系统具有有限的像素阵列。 </li><li>封面＃1.5盖玻片，18毫米（很干净）组织。粘“顶”盖玻片的幻灯片贴有最小的凡士林组织侧翼滑倒，。 </li><li>顶端盖玻片（如果您使用的HL - 6）的1.3379折射率嘉吉公司自订的目标油价下降和翻转本次大会。 </li><li>位置显微镜，油面朝着目标的组织大会，并集中你的目标。 安装在灌注腔组织。 </li><li>胶水1.5盖玻片，形成了在RC 20室的地板。在商会组织的地位，每RC - 20指令。使用一块耐特网格，而不是一个盖玻片形成室的屋顶。 </li></ol>第5部分：代表性的成果： <ol><li> 由内而外的组织准备清扫序列 ： <img src="/files/ftp_upload/1154/fig1.jpg" alt="图1" /> W3的幼虫，洗净（“桶状”的表型）（5.1.1）。 W3的幼虫，后2段解剖。你可以看到肥胖的身躯推反体壁的压力（箭头）的体腔。尽量减少内脏“喷发”，通过了一个小洞，在体壁夹层前1分钟（5.1.2）。准备与内脏解剖之前，内而外的准备（5.1.3）。预习，大多外翻，针管腔内的预习（5.1.4）（箭头）。肌肉是现在在外面的角质层内，与一些脂肪体和连接（5.1.5）trachioles。完全解剖一个与几乎所有的脂肪体和trachioles解剖（5.1.6）的组织准备。 <img src="/files/ftp_upload/1154/fig2a.jpg" alt="图2" width="600" height="200" /></li><li>活动标签使用抗- HRP在幼虫NMJ。一个代表W3的幼虫神经肌肉突触与神经胶质延伸（内而外的预习）。运动神经元布顿终端与主要反身对HRP（洋红色），这是结合Cy5的标记。胶质进程标记GFP（绿色）。 <img src="/files/ftp_upload/1154/fig2b.jpg" alt="图3" /></li><li>荧光Perisynaptic空间可视化与Alexa的680葡聚糖在里面了准备。膜有针对性的GFP标记的胶质过程（绿色）。肌肉表面的突触后的SSR（蓝色）与DsRed的标记。 Alexa的右旋糖酐（红色）的形式集中在细胞外的地区。葡聚糖染料形式在perisynaptic空间甜甜圈形池。葡聚糖标签和DsRed的标记的SSR以灰度显示。请注意多个甜甜圈形染料池，突出perisynaptic空间（箭头）。 </li></ol>

<ol>
	<li>Macleod, G. T., Marin, L., Charlton, M., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+Vesicles:+Test+for+a+role+in+presynaptic+Calcium+regulation.">Synaptic Vesicles: Test for a role in presynaptic Calcium regulation.</a> J. Neurosci. 24, 2496-2505 (2004).</li><li>Macleod, G. T., Hegstro, M., Wojtowicz, M., Charlton, M. P., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+Calcium+signals+in+Drosophila+motor+neuron+terminals.">Fast Calcium signals in Drosophila motor neuron terminals.</a> J. Neurophysiology. 88, 2659-2663 (2002).</li><li>Morales, M., Ferrus, A., Martinez-Padron, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+calcium-channel+currents+in+normal+and+csp+mutant+Drosophila+peptidergic+terminals.">Presynaptic calcium-channel currents in normal and csp mutant Drosophila peptidergic terminals.</a> Eur J Neurosci. 11, 1818-1826 (1999).</li><li>Stork, T., Engelen, D., Krudewig, A., Silies, M., Bainton, R. J., Kla&#168;mbt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+and+Function+of+the+Blood&#8211;Brain+Barrier+in+Drosophila.">Organization and Function of the Blood&#8211;Brain Barrier in Drosophila.</a> J. Neurosci. 28, 587-597 (2008).</li></ol>

此过程允许长期住标记的蛋白和细胞进程的成像。 原位组织准备，我们描述了一个完整的和运作的中枢神经系统，百佳和反射电路。该组织准备已超过标准的幼虫蝇肌肉协议，幼虫体壁的肌肉被拉伸（当它被固定）优势。拉伸可以扭曲突触形态和触发反射的基础收缩。我们出预习内机械性能稳定，并且有特别好的光学，促进细胞的变化实时高分辨率分析。此外里出来准备存活一个小时，并...

<table><tbody><tr><td>HL-6: Artifical Drosophila hemolymph, with 5 mM L-glutamate added, and 2 mM Calcium.</td><td>Reagent</td><td>N/A</td><td>NA</td><td>5 mM L-glutamate blocked muscle contractions. We used Molecular grade L-Glutamate (Sigma).2 mM Calcium is close to physiological Calcium levels in natural larval hemolymph.References: Macleod et al 2002 and Macleod 2004</td></tr><tr><td>Dextran, Alex Fluor 680; 10,000 MW, anionic, fixable</td><td>Reagent</td><td>Molecular Probes, Life Technologies</td><td>D34680</td><td>Use a small volume perfusion chamber to keep the total volume of dye low</td></tr><tr><td>Anti-HRP-CY5 conjugate (goat)</td><td>Reagent</td><td>Jackson ImmunoResearch</td><td>123-175-021</td><td>Dilute 2.0 mg into 1 ml ddH2O; aliquot into 4 microliter aliquots. Freeze at &amp;#8211;20C. Dilute one aliquot into 100 microliters of HL-6</td></tr><tr><td>Alexa 647 antibody labeling kit</td><td>Reagent</td><td>Molecular Probes, Life Technologies</td><td>A10475</td><td>We prepared a total of 80 micro liters of conjugated primary antibody, and stored as 2 microliter aliquots. We diluted each aliquot into 100 microliter of HL-6 for labeling.</td></tr><tr><td>Custom Formulated Objective Oil, refractive index 1.3379</td><td>Reagent</td><td>Cargill Labs</td><td>Custom Formulated</td><td></td></tr><tr><td>Ultra Fine Forceps</td><td>Tool</td><td>Fine Science Tools</td><td>11252-23 or 11295-20</td><td></td></tr><tr><td>Spring scissors</td><td>Tool</td><td>Fine Science Tools</td><td>91500-09</td><td></td></tr><tr><td>Ultra fine clipper scissors</td><td>Tool</td><td>Fine Science Tools</td><td>15200-00</td><td></td></tr><tr><td>Perfusion Chamber RC 20 Series</td><td>Tool</td><td>Warner Instruments</td><td>64-02222</td><td></td></tr><tr><td>Spinning Disc confocal</td><td>Microscope</td><td>Quorum Technologies</td><td>Quorum Wave FX</td><td>Mounted on a Leica DMI6000 Inverted Microscope</td></tr></tbody></table>

visualizing the live drosophila glial-neuromuscular junction with fluorescent dyes

我们的项目确定在发展中的幼虫飞神经肌肉突触的绿色荧光蛋白标记的胶质结构。为了看住胶质细胞神经​​肌肉突触的发展，我们开发了一个幼虫的组织编制了完整的幼虫生活的功能，但也有良好的光学性能。这个新的编制也不允许灌流液进入突触。我们用蝇蛆，沉浸在人工血淋巴，寒心他们放宽了其正常的有节奏的身体收缩。下一步，我们解剖了每个动物的后段，并用钝器昆虫针推通过体腔口部位落后。这外翻幼虫体壁，就像把袜子内而外。我们完成了超细清扫剪刀剥离，从而暴露了体壁肌肉内脏的一面。在NMJ的胶质结构对膜有针对性的绿色荧光蛋白，胶质细胞特异性启动子的控制下。突触后膜，肌肉中的SSR（Subsynaptic Reticula）表示突触针对性DsRed的。我们需要清楚地标记的运动神经元终端突触的第三部分。要做到这一点，我们采用的主要抗体，以辣根过氧化物酶结合到远红外发光flurophore。 perisynaptic空间之间的运动神经元终端和SSR的染料扩散性能测试，我们采用一个大型的葡聚糖分子的共轭远红外发光flurophore和收集到的图像解决方案。

Watch this Scientific Journal Video about Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes at JoVE.com

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

我们描述的神经胶质细胞神经​​肌肉突触的结构特点，以一种新颖的由内而外的活飞使用共聚焦显微镜荧光染料的幼虫的组织准备。我们辣根过氧化物酶的荧光抗体标记的活神经元的终端，也可视荧光右旋糖酐perisynaptic空间。

可视化的Live果蝇胶质细胞神经​​肌肉交界处，与荧光染料

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

visualizing-live-drosophila-glial-neuromuscular-junction-with

Research

Education

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Unit 1

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Skeletal Muscle Relaxants

SCIENTISTS IN ACTION

10.8K 次观看.  University of British Columbia - UBC. 我们描述的神经胶质细胞神经​​肌肉突触的结构特点，以一种新颖的由内而外的活飞使用共聚焦显微镜荧光染料的幼虫的组织准备。我们辣根过氧化物酶的荧光抗体标记的活神经元的终端，也可视荧光右旋糖酐perisynaptic空间。

视频: Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

在果蝇幼虫分部轴突在体内的突触小泡的可视化

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

科研

神经科学

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 ...

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

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

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

The majority of work on the neuronal specification has been carried out in genetically and physiologically tractable models such as C. elegans, Drosophila larvae, and fish, which all engage in undulatory movements (like crawling or swimming) as their primary mode of locomotion. However, a more sophisticated understanding of the individual motor neuron (MN) specification&#8212;at least in terms of informing disease therapies&#8212;demands an equally tractable system that better models the complex appendage-based locomotion schemes of vertebrates. The adult Drosophila locomotor system in charge of walking meets all of these criteria with ease, since in this model it is possible to study the specification of a small number of easily distinguished leg MNs (approximately 50 MNs per leg) both using a vast array of powerful genetic tools, and in the physiological context of an appendage-based locomotion scheme. Here we describe a protocol to visualize the leg muscle innervation in an adult fly.

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

Here we describe a protocol to visualize the axonal targeting with a florescent protein in adult legs of Drosophila by fixation, mounting, imaging, and post-imaging steps.

通过成人角质层可视化果蝇腿运动神经元轴突

The majority of work on the neuronal specification has been carried out in genetically and physiologically tractable models such as C. elegans, Drosophila ...

发育生物学

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes

We describe a technique for retrograde labeling of motor neurons in Drosophila. We use an oil-dissolved lipophilic dye and deliver a small droplet to an embryonic fillet preparation by a microinjector. Each motor neuron whose membrane is contacted by the droplet can then be rapidly labeled. Individual motor neurons are continuously labeled, enabling fine structural details to be clearly visualized. Given that lipophilic dyes come in various colors, the technique also provides a means to get adjacent neurons labeled in multicolor. This tracing technique is therefore useful for studying neuronal morphogenesis and synaptic connectivity in the motor neuron system of Drosophila.

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes

We describe a method for retrograde tracing of the Drosophila embryonic motor neurons using lipophilic fluorescent dyes.

使用亲脂荧光染料的果蝇胚胎运动神经元的逆行追踪

We describe a technique for retrograde labeling of motor neurons in Drosophila. We use an oil-dissolved lipophilic dye and deliver a small droplet to an ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

在完整的果蝇幼虫体内的亚细胞分辨率成像

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. Furthermore, despite progress in understanding postsynaptic MTs, much less is known about the contributions of presynaptic MTs to neuronal morphogenesis. In particular, studies of in vivo MT dynamics in Drosophila sensory dendrites yielded significant insights into polymer-level behavior. However, the technical and analytical challenges associated with live imaging of the fly neuromuscular junction (NMJ) have limited comparable studies of presynaptic MT dynamics. Moreover, while there are many highly effective software strategies for automated analysis of MT dynamics in vitro and ex vivo, in vivo data often necessitate significant operator input or entirely manual analysis due to inherently inferior signal-to-noise ratio in images and complex cellular morphology. &#160;To address this, this study optimized a new software platform for automated and unbiased in vivo particle detection. Multiparametric analysis of live time-lapse confocal images of EB1-GFP labeled MTs was performed in both dendrites and the NMJ of Drosophila larvae and found striking differences in MT behaviors. MT dynamics were furthermore analyzed following knockdown of the MT-associated protein (MAP) dTACC, a key regulator of Drosophila synapse development, and identified statistically significant changes in MT dynamics compared to wild type. These results demonstrate that this novel strategy for the automated multiparametric analysis of both pre- and postsynaptic MT dynamics at the polymer-level significantly reduces human-in-the-loop criteria. The study furthermore shows the utility of this method in detecting distinct MT behaviors upon dTACC-knockdown, indicating a possible future application for functional screens of factors that regulate MT dynamics in vivo. Future applications of this method may also focus on elucidating cell type and/or compartment-specific MT behaviors, and multicolor correlative imaging of EB1-GFP with other cellular and subcellular markers of interest.&#160;

This study presents a noninvasive intravital neuronal imaging strategy combined with a new software strategy to achieve automated, unbiased tracking and analysis of in vivo microtubule (MT) plus-end dynamics in the sensory dendrites and the neuromuscular junctions of Drosophila.

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. ...

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.

可视化的Live果蝇胶质细胞神经肌肉交界处，与荧光染料

摘要

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此视频中的章节

在果蝇幼虫分部轴突在体内的突触小泡的可视化

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免疫

通过成人角质层可视化果蝇腿运动神经元轴突

使用亲脂荧光染料的果蝇胚胎运动神经元的逆行追踪

在完整的果蝇幼虫体内的亚细胞分辨率成像

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

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

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络

可视化的Live果蝇胶质细胞神经​​肌肉交界处，与荧光染料

摘要

探索更多视频

此视频中的章节

在果蝇幼虫分部轴突在体内的突触小泡的可视化

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免疫

通过成人角质层可视化果蝇腿运动神经元轴突

使用亲脂荧光染料的果蝇胚胎运动神经元的逆行追踪

在完整的果蝇幼虫体内的亚细胞分辨率成像

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

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

使用 果蝇 幼虫神经肌肉接头和肌肉细胞可视化微管网络

可视化的Live果蝇胶质细胞神经肌肉交界处，与荧光染料

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

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络