我们感谢熊莹博士对手稿的讨论和评论。这项工作得到了中国国家科学基金委员会（NSFC）对C.M.（31500839）的资助。

1.幼虫解剖
注意:解剖溶液血淋巴样盐水 （HL3.1）18 和固定溶液 4% 多聚甲醛 （PFA）19,20 在室温下使用，因为当温度过低时微管会解聚。
<ol><li>用长钝镊子挑选一只流浪的 3龄 幼虫。用HL3.1洗涤，并将其放在立体显微镜下的解剖皿上。 注:徘徊的3龄 幼虫由分枝的前螺旋体识别，并在约96小时（25°C）21时在管周围爬行。</li>
	<li>将幼虫的背侧或腹侧朝上放置。通过两个长气管识别背侧，通过腹部齿带识别腹侧。<ol><li>根据要观察的组织，选择背侧或腹侧解剖。这将允许更精确、更详细地了解感兴趣的特定区域。对于NMJ和肌肉细胞观察，分别切开幼虫背侧和腹侧区域。</li>
	</ol></li>
	<li>别住嘴钩和尾巴。调整销钉以保持幼虫处于伸展状态。在幼虫中加入一滴HL3.1，以防止其干燥。 注意:不含 Ca2+ 的 HL 3.1 可用于最大限度地减少解剖过程中肌肉的收缩。</li>
	<li>用解剖剪刀在靠近后端的地方做一个小的横向切口，但不要剪掉后端。然后沿腹侧中线向前端切开。</li>
	<li>在幼虫的四个角上插入四个昆虫针。重新调整昆虫针，使幼虫在各个方向上最大限度地伸展。</li>
	<li>使用镊子切除内脏，同时不损伤肌肉。</li>
</ol>2. 固定
<ol><li>加入 100 μL PFA （4%） 将胴体浸泡 40 分钟，同时胴体仍固定在解剖皿上。 注意: 采取有效的保护措施以避免直接接触皮肤或吸入，因为 PFA 是一种危险。</li>
	<li>卸下针脚并将样品转移到 2 mL 微量离心管中。用含有0.2%Triton X-100（0.2%PBST）的1x磷酸盐缓冲盐水冲洗PFA，以15rpm在脱色振荡器上填充2mL微量离心管10分钟。重复洗涤过程 5 次。</li>
</ol>3. 免疫细胞化学
<ol><li>将胴体浸入封闭剂（5%山羊血清在0.2%PBST中）中，并在室温下封闭40分钟。</li>
	<li>除去封闭剂，并用200μL的一抗（例如，抗α-微管蛋白，1:1000;抗Futsch，1:50）在4°C下用0.2%PBST稀释过夜。通过用单克隆抗α-微管蛋白免疫染色来观察肌肉微管。Anti-Futsch可以间接反映NMJ中的微管形态。</li>
	<li>一抗孵育后，用0.2%PBST洗涤幼虫10分钟，重复5次。</li>
	<li>将幼虫与200μL二抗（例如，山羊抗Mouse-488,1:1000）在室温下用0.2%PBST稀释在黑暗中孵育1.5小时。</li>
	<li>接下来，在黑暗20,22中对微管进行染色时，将浓度为1:1000的核染料如TO-PRO（R）3碘化物（T3605）加入孵育管中30分钟。</li>
	<li>用 0.2% PBST 冲洗掉二抗和核染料 10 分钟。在黑暗中重复 5 次。</li>
</ol>4. 安装
<ol><li>将幼虫胴体置于载玻片上的0.2%PBST中，并在立体显微镜下进行调整。确保幼虫胴体的内表面朝上，并且所有幼虫胴体都按所需排列。</li>
	<li>用擦拭物吸收多余的PBST溶液，然后轻轻加入一滴抗淬灭封固剂。</li>
	<li>在载玻片上放置盖玻片，缓慢而轻柔地覆盖解剖的幼虫，以避免气泡。</li>
	<li>在盖玻片周围涂抹指甲油。将载玻片放在黑暗空间中以减少荧光衰减。</li>
</ol>5. 图像采集
<ol><li>要获取图像，请使用激光扫描共聚焦显微镜，选择60倍油浸物镜（数值孔径1.42）或类似物镜，并根据实验调整激光功率和波长。</li>
	<li>要识别 NMJ，请在 A3 段的肌肉 4 中捕获图像（如图 1F 中的位置所示）。选择 488 nm 激光以激活 α-微管蛋白或 Futsch，选择 543 nm 以激活 HRP 成像轨迹。将参数调整为 800 像素 x 800 像素的帧大小、2.0 的数码变焦和 0.8 μm 的成像间隔（图 2）。</li>
	<li>为了识别肌肉中的微管，捕获A3-A5段中肌肉2的图像（ 如图1G中的位置所示），因为它具有较少的气管分支。选择 488 nm 激光器激活 α-微管蛋白，选择 635 nm 激光器激活 T3605 成像轨道。将参数调整为1024像素x 1024像素的帧大小，3.0的数码变焦和肌肉细胞中0.4μm的成像间隔（图3）。</li>
</ol>

观察 果蝇 神经肌肉接头和肌肉细胞中的微管网络

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作者没有什么可透露的。

这里描述了用于 果蝇 幼虫神经肌肉接头和肌肉细胞的解剖和免疫染色的方案。有几个要点需要考虑。首先，在解剖过程中，避免观察到的肌肉受伤至关重要。在切除内脏之前，可能值得固定鱼角，以防止镊子和肌肉直接接触。为避免肌肉损伤或与幼虫表皮分离，重要的是要确保在洗涤和孵育过程中振荡器的速度不超过 15 rpm。此外，必须精确控制皮肤延伸，以确保清晰、完整地拍摄 NMJ 形?...

微管（MTs）作为细胞骨架的结构成分之一，在细胞分裂、细胞生长和运动、细胞内运输和维持细胞形状等多种生物过程中发挥着重要作用。微管动力学和功能通过与其他蛋白质的相互作用进行调节，例如 MAP1、MAP2、Tau、Katanin 和驱动蛋白 1,2,3,4,5。
在神经元中，微管对于轴突和树突的发育和维持至关重要。微管异常会导致功能障碍，甚至导致神经元死亡。例如，在阿尔茨海默氏症患者的大脑中，Tau 蛋白过度磷酸化会降低微管网络的稳定性，导致神经系统异常6。因此，检查微管网络将有助于理解神经发育和神经系统疾病的发病机制。
神经肌肉接头（NMJ）是在运动神经元轴突末梢和肌纤维之间形成的外周突触，是研究突触结构和功能的优秀而强大的模型系统7。Futsch 是果蝇中的一种蛋白质，与哺乳动物中发现的微管结合蛋白 MAP1B 同源8。它仅在神经元中表达，并在 NMJ 突触按钮的发育中发挥作用 8,9。在野生型中，通过用抗Futsch进行免疫染色来观察沿NMJ过程中心运行的丝状束。当到达 NMJ 的末端时，该束能够形成由微管组成的环或失去其丝状结构，从而导致弥漫性和点状外观10。微管环与暂停的生长锥有关，这表明微管阵列是稳定的11。因此，我们可以通过 Futsch 染色间接确定 NMJ 中微管的稳定发育。果蝇幼虫中大尺寸的肌肉细胞可以清晰地观察微管网络。通过分析微管的密度和形状，可以发现影响微管网络稳定性的因素。同时，可以将肌肉细胞的微管网络状态与NMJ的结果进行交叉验证，以获得更全面的结论。
许多协议已被用于研究微管的网络和动力学。然而，这些研究通常集中在体外研究12,13,14,15,16。或者，一些体内实验已经使用电子显微镜来检测细胞骨架17。根据荧光标记的抗体或化学染料与蛋白质或DNA的特异性结合，这里介绍的方法允许在体内单个神经元水平上检测NMJ中的微管网络，结果通过肌肉细胞的观察得到证实。当与果蝇黑腹果蝇中可用的强大遗传工具结合使用时，该方案是简单、稳定和可重复的，能够对体内神经系统中微管网络调节蛋白的作用进行各种表型检查和遗传筛选。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor Plus 405 phalloidin</td>
			<td>invitrogen</td>
			<td>A30104</td>
			<td>dilute 1:200</td>
		</tr>
		<tr>
			<td>Enhanced Antifade Mounting Medium</td>
			<td>Beyotime</td>
			<td>P0128M</td>
			<td></td>
		</tr>
		<tr>
			<td>FV10-ASW confocal microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse antibody, Alexa Fluor 488 conjugated</td>
			<td>Thermo Fisher</td>
			<td>A-11001</td>
			<td>dilute 1:1,000</td>
		</tr>
		<tr>
			<td>Laser confocal microscope LSM 710</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Scissors</td>
			<td>66vision</td>
			<td>54138B</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-Futsch antibody</td>
			<td>Developmental Studies Hybridoma Bank&#160;&#160;</td>
			<td>22C10</td>
			<td>dilute 1:50</td>
		</tr>
		<tr>
			<td>Mouse anti-&#945;-tubulin antibody</td>
			<td>Sigma</td>
			<td>T5168</td>
			<td>dilute 1:1,000</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Wako</td>
			<td>168-20955</td>
			<td>Final concentration: 4% in PB Buffer</td>
		</tr>
		<tr>
			<td>Stainless Steel Minutien Pins</td>
			<td>Entomoravia</td>
			<td></td>
			<td>0.1mm Diam</td>
		</tr>
		<tr>
			<td>Stereomicroscope SMZ161</td>
			<td>Motic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>stereoscopic fluorescence microscope BX41</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Texas Red-conjugated goat anti-HRP</td>
			<td>Jackson ImmunoResearch</td>
			<td></td>
			<td>dilute 1:100</td>
		</tr>
		<tr>
			<td>TO-PRO(R) 3 iodide</td>
			<td>Invitrogen</td>
			<td>T3605</td>
			<td>dilute 1:1,000</td>
		</tr>
		<tr>
			<td>Transfer decoloring shaker TS-8</td>
			<td>Kylin-Bell lab instruments</td>
			<td>E0018</td>
			<td></td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>BioFroxx</td>
			<td>1139</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers&#160;</td>
			<td>dumont</td>
			<td>500342</td>
			<td></td>
		</tr>
	</tbody>
</table>

using drosophila larval neuromuscular junction and muscle cells to visualize microtubule network

微管网络是神经系统的重要组成部分。许多微管调节蛋白的突变与神经发育障碍和神经系统疾病有关，如微管相关蛋白Tau对神经退行性疾病，微管切断蛋白Spastin和Katanin 60分别引起遗传性痉挛性截瘫和神经发育异常。检测神经元中的微管网络有利于阐明神经系统疾病的发病机制。然而，神经元的小尺寸和轴突微管束的密集排列使得微管网络的可视化具有挑战性。在这项研究中，我们描述了一种解剖幼虫神经肌肉接头和肌肉细胞的方法，以及α-微管蛋白和微管相关蛋白Futsch的免疫染色，以可视化果蝇中的微管网络。神经肌肉接头使我们能够观察突触前和突触后的微管，果蝇幼虫中大尺寸的肌肉细胞可以清晰地观察微管网络。在这里，通过在黑腹果蝇中突变和过表达Katanin 60，然后检查神经肌肉接头和肌肉细胞中的微管网络，我们准确地揭示了Katanin 60在神经发育中的调节作用。因此，结合果蝇黑腹果蝇强大的遗传工具，该方案极大地促进了微管网络调节蛋白在神经系统中的作用的遗传筛选和微管动力学分析。

我们演示了可视化神经肌肉接头 （NMJ） 和肌肉细胞中微管网络的分步程序。根据示意图（图1A-E）解剖后，进行免疫染色，随后在激光共聚焦显微镜或立体荧光显微镜下观察和收集图像（图1F，G）。
NMJ的突触前后微管组织都可以用抗α微管蛋白标记，通过选择激光扫描共聚焦显微镜的层厚，可以显示不同?...

Watch this Scientific Journal Video about Understanding Microtubule Network in Drosophila Neuromuscular Junctions at JoVE.com

作者聚焦:了解果蝇神经肌肉接头中的微管网络 

在这里，我们提出了一个详细的方案来可视化神经肌肉接头和肌肉细胞中的微管网络。结合 黑腹果蝇强大的遗传工具，该方案极大地促进了微管网络调节蛋白在神经系统中的作用的遗传筛选和微管动力学分析。

使用 果蝇 幼虫神经肌肉接头和肌肉细胞可视化微管网络

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with ...

目前，微管动力学是通过微管结合蛋白（如EB1）间接观察到的。由于微管蛋白异二聚体在细胞质中含量丰富，直接折叠，相邻的微管蛋白异二聚体缺乏体内微管网络的动力学。我们的方案可视化了神经肌肉突触和肌肉细胞中的微管。当与黑腹果蝇中可用的强大遗传工具相结合时，可以进行各种表型检查和遗传筛选，以发现微管网络调节蛋白在体内神经系统中的作用。我们的研究重点是细胞骨架与神经发育之间的关系，以及它对神经系统疾病的影响。我们希望通过强调细胞骨架调节蛋白对神经元的影响来发现神经发育和神经系统疾病孤雌生殖的机制。

using-drosophila-larval-neuromuscular-junction-muscle-cells-to

Research

JoVE Journal

Neuroscience

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

1.9K 次观看.  Hubei University. 目前，微管动力学是通过微管结合蛋白（如EB1）间接观察到的。由于微管蛋白异二聚体在细胞质中含量丰富，直接折叠，相邻的微管蛋白异二聚体缺乏体内微管网络的动力学。我们的方案可视化了神经肌肉突触和肌肉细胞中的微管。当与黑腹果蝇中可用的强大遗传工具相结合时，可以进行各种表型检查和遗传筛选，以发现微管网络调节蛋白在体内神经系统中的作用。我?...

视频: 作者聚焦:了解果蝇神经肌肉接头中的微管网络 

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

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 ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

用线性琼脂糖渠道研究<em&gt;果蝇</em&gt;幼虫爬行行为

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

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 ...

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