这项工作得到了加拿大卫生研究所（赠款编号澳门币38040）向R.K.、肌肉萎缩协会（美国）向R.K.、SMA家庭向R.K.和L.M.M、SMA信托基金向T.H.G.和肌肉萎缩运动向T.H.G.提供的赠款的支持。L..M.M是加拿大多发性硬化症学会博士后奖学金的接受者，R.K.是渥太华大学健康研究主席的接受者。

所有程序都应遵守该机构规定的动物护理标准。
1. 从鼠标解剖腹部肌肉
<ol><li>启动前，组成4%的甲醛（PFA）。注意：始终将 PFA 放在烟罩内，并佩戴适当的防护设备。</li>
	<li>通过批准的方法对小鼠实施安乐死。 注： 视频中显示的鼠标因过量的二氧化碳 和颈椎脱位而安乐死。这只老鼠是CD1/C57Bl6混合背景上一只4周大的野生型鼠标。这只老鼠是在渥太华大学的动物设施中从最初购买的老鼠身上培育的。</li>
	<li>在臀部水平的皮肤上做一个初始切口，并在小鼠周围一直切开皮肤。</li>
	<li>剥去皮肤，向上拉，直到你达到前肢的水平。</li>
	<li>切开臀部水平的腹部肌肉，继续切口，直到你到达椎柱。</li>
	<li>此时，开始向上切开肌肉和肋骨，直到到达上肢。</li>
	<li>直截，直到到达另一侧的椎柱。</li>
	<li>向下切，直到到达开始的点。</li>
	<li>从下面释放隔膜（注意不要损坏TVA肌肉），并将其放置在磷酸盐缓冲盐水（PBS）中，配有0.2毫米解剖别针的SYLGARD涂层解剖盘。</li>
	<li>将肋骨笼和菜中相关的肌肉固定在盘子中，表面侧向上，确保肌肉完全伸展。</li>
	<li>倒掉 1 倍 PBS，替换为 4% PFA。</li>
	<li>盖上盖子，在室温下在摇动平台上离开15分钟。</li>
	<li>在室温下用1倍PBS清洗3次10分钟。</li>
</ol>*在这一点上，固定的肌肉可以留在冰箱过夜，然后再进行解剖。
2. TVA 肌肉的隔离
<ol><li>要进行 TVA 肌肉的隔离，在解剖显微镜下，首先切开最后几根肋骨水平的外部斜肌（见 图 1， 了解注释指南）。</li>
	<li>切下直线旁边线阿尔巴（注意不要切通过下面的TVA），直到你达到内部斜肌的开始。</li>
	<li>然后切开，从下面的TVA的上部释放过度 的直肠腹肌 和外部斜面肌肉。</li>
	<li>从TVA肌肉中取出血管和任何过度脂肪。</li>
	<li>从过度拉扯的最后一根肋骨中释放肌肉。</li>
	<li>切开肌肉边缘，取出到包含 PBS 的 24 井板。</li>
</ol>3. TVA 肌肉的免疫荧光标签和显微镜
对于所有后续步骤，使用细尖的移液器取出液体，并将板留在摇动平台上。除非另有说明，否则所有后续步骤均在室温下执行。
<ol><li>在含有 1：1，000 荧光标记的双体毒素的 PBS 中孵化 30 分钟，以标记 NMJ。这可以在黑暗的房间里或箔下完成。</li>
	<li>通过在PBS中添加300微克每口2%的Triton X-100油井，使肌肉渗透，并在摇动平台上离开30分钟。</li>
	<li>在阻塞试剂中孵化（4% BSA，PBS 中 1% 特里顿）30 分钟。</li>
	<li>在 4 °C 过夜中孵育含有主要抗体的阻塞试剂（神经丝 1：100;突触囊蛋白 2，1：250）。</li>
	<li>在1倍PBS中清洗3次。</li>
	<li>在含有 1：250 二次抗体的 PBS 中孵化 2-4 小时。</li>
	<li>在1倍PBS中清洗3次。</li>
	<li>使用足够的荧光安装介质和盖片将肌肉安装到玻璃滑梯上。</li>
	<li>最好使用标准表观显微镜上的双带通滤镜查看幻灯片。</li>
	<li>NMJs 最好使用 z 系列投影在配备至少 40 倍目标的对焦显微镜上进行成像。</li>
</ol>

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作者没有什么可透露的。

在这段视频中，我们详细介绍了从小鼠身上解剖TVA肌肉以及肌肉内NMJs全山免疫荧光标签的协议。我们还提供的数据表明，这种肌肉可用于分析SMA小鼠模型中的神经肌肉结点病理学。
这项技术的成功取决于许多因素。下面概述了一些最常见的问题。首先：免疫化学污渍不良。原因有很多，其中最常见的一个是使用与本协议中列出的试剂不同的试剂。高质量的电子显微镜级 PFA 对于确保良好的染色非常重要，本协议中列出的抗体选择也非常重要。此外，在较老的动物（即>3个月），获得良好的质量染色可能更加困难。这是因为肌肉周围的筋膜厚度增加，外部斜面和 转子之间的 脂肪积累增加。在开始免疫荧光之前，去除脂肪很重要。它可能也有必要剥离一些覆盖肌肉的筋膜，这可能会变厚。有时很难从肌肉中去除筋膜和脂肪，而不会对肌肉纤维造成一些损伤，并破坏内侧的内侧模式。但是，如果仔细执行此技术，则至少可以从 1 岁以下的小鼠身上获得高质量的染色。在幼鼠（即小于3个月大）中，没有必要进行任何肌肉纤维的戏弄或分离。其次：在解剖和染色后很难找到NMJ。这通常是因为解剖没有在最后一根肋骨下面延伸。大多数NMJ位于最后一根肋骨的正下方，因此必须小心确保肌肉的这一部分被纳入解剖。第三：将EO肌肉粘在TVA肌肉上。当个人试图将解剖范围扩展到内部斜面 （IO） 肌肉水平以下时，这通常是一种抱怨。IO 也存在的 TVA 肌肉区域更难分析，因为很难区分哪种肌肉是哪种肌肉。因此，我们通常只是解剖TVA肌肉最优越的部分。在这个水平上，EO 和 TVA 肌肉之间没有粘性，因此这应该不是一个严重的问题。
与血管外肌肉相比，使用TVA肌肉的一个重要障碍是手术操作或注射物质的可及性。这些类型的实验对于研究给定肌肉中的NMJ生理学至关重要。虽然TVA肯定比更常用的肌肉，如 头骨前 或 胃内分，更容易获得，以前的工作已经表明，有可能通过手术损伤的间骨神经18使TVA脱神经。我们最近还使用这种肌肉对全身麻醉下的物质进行局部管理（未公布的数据）。虽然这些实验可以代表一个适度的技术挑战，这项工作表明，他们是可行的，从而扩大这种肌肉的效用分析NMJ在病理和生理操纵。
TVA 肌肉是位于全身的众多薄扁平肌肉之一，可用于对内向模式进行全安装分析。其他肌肉包括一组颅骨肌肉，由来自脑干面部核心的运动神经元内侧，包括 高悬浮器奥里斯龙格斯， 血管优越，和 加法器奥里斯朗格斯，解剖之前已经描述14，19。此外，TVA肌肉周围的肌肉，包括EO，IO和 直肠腹膜，也可以标记和用于NMJ分析。对于小鼠模型中的 NMJ 病理学进行综合分析，必须考虑位于全身的一些肌肉，而不是将分析限制在单个肌肉上。这表现在运动神经元疾病的小鼠模型中，其中不同肌肉之间NMJ病理学水平存在显著的异质性。这种肌肉间变异性是研究运动神经元脆弱性机制时极其有价值的工具，因此将分析限制在单个肌肉上会显著降低研究的潜力。

神经肌肉结 （NMJs） 是下运动神经元和骨骼肌纤维之间的突触连接。他们传统上被认为是一个三方突触，由神经元（先天性末梢），肌肉纤维（后突触终端）和终端施万细胞1组成。NMJs似乎是早期和重要的病理学目标在一系列运动神经元疾病和小鼠模型2，3。典型的症状包括衰减，其中运动端板变得缺乏先天性内向，前突末端肿胀，以及NMJ形态4-11的复杂性降低。补偿性响应也可以注意到，其中包括终端和节点发芽，其中轴突过程从剩余的突触终端或内分节延伸到重新插入的解压端板12，13。由于突触活性和NMJ形态学之间的紧密相关性，从NMJ形态学分析中可以获得大量有关运动神经元的功能状态的信息。由于NMJ的丧失经常是神经肌肉病理学4，10的最初方面之一，内在程度的定量可以提供有关病理学进展和治疗干预的潜在效果的重要信息。此外，由于NMJ损失是病理进展的重要一步，开发能够稳定联系和鼓励再生的治疗方法可能会产生显著的好处。
在分析NMJ形态时，肌肉选择非常重要。一些主要考虑因素可能包括肌肉纤维类型、身体状况和与人类状况的比较分析。此外，如果需要注射物质或创伤性神经损伤等操作，实验性可及性也很重要。一般来说，最好分析一系列肌肉定位在整个身体反映了一系列运动单位亚型。然而，肌肉选择往往受到解剖方便的影响。因此，NMJ分析通常只对胃内膜等大型血管肌肉进行。为了在这种肌肉中获得良好的 NMJ 染色，通常需要断断或机械破坏肌肉纤维。因此，内向模式可能中断，对内向模式、发芽和衰减的全面和高质量的分析往往受到损害。另一种方法是使用不需要分割的薄扁平肌肉，可以完整地染色和安装，从而全面概述肌肉的整个内脏。有许多肌肉可用于这样的分析，包括一组颅肌，（包括悬浮器奥里斯龙格斯，血管优越，和加法器奥里斯朗格斯）14，胸肌（如.三角形斯特尼）15， 和腹部 （如横向腹部肌 （TVA） 肌肉。使用这种肌肉的主要障碍是解剖这些肌肉而不需要任何损伤所需的技术专长。
在这段视频中，我们提供了一个协议，从小鼠身上解剖和执行TVA肌肉的免疫荧光标记，以便全面分析内向模式和NMJ形态。TVA肌肉是一种以缓慢抽搐为主的肌肉，由腹部肌肉的最深处组成，内侧由下层间神经组成。先前的工作已经表明，它一直非常容易受到病理学在一些小鼠模型的儿童运动神经元疾病脊髓肌肉萎缩（SMA）和其他小鼠模型的早期发病运动神经元退化4，16。因此，我们建议TVA是进行NMJ分析外周神经病变的有用肌肉。

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			<td height="21" style="height:21px;width:316px;">Paraformaldehyde Aqueous Solution (16% )</td>
			<td style="width:259px;">Electron Micropscopy Sciences</td>
			<td style="width:216px;">15700</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Dumont #5 Forceps</td>
			<td style="width:259px;">Fine Science Tools</td>
			<td>11251-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Angled Sprung Scissors</td>
			<td style="width:259px;">Fine Science Tools</td>
			<td>15006-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Fine Scissors - ToughCut</td>
			<td style="width:259px;">Fine Science Tools</td>
			<td>14058-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">SYLGARD 184 Silicone Elastomer Kit</td>
			<td style="width:259px;">Dow Corning</td>
			<td style="width:216px;">dependant on local supplier</td>
			<td>Use this to make dissection dish for pinning out muscle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Minutien Pins</td>
			<td style="width:259px;">Fine science tools</td>
			<td>26002-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">&#945;-Bungarotoxin, Alexa Fluor 488 Conjugate</td>
			<td style="width:259px;">Invitrogen</td>
			<td style="width:216px;">B-13422</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Albumin from Bovine Serum</td>
			<td style="width:259px;">Sigma Aldrich</td>
			<td style="width:216px;">A4503</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:316px;">Neurofilament Primary antibody (2H3), Supernatant</td>
			<td style="width:259px;">Developmental Studies Hybridoma Bank</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:316px;">SV2 Primary antibody (SV2), Supernatant</td>
			<td style="width:259px;">Developmental Studies Hybridoma Bank</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Mouse IgG (H+L)</td>
			<td style="width:259px;">Jackson ImmunoResearch</td>
			<td>115-166-003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Fluorescence Mounting Medium</td>
			<td style="width:259px;">Dako</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Slides (Superfrost Plus; White)</td>
			<td style="width:259px;">Fisher</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Coverslips</td>
			<td style="width:259px;">Fisher</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Triton X-100</td>
			<td style="width:259px;">Sigma Aldrich</td>
			<td style="width:216px;">T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">CD1/C57Bl6 mouse</td>
			<td style="width:259px;">Jackson Labs</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

dissection of the transversus abdominis muscle for whole-mount neuromuscular junction analysis

神经肌肉结点形态分析可以对给定运动神经元的生理状态有重要的洞察力。与传统上使用的较厚的肌肉（如后肢肌肉）的肌肉（例如） 胃肠）。细小的肌肉可以全面概述给定肌肉的整个内向模式，这反过来又允许识别有选择地脆弱的运动神经元池。这些肌肉还允许分析参数，如运动单元大小、轴向分支和末端/节点发芽。使用这种肌肉的一个常见障碍是获得技术专长来解剖它们。在这段视频中，我们详细介绍了解剖幼鼠 跨性别abdominis （TVA） 肌肉和进行免疫荧光以可视化轴突和神经肌肉结 （NMJs） 的协议。我们证明，这项技术提供了TVA肌肉内向模式的完整概述，并可用于研究NMJ病理学在小鼠模型的儿童运动神经元疾病，脊髓肌肉萎缩。

上述协议指导 TVA 肌肉的隔离和染色，用于 NMJ 分析。这允许对肌肉内向模式进行全安装分析，以及对 NMJ 形态的高分辨率分析 （图2）。该技术可成功应用于SMA4，17（图3）等运动神经元疾病的小鼠模型中揭示NMJ病理学。在SMA的小鼠模型中，病理学存在显著的肌肉内变异性，但TVA肌肉一直受到严重影响。这可以通过运动端板的衰减、前突末梢神经丝的积累和终端发芽（图3）来证明这一点。由此可见，上述技术是全面概述小鼠模型NMJ病理学的有力方法。
<img alt="Figure 1" fo:content-width="4in" fo:src="/files/ftp_upload/51162/51162fig1highres.jpg" src="/files/ftp_upload/51162/51162fig1.jpg" width="600px"> 图1。小鼠腹部肌肉概览。 （A） 图像显示胸腔笼与附着腹部肌肉，已被从最近安乐死的老鼠，并固定在解剖盘表面侧向上（A）。（B） A 中的解剖已注释，以标记腹部肌肉的大致边界。表面腹部肌肉可以在解剖的左侧看到，包括外部斜面（蓝色勾勒）和 直肠 abdominis（ 红色勾勒）。在解剖的右侧，表面肌肉已被移除，允许可视化 横向腹部 肌肉（以绿色勾勒）和内部斜肌（黄色勾勒）。此协议的目的是直接解剖 TVA 肌肉的上部，如实绿色三角形所示。 <a href="https://www.jove.com/files/ftp_upload/51162/51162fig1highres.jpg" target="_blank">单击此处查看更大的图像</a>。
<img alt="Figure 2" fo:content-width="4in" fo:src="/files/ftp_upload/51162/51162fig2highres.jpg" src="/files/ftp_upload/51162/51162fig2.jpg" width="600px"> 图2。TVA 肌肉神经肌肉结点的全安装概述。图像显示的例子NMJs从TVA肌肉可视化与免疫荧光染色神经丝 （NF; 绿色）， 突触囊蛋白 2 （SV2; 绿色） 和双体毒素 （BTX; 红色）.图像是蒙太格荧光显微图，显示整个肌肉（A）或对焦图像，显示组（B）或单独（C）NMJs. 比例杆 = 800μm （A）、70μm（B）、25μm（C）。<a href="https://www.jove.com/files/ftp_upload/51162/51162fig2highres.jpg" target="_blank">单击此处查看更大的图像</a>。
<img alt="Figure 1" fo:content-width="4in" fo:src="/files/ftp_upload/51162/51162fig3highres.jpg" src="/files/ftp_upload/51162/51162fig3.jpg" width="600px"> 图3。TVA肌肉中的神经肌肉结路病理学来自SMA的小鼠模型。对焦显微图显示来自TVA肌肉的NMJ，从控制（Smn2B/+）或SMA鼠标模型（Smn2B/-）中可视化，用于神经丝质（NF;绿色）、突触囊蛋白2（SV2;绿色）和双体糖毒素（BTX;红色）。请注意，虽然在对照小鼠中可以观察到正常的 NMJ 形态，但在 Smn2B/- 小鼠的 TVA 肌肉中，有充分解热（白色箭头）、部分衰减（紫色箭头）终端发芽（蓝色箭头）和前突状肿胀（黄色箭头）的证据。后突触端板也不太复杂，反映了一个明显不太成熟的表型。缩放条 = 50μm. <a href="https://www.jove.com/files/ftp_upload/51162/51162fig3highres.jpg" target="_blank">单击此处查看更大的图像</a>。

Watch this Scientific Journal Video about Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis at JoVE.com

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

在这段视频中，我们演示了解剖小鼠 跨性别腹肌 的方案，并使用免疫荧光和显微镜来可视化神经肌肉结。

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 ...

dissection-transversus-abdominis-muscle-for-whole-mount-neuromuscular

Research

JoVE Journal

Neuroscience

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

11.4K 次观看.  Ottawa Hospital Research Institute. 在这段视频中，我们演示了解剖小鼠 跨性别腹肌 的方案，并使用免疫荧光和显微镜来可视化神经肌肉结。

视频: Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

解剖鼠标眼的视网膜色素上皮的全山

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

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 ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

重复性小鼠坐骨神经挤压和再生的后续评估整装肌肉分析

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

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 ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

全山解剖和成年小鼠耳蜗的免疫

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

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 ...

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 ...

全山神经肌肉交界分析的 横向解剖阿布多米尼斯 肌肉

全山神经肌肉交界分析的横向解剖阿布多米尼斯肌肉