描述该项目是从国家儿童健康和人类发展研究所的支持标记的内容完全是作者的责任，并不一定代表的国家儿童健康和人类发展或研究所的官方意见，赠款数R01HD050725美国国立卫生研究院。我们感谢他们的手稿和视频输入Godenschwege实验室以及芭芭拉Schreader成员。

1。腹神经索的剖析可在室温下进行解剖和染料的GF灌装。对于所有清扫步骤冷（4℃）生理盐水应使用减慢细胞代谢和维持神经元存活更长的时间。 <ol><li>麻醉与CO 2成蝇（4天或以上），或阴寒他们，直到他们被固定在冰上。麻醉用冰块将需要更多的时间（最多30分钟），但将保持更长的时间固定的苍蝇。 </li><li>放置一个飞在Sylgard涂层的培养皿。剥离需要使用高倍率（80 - 100X）夹层体视显微镜。 </li><li> Vannas剪刀，删除的腿，喙，和翅膀。 </li><li>使用粗钳和罚款昆虫引脚（minutien引脚）到东方飞背侧Sylgard菜。第一脚的腹部，然后放在颈部两侧的两个引脚，后面的头部，使颈部​​轻微伸和访问（见图2）动物略有拉长。 </li><li>淹没飞鲜，冷的果蝇生理盐水。最常用的果蝇salines足够的清扫，但是在这里我们使用谷和奥多德生理盐水，2006 年 4 。气泡环绕飞行。取出用注射器中的空气。 </li><li>到腹部的背端短，外侧切口。在切口开始，沿背中线的腹部朝向胸部浅，直切。然后继续在胸部浅，尽可能准确的纵向切割，沿中线走向的结缔组织。这是至关重要的，使削减浅，以不损害VNC（图1）。 </li><li>使用两个细虫针小心地打开胸廓。传播除了身体一半使用的引脚和地方引脚通过飞行肌（图2）。胸部不应该进一步开放，描绘在图2，以避免角质层翻录。为了确保直线方向的VNC引脚应放置在相同的前每边后位。 </li><li>染料注射和适当的成像到VNC可能会妨碍内脏器官。因此，使用镊子和剪刀除去胃，肠，心脏，生殖器官。拉出的VNC双方唾液腺躺在。仔细去除浮动的组织和脂肪的新鲜生理盐水冲洗剥离。 </li><li>删除从下面的宫颈结缔组织（CVC）脂肪体。肥胖的身体是最好的高倍率可见。用细镊子，要认真把握组织从侧面，并将其拉出。 </li></ol> 5至10分钟内，可以进行剥离。通过使用新鲜，冷盐水的解剖，完好的神经系统将仍然活着的至少一个小时。 2。细胞内的绿表填写这是推荐的夹层填充后尽快执行染料。 <ol><li> Sylgard菜放在一个固定的阶段（图3）和10倍的空中目标的直立显微镜下解剖飞。中心对染料填充电极支架定位飞后结束的样品。使用的CVC微分干涉对比（DIC）的模式和重点。政府飞行服务队的大轴突应CVC背表面清晰可见。 </li><li>在去离子水的浓度为1％稀释路西法黄河CH达丽星盐。拉硼硅玻璃电极丝电阻约。 60-80MΩ。玻璃电极的尖端，应该是长期的和尖锐的。电极尖端填充结束对面放置电极，电极尖端到路西法黄色溶液约1％路西法黄色溶液。 30-60秒。然后回填3M氯化锂与电极使用汉密尔顿注射器留下的染料和LiCl之间的气泡。连接染料填充电极头阶段。 </li><li>氯涂层银线接地电极插入生理盐水和地方染料填充电极上面的飞并行使用3D液压显微到VNC。应连接到放大器，允许为了传递电流从电极释放消极或积极收取染料通过超极化电流，分别染料填充电极。 </li><li>切换到40倍的水浸泡镜片和染料填充电极的尖端降低到政府飞行服务队之一。 GF轴突奠定接近的背水面的CVC。使用向前（向头部方向）运动的GF插入电极。 </li><li>更改您的啶光的光源，并切换到路西法黄河滤波器。应用到染料填充电极极化与放大器注入了GF路西法黄河电流。染料注入量可以控制日E的电流的大小，以及通过的电流通过时间。为了防止蔓延的注射部位或损坏的GF染料，它是推荐使用超过1-5分钟，低电流（约3-5 NA）注入。 </li><li>应用电流，直到在VNC的GF终端可以清楚地看到（图4A）。对于跨突触的突触后神经元的染色，继续传递，直到染料也可以看到在突触后神经元的电流。关闭当前和删除的染料填充电极。 </li></ol> 3。代表性的成果： 代表染料之一玻纤填充路西法黄河如图4A所示。作为尼康Elements软件的形象堆栈使用现场摄像头安装在显微镜上的图像被收购。在此示例中电流通过电极约2分钟。彻底填补了GF，在胸部的大型玻纤终端以及标记（图4A，椭圆形）。没有突触后神经元可见，在这种情况下，尽管路西法黄河是小到足以通过缝隙连接。如果染料是没有注入足够长的GF然后跨突触的突触后神经元的填充可能不会被看到，因为被检测到的信号太弱。政府飞行服务队（图4B，箭头）神经元突触后可以可视化，更可靠的使用更小的神经示踪剂，如Neurobiotin。在2米，为2分钟的去极化电流的醋酸钾溶液注入的7％Neurobiotin，是足够标签突触后神经元的强烈。 Neurobiotin可以使用耦合到不同波长的染料的链霉亲和的共聚焦显微镜观察。由于Neurobiotin是不是一种荧光染料，罗丹明-葡聚糖或Alexa的肼在同一解决方案的合作注入，可用于监测一个成功的注入到GF（图4C，从Boerner和Godenschwege，2010年5月） 。 <img src="/files/ftp_upload/3080/3080fig1.jpg" alt="图1" /> 图1原理图绘制一只苍蝇横向观看。胸部是显示一纵形切口，以可视化的内部结构。在清扫，背纵向飞行肌（DLMS，蓝）不损害腹神经索（VNC，深红色），位于腹侧的飞行肌肉被切断。 （绿色）肠道在于上面的VNC。 <img src="/files/ftp_upload/3080/3080fig2.jpg" alt="图2" /> 图2：一个解剖飞背视图。两个引脚放在头后面的位置飞。通过飞行肌（DLM），贴引脚按住胸部打开。内部器官被拆除，露出腹侧神经线（VNC）。 <img src="/files/ftp_upload/3080/3080fig3.jpg" alt="图3" /> 图3。染料填充设置。 <img src="/files/ftp_upload/3080/3080fig4.jpg" alt="图4" /> 图4。）Z轴投影显示玻纤填充与路西法黄河的部分后的VNC 。图像被收购后，立即染料与现场摄像头灌装。在野生型动物的终端（椭圆）在胸部清晰可见。二）一个GF染料的共聚焦图像填充Neurobiotin在野生型飞。政府飞行服务队（箭头）和政府飞行服务队（箭头元首）的神经元突触后标记。 Neurobiotin是可视化与链霉亲和耦合，以Cy3标记。三）野生型政府飞行服务队共聚焦图像充满Alexa555。

<ol>
	<li>Power, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thoracico-abdominal+nervous+system+of+an+adult+insect+Drosophila+melanogaster.">The thoracico-abdominal nervous system of an adult insect Drosophila melanogaster.</a> J. Comp. Neurol. 88, 347-409 (1948).</li><li>Allen, M. J., Godenschwege, T. A., Tanouye, T. A., Phelan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+an+escape:+development+and+function+of+the+Drosophila+giant+fibre+system.">Making an escape: development and function of the Drosophila giant fibre system.</a> Semin. Cell Dev. Biol. 17, 31-41 (2006).</li><li>Boerner, J., Duch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Average+shape+standard+atlas+for+the+adult+Drosophila+ventral+nerve+cord.">Average shape standard atlas for the adult Drosophila ventral nerve cord.</a> J. Comp. Neurol. 518, 2437-2455 (2010).</li><li>Gu, H., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+synaptic+transmission+in+adult+Drosophila+Kenyon+cells+in+sity.">Cholinergic synaptic transmission in adult Drosophila Kenyon cells in sity.</a> J. Neurosci. 26, 265-272 (2006).</li><li>Boerner, J., Godenschwege, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+for+the+Drosophila+ventral+nerve+cord+standard+in+neuronal+circuit+reconstruction+and+in-depth+analysis+of+mutant+morphology.">Application for the Drosophila ventral nerve cord standard in neuronal circuit reconstruction and in-depth analysis of mutant morphology.</a> J. Neurogent. 24, 158-1567 (2010).</li><li>Godenschwege, T. A., Kristiansen, L. V., Uthaman, S. B., Hortsch, M., Murphey, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conderved+role+for+Drosophila+Neuroglian+and+human+L1-CAM+in+central-synapse+formation.">A conderved role for Drosophila Neuroglian and human L1-CAM in central-synapse formation.</a> Curr. Biol. 16, 12-23 (2006).</li><li>Uthaman, S. B., Godenschwege, T. A., Murphey, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechanism+distinct+from+highwire+for+the+Drosophila+ubiquitin+conjugase+bendless+in+synaptic+growth+and+maturation.">A mechanism distinct from highwire for the Drosophila ubiquitin conjugase bendless in synaptic growth and maturation.</a> J. Neurosci. 28, 8615-8623 (2008).</li><li>Storkebaum, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dominant+mutations+in+the+tyrosyl-tRNA+synthetase+gene+recapitulate+in+Drosophila+features+of+human+Charcot-Marie-Tooth+neuropathy.">Dominant mutations in the tyrosyl-tRNA synthetase gene recapitulate in Drosophila features of human Charcot-Marie-Tooth neuropathy.</a> Proc. Natl. Acad. Sci. USA. 106, 11782-11787 (2009).</li><li>Allen, M. J., Godenschwege, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+recordings+from+the+Drosophila+giant+fiber+system+(GFS).">Electrophysiological recordings from the Drosophila giant fiber system (GFS).</a> Cold Spring Harb. Protoc. 2010, pdb.prot5453-pdb.prot5453 (2010).</li><li>Augustin, H., Allen, M. J., Partridge, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+Recordings+from+the+Giant+Fiber+Pathway+of+D.+melanogaster.">Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster.</a> J. Vis. Exp. , (2011).</li></ol>

没有从果蝇体内提取的神经系统，可以解剖。这有两个好处，第一，夹层导致神经系统的损害不大，并保持连接第二，大多数神经肌肉和感觉器官。表演解剖描述的那样，准备向前伸直政府飞行服务队的染料标记的样品。为方便起见，政府飞行服务队的运动神经元突触后留附着的肌肉。轴突，因此，没有损坏，保持神经元的存活更长的时间。此外，对神经系统的损害是可能扰乱间隙连接功能，从?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td> Sylgard 184有机硅弹性体套件</td><td>道康宁</td><td></td><td></td></tr><tr><td>解剖镜下</td><td> AmScope </td><td> SM - 2TZ </td><td></td></tr><tr><td> Vannas剪刀超细</td><td>学术仪器</td><td> VS1023 </td><td></td></tr><tr><td>杜蒙钳Dumontstar 55 </td><td>精细科学的工具</td><td> 11295-51 </td><td></td></tr><tr><td>奥斯特利茨昆虫引脚</td><td>精细科学的工具</td><td> 26002-10 </td><td>直径0.1毫米</td></tr><tr><td>硼硅玻璃电极</td><td>世界精密仪器</td><td> 1B100F - 4 </td><td></td></tr><tr><td>垂直移液器拖轮700C </td><td>大卫Kopf仪器</td><td>型号700C </td><td></td></tr><tr><td>路西法黄河CH达丽星盐</td><td>西格玛</td><td> L0259 </td><td> 1％H 2 O </td></tr><tr><td>固定舞台直立显微镜和相机</td><td>尼康</td><td> FN - 1和DS - U2的相机</td><td></td></tr><tr><td>计划Fluor公司10X空气目的</td><td>尼康</td><td>原讼法庭计划Fluor公司10X NA 0.3的WD16毫米</td><td></td></tr><tr><td>福陆公司40X水浸渍目的</td><td>尼康</td><td>原讼法庭福陆公司40X W NA 0.80的WD2.0毫米</td><td></td></tr><tr><td> 3D液压微操作机器人</td><td> Narishige国际</td><td> MW0 - 3型</td><td></td></tr><tr><td>放大器</td><td>入门仪器公司</td><td>型号5A </td><td></td></tr></tbody></table>

whole mount preparation of the adult drosophila ventral nerve cord for giant fiber dye injection

为了分析神经元轴突和树突状的形态，这是必须获得准确的神经元结构的标签。准备以及标记的样品几乎没有组织损伤，使我们能够分析细胞形态和相互比较个别样品，从而使突变异常的识别。 在证明清扫方法的神经系统仍然主要是里面的成人飞。通过背侧切口，腹部和胸部开了，大部分内脏器官都将被删除。只有背侧，腹侧神经线（VNC）和宫颈结缔组织（CVC）的巨型纤维（GFS）1大轴突外露，而大脑含有的GF胞体和树突仍然完整的头2 。在这个准备，最神经的VNC应保持连接到他们的肌肉。 剥离后，细胞内的巨型纤维（GF）用荧光染料填充证明。 CVC GF轴突位于背表面，从而可以很容易地与微分干涉对比（DIC）的光学显微镜下可视化。这使得在此网站与染料的GF轴突注射标签VNC的轴突和他们的终端包括整个的GF。该方法结果可靠和强大的政府飞行服务队的允许填充啶显微镜后，立即进行成像神经元染色。另外，使用的标准的免疫组织化学程序 3适用于高分辨率的共聚焦显微镜，荧光信号可增强。

Watch this Scientific Journal Video about Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection at JoVE.com

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

一个<em&gt;在体内</em&gt;成人解剖<em&gt;果蝇</em&gt;腹神经索（VNC）是证明。这种特殊的解剖方法会导致损伤小到VNC允许的高分辨率成像的荧光染料的巨纤维神经元的后续标签。

成人整装准备果蝇巨纤维染料注射腹侧神经索

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

whole-mount-preparation-adult-drosophila-ventral-nerve-cord-for-giant

Research

JoVE Journal

Neuroscience

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

18.9K 次观看.  Florida Atlantic University. 一个在体内成人解剖果蝇腹神经索（VNC）是证明。这种特殊的解剖方法会导致损伤小到VNC允许的高分辨率成像的荧光染料的巨纤维神经元的后续标签。

视频: Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

在全安装的胚胎神经系统的可视化果蝇胚胎

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

科研

神经科学

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

从巨纤维通路的电生理记录 D。果蝇</em

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

电检测屏幕的配对Nanoinjection和生物活性化合物的使用果蝇巨人纤维系统

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

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 ...

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1).
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however, other options have been discussed as well5.
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.

DII的标签DRG神经元的研究轴突分支在小鼠胚胎脊髓整装准备

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

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 ...

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 ...

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional afferent fibers monitor other characteristics of the muscle environment, including metabolite buildup, temperature, and nociceptive stimuli. Overall, abnormal activation of sensory neurons can lead to movement disorders or chronic pain syndromes. We describe the isolation of the extensor digitorum longus (EDL) muscle and nerve for in vitro study of stretch-evoked afferent responses in the adult mouse. Sensory activity is recorded from the nerve with a suction electrode and individual afferents can be analyzed using spike sorting software. In vitro preparations allow for well controlled studies on sensory afferents without the potential confounds of anesthesia or altered muscle perfusion. Here we describe a protocol to identify and test the response of muscle spindle afferents to stretch. Importantly, this preparation also supports the study of other subtypes of muscle afferents, response properties following drug application and the incorporation of powerful genetic approaches and disease models in mice.

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons are involved in proprioceptor signaling and also report on metabolic state and injury related events. We describe an adult mouse in vitro muscle-nerve preparation for studies on stretch-activated muscle afferents.

一<em&gt;在体外</em&gt;成年小鼠肌肉，神经准备留学肌肉传入放电的放电特性

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional ...

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 ...

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to network properties. We show how to prepare viable oblique slices of the spinal cord of young mice (P2 - P11). In this preparation, the motoneurons retain&#160;their axons coming out from the ventral roots of the&#160;spinal cord. Stimulation of these axons elicits back-propagating action potentials invading the motoneuron&#160;somas and exciting the motoneuron&#160;collaterals within the spinal cord. Recording of antidromic action potentials is an immediate, definitive and elegant way to characterize motoneuron identity, which surpasses&#160;other identification methods. Furthermore, stimulating the motoneuron collaterals is a simple and reliable way to excite the collateral targets of the motoneurons within the spinal cord, such as other motoneurons or Renshaw cells. In this protocol, we present antidromic recordings from the motoneuron somas as well as Renshaw cell excitation, resulting from ventral root stimulation.

We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

斜脊髓切片用于神经前根刺激的制备

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...