作者希望感谢阿利斯泰尔Garratt博士（最大德尔布吕克中心，柏林）有益的意见。这项工作是支持的协作研究中心（SFB665）德国研究会（DFG）。

1。解剖过程 注 :小鼠实验使用应遵循正式批准的关心和使用实验动物的指导方针。 <ol><li>前的准备，建立您的解剖显微镜，并打下了清扫，包括大，小剪刀，大齿镊，弯钳和四套杜蒙五号解剖钳（其中有两个内抛光技巧）所需的手术器械（详情参见特定的试剂和设备表）。在100毫米的Sylgard涂层的培养皿放在一张滤纸。倒入冷PBS在12孔板，100毫米的培养皿和12毫升管水井和留在冰上。移液器2毫升固定缓冲区（4％多聚甲醛PBS，pH值7.4），以及在每个第二个12孔板置于冰上。 </li><li>麻醉后，牺牲在E12.5（E0.5指定检测阴道塞）定时怀孕大坝，放置鼠标3张纸巾，用70％乙醇浸泡腹部腹面的一面向上。 </li><li>打开腹部盆腔腔，分离双侧子宫角和转移到冰冷PBS 100毫米的菜肴之一。 </li><li>根据微观控制，使子宫壁与小，直剪刀纵形切口，切离每个胎盘羊膜囊。 </li><li>从每个胚胎羊膜囊剥离，剪断脐带。 </li><li>使用小剪刀，杀头的胚胎和基因组DNA的元首或其中的一部分预留是否应要求基因分型。随后，转让torsi的12孔板，用冷PBS各自的水井。 </li><li>湿的PBS几滴在Sylgard培养皿滤纸和纸张上的位置与胚胎躯干背侧。为了更好地稳定，整顿它的尾巴和四肢远离身体。 </li><li>工作约16X放大倍率解剖显微镜下，仔细地捏皮肤和胚胎脊髓以上的两双细尖镊子轻轻撕除。在中间开始，首先对尾部进行，然后从中间恢复对前侧。 </li><li>湿胚胎不时有两个或三个滴的PBS，以防止它干燥。 </li><li>为了防止，DRG的teared从脊髓时，它是从胚胎中删除分离的背根神经节和周围的软骨脊柱脊髓内抛光技巧罚款镊子刀片横向滑动运动。在胚胎的右侧中间开始，您对尾的方式工作，然后到前结束。之后，重复对方的过程。要小心，不要撕掉关联的DRG。 </li><li>从胚胎分离完全放松的脊髓，拿起其cervic人用细镊子，拉出来一件对尾鳍结束。 </li><li>附加DRG的淹没孤立的脊髓以及在洋溢着固定缓冲区的12孔板进行准备从剩余胚胎的脊髓。 </li><li>修复脊髓冰至少有两个小时。 </li></ol> 2。 DII - DRG神经元的标签<ol><li>对于每一个被标记的脊髓，准备与微管拉拔两个玻璃针。我们使用一个萨特模式的P - 97电极拉马（程序设置:1热= 625，VEL = 35，时间= 175; 2 = 600，VEL = 25时间= 175;热加热，VEL = 630 =。 30，时间= 150）。 </li><li>根据微观控制每个玻璃针的尖端沾在5％（W / V）溶液中乙醇的DII的三至五倍。乙醇的蒸发，创建一个针上一层薄薄的DII晶体。 </li><li>一个放大了约40倍的解剖显微镜下工作，将其V脊髓entral方幻灯片上。使用弹簧剪刀，打开其整个长度的线，通过floorplate切割，让后来安装在一个倒置的开卷模式的准备（见C节）腹侧方。 </li><li>与脊髓背侧幻灯片上的位置。然后手动的方法与玻璃针DII覆盖微观可视化的提示下，仔细皮尔斯每脊髓一侧的第二个DRG的电源线。 DII几乎没有痕迹应在刺穿背根神经节可见，以确保只有少数的神经元的标签。为第二针电源线的另一端​​。针对只有每秒DRG避免来自邻国的DRG，这可能会使个别的轴突的分化标记的轴突预测的重叠。 </li><li>在室温下至少六个小时或4℃过夜，返回脊髓固定缓冲区，并在黑暗中孵育° C至染料DI沿着轴突ffuse质膜内。允许抵押品增长到脊髓的分析，如果需要一个较长的潜伏期（最多两天在4 ° C）。 </li></ol> 3。安装和微观分析<ol><li>以下染料扩散，位置单一脊髓背侧向下一个盖玻片的PBS下降。小心附加DRG的方向正确，横向和不混杂。吸干多余的液体和扁平化，在一个倒置的开卷模式，使用镊子线。 </li><li>安装在显微镜幻灯片，用PBS的准备。 </li><li>为了避免不必要的背景染色荧光标记的轴突预测的微观分析增加，应进行一天的安装。直到然后继续在黑暗中的幻灯片在4 ° C。 </li></ol> 4。代表性的成果: 脊髓鼠标接收传入公关ojections从8对颈椎，胸椎13对，共60脊神经节的腰椎和骶部的DRG 4对5对。一些培训后与脊髓仍然附着的DRG的大部分可以从胚胎中分离下五分钟。此过程适用于隔离附带的DRG从E11.5到E13.5小鼠胚胎脊髓。然而，最好的结果是实现了从E12.5。这里所描述的标签程序示范结果如图3所示。在整个长度的脊髓背根神经节的标签，然后可以用来量化在不同的椎体水平（ 图 4）轴突分支行为。 <img alt="图1" src="/files/ftp_upload/3667/3667fig1.jpg" /> 图1。计划描绘轴突分支的两个主要模式。 （一）分支，可能会导致分叉的生长锥的活动 - 在此间表示 - 以及复杂的终端乔木或（二）抵押物形成的轴突轴（间质性分支）。后一种模式是占主导地位的分支预测皮质和丘脑皮层的轴突6,7类型。 <img alt="图2" src="/files/ftp_upload/3667/3667fig2.jpg" /> 图2 DRG神经元传入的预测显示成胚胎脊髓轴突分支的两种类型:（1）在分叉DREZ的女儿分支络脉间质性分支（2）等待期后的形式轴突第一家分行。 <img alt="图3" src="/files/ftp_upload/3667/3667fig3.jpg" /> 图3小鼠胚胎DRG神经元的单性轴索轨迹的可视化。 （一）附有背根神经节脊髓准备从E12.5小鼠胚胎。 （比例尺，1毫米。）（BD）DII标记的野生型小鼠在增加放大倍率DRG的背视图显示。在B每秒DRG的是labeleD通过DII。荧光图像是倒立，尾鳍是在左，C和D，横向是在底部。在C语言中少数的轴突标记，并在更高的放大倍率的T状分支的存在可以在脊髓DREZ确定。 （比例尺，250微米（二），100微米（C）和50微米（四）。） <img alt="图4" src="/files/ftp_upload/3667/3667fig4.jpg" /> 图4 T形分支，单延髓或尾鳍轮流在野生型和C型利钠肽（CNP）缺陷小鼠在E13.5的量化。计算单个轴突的数字括号内为各基因型的不同主干水平。 C型或R匝 - 仅在尾鳍或延髓方向的增长，分别。 <img alt="图5" src="/files/ftp_upload/3667/3667fig5.jpg" /> 图5一个的cGMP信号通路触发脊髓DREZ感觉轴突分叉。 （a）计划CNP的配体，受体鸟苷酸环化酶Npr2和丝氨酸/苏氨酸激酶在胚胎DRG神经元cGKIα组成的cGMP信号通路。 Npr2从GTP生成cGMP的应CNP的刺激。 （二）投资收益跟踪单在野生型和CNP缺陷小鼠背根神经节神经元的轴突。 （比例尺，25微米）。

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作者什么都没有透露。

刻板的投影模式，轻松结合DII标签使用固定的组织编制，包括轴突分支形成两种类型的附有DRG的一个有利的模型来研究神经轴突分支胚胎脊髓。 DII分钟，使用镀膜玻璃针的应用程序允许 - 背根神经节的体积标签 - DRG神经元的小群体的可视化，从而个人轴突及其分支模式的分析对比。所描述的方法可以进一步改善iontophoretic注射的亲脂性的示踪从而进一步降低标记的神经元 8 。然而，这将是更费时。 DII单个轴突在胚胎脊髓整装准备轨迹标签是在一个cGMP的信号级联，包括配体的建筑噪音许可证，其受体Npr2鉴定，和cGMP依赖性激酶cGKIα -调节一种类型的轴突分支:背根神经节神经元在DREZ 4,5,9-12分叉。打开对比野生型的，在cGMP的信号突变体背根神经节神经元的轴突分叉DREZ（ 图 5），而不是只在一个方向。这些研究也显示，抵押品的形成不是在cGMP的信号表明，这DRG神经元中观察到的分支形成的第二种类型是不同的规管4突变体的影响。 另外，单一的背根神经节轴突的分支行为也可能对DRG 神经元13或含有荧光标记的组合，它允许一个14单个神经元之间的区分只在很小的子集的荧光蛋白的转基因小鼠线进行研究。然而，跨品种的突变小鼠模型的荧光鼠标线分析，必须考虑到。此外，由于使用的荧光蛋白在转基因小鼠线的表达的启动子（Thy1）开始只在产后阶段，使得它很难找出是否观察到的分支表型是胚胎发育过程中或稍后变性而受损的分支形成过程，而不是。相比之下，DII标签的胚胎背根神经节轴突分支发生时的优势，研究当时的轴突分支和其在适当的鼠标突变体的破坏过程。 附有DRG的胚胎脊髓的筹备工作可能会进一步用于其他标签程序（整装原位杂交，LacZ基因的转基因小鼠AP染色），收集脊髓和背根神经节组织RNA隔离，或改编时无菌条件下，组织培养实验和下游应用，如钙成像或电测量。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>体视显微镜STEMI刚果（金） </td><td>蔡司</td><td></td><td></td></tr><tr><td>磷酸盐缓冲液（PBS） </td><td> Biochrom公司</td><td> L182 - 50 </td><td></td></tr><tr><td>多聚甲醛</td><td>默克公司</td><td> 8.18715.1000 </td><td></td></tr><tr><td>标准的手术剪</td><td>精细科学工具</td><td> 14001-13 </td><td></td></tr><tr><td>齿标准钳</td><td>精细科学工具</td><td> 11021-14 </td><td></td></tr><tr><td>超等虹膜剪刀</td><td>精细科学工具</td><td> 14088-10 </td><td></td></tr><tr><td>弯钳</td><td>精细科学工具</td><td> 11003-13 </td><td></td></tr><tr><td>杜蒙五号罚款提示钳</td><td>精细科学工具</td><td> 11254-20 </td><td></td></tr><tr><td>杜蒙5号镜面钳</td><td>精细科学工具</td><td> 11252-23 </td><td></td></tr><tr><td> Vannas，蒂宾根弹簧剪刀</td><td>精细科学工具</td><td> 15008-08 </td><td></td></tr><tr><td>滤纸</td><td> Fisher Scientific则</td><td> FB59041 </td><td></td></tr><tr><td> Sylgard 184 </td><td>世界Precission仪器</td><td> SYLG184 </td><td></td></tr><tr><td> 100毫米的培养皿</td><td>格雷纳</td><td> 663102 </td><td></td></tr><tr><td> 12毫升的聚丙烯管</td><td>卡尔罗斯有限公司</td><td> ECO3.1 &lt;/ TD&gt; <td></td></tr><tr><td> 12孔培养板</td><td>碧迪</td><td> 35-3043 </td><td></td></tr><tr><td>乙醇</td><td>默克公司</td><td> 1.00983.2500 </td><td></td></tr><tr><td>火焰山/布朗微量拉马的P - 97 </td><td>萨特仪器有限公司</td><td></td><td></td></tr><tr><td>硼硅玻璃毛细管</td><td>哈佛仪器</td><td> 30-0066 </td><td></td></tr><tr><td> DII（1,1&#39; -双十八烷基- 3，3,3&#39;，3&#39; -甲基- indocarbocyanine高氯酸盐） </td><td> Sigma - Aldrich公司</td><td> 468495 </td><td></td></tr><tr><td>显微镜载玻片SuperFrost加</td><td>卡尔罗斯有限公司</td><td> H867.1 </td><td></td></tr><tr><td>玻璃盖玻片</td><td>卡尔罗斯有限公司</td><td> 1870.2 </td><td></td&gt; </tr></tbody></table>

dii-labeling of drg neurons to study axonal branching in a whole mount preparation of mouse embryonic spinal cord

在这里，我们提出了一个技术标签成胚胎脊髓DRG神经元的小团体的运动轨迹，通过扩散染色使用的亲脂示踪剂1,1&#39; -双十八烷基- 3，3,3&#39;，3&#39; - tetramethylindocarbocyanine高氯酸盐 （DII）1 。与哪些基因突变的鼠标线的野生型轴索途径的比​​较，使测试的候选蛋白在轴突分支的控制，这是一种在神经系统的布线必要机制的职能作用。轴突分支使单个神经元的连接多个目标，从而提供了并行处理信息的物理基础。可区别于终端树枝状的轴突生长的中间目标地区产生影响。此外，取决于是否从生长锥的活动（分裂或延迟文胸分支的结果，可分为轴突分支形成不同的模式nching），或从萌芽进程中的轴突轴络脉称为间质性分支 2（ 图1） 。 中央从背根神经节神经元的预测提供有用的实验系统研究轴突分支两种类型:当其传入轴突之间达成胚胎天，10至13（E10 - E13）背根进入区（DREZ）脊髓显示一个千篇一律的图案T或Y形分叉。产生两个女儿轴突在延髓或尾鳍的方向，然后进行，分别于背侧缘的线，只有经过等待期络脉从这些干轴突发芽渗透灰质（间质性分支）和项目，以在特定的中继神经元椎板脊髓他们进一步arborize（终端分支）3。 DII追查发现生长锥的脊髓背根进入区，似乎是在公关表明分叉分裂ocess是造成分裂生长锥4（ 图2），然而，其他的选项，以及5讨论。 第一视频演示如何剖析DRG的附加的E12.5小鼠的脊髓。适用于录制后的少量标本的DII DRG的使用玻璃毛细管拉针。孵化步骤后，标记的脊髓是安装作为一个倒置的开卷准备，用荧光显微镜来分析单个轴突。

Watch this Scientific Journal Video about DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord at JoVE.com

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

啮齿类动物脊髓感觉传入到定型预测提供了一个方便的实验系统研究轴突通过追踪单个轴突分支。

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 ...

dii-labeling-drg-neurons-to-study-axonal-branching-whole-mount

Nanyang Technological University

Research

JoVE Journal

Neuroscience

24.8K 次观看.  Max Delbrück Center for Molecular Medicine. 啮齿类动物脊髓感觉传入到定型预测提供了一个方便的实验系统研究轴突通过追踪单个轴突分支。

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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

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 ...

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

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

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

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

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 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 Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

一个<em&gt;离体</em&gt;激光诱导脊髓损伤模型，以评估轴索变性的机制在实时

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

第一次混合胶质细胞培养的成年小鼠脊髓组织准备

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

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 ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

斑马鱼<em&gt;原位</em&gt;脊髓制备用于脊髓感觉和运动神经元的电生理记录

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

胚胎运动神经元轴突投影模式的可视化<em&gt;果蝇</em

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

拉米内切手术和脊髓窗口植入小鼠

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...