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
Summary
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。
Abstract
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' -双十八烷基- 3，3,3'，3' – 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。
Protocol
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。 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?…
Discussion
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…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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）。
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| Stereomicroscope Stemi DRC | Zeiss | ||
| Phosphate-buffered solution (PBS) | Biochrom AG | L182-50 | |
| Paraformaldehyde | Merck | 8.18715.1000 | |
| Standard surgical scissors | Fine Science Tools | 14001-13 | |
| Toothed standard forceps | Fine Science Tools | 11021-14 | |
| Extra fine iris scissors | Fine Science Tools | 14088-10 | |
| Curved forceps | Fine Science Tools | 11003-13 | |
| Dumont No.5 fine tips forceps | Fine Science Tools | 11254-20 | |
| Dumont No.5 mirror finish forceps | Fine Science Tools | 11252-23 | |
| Vannas-Tübingen spring scissors | Fine Science Tools | 15008-08 | |
| Filter paper | Fisher Scientific | FB59041 | |
| Sylgard 184 | World Precission Instruments | SYLG184 | |
| 100-mm Petri dishes | Greiner | 663102 | |
| 12-ml polypropylene tube | Carl Roth GmbH | ECO3.1 | |
| 12-well culture plate | Becton Dickinson | 35-3043 | |
| Ethanol | Merck | 1.00983.2500 | |
| Flaming/Brown micropipette puller P-97 | Sutter Instrument Co. | ||
| Borosilicate glass capillaries | Harvard Apparatus | 30-0066 | |
| DiI (1,1′-Dioctadecyl-3,3,3′,3′-tetramethyl–indocarbocyanine perchlorate) | Sigma-Aldrich | 468495 | |
| Microscope slides SuperFrost Plus | Carl Roth GmbH | H867.1 | |
| Glass cover slips | Carl Roth GmbH | 1870.2 |
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Tags
DiI-labelingDRG NeuronsAxonal BranchingWhole Mount PreparationMouse Embryonic Spinal CordDiffusive StainingLipophilic Tracer11′-dioctadecyl-333′3′-tetramethylindocarbocyanine Perchlorate (DiI)Axonal PathwaysWild-typeMutated Mouse LinesFunctional RoleCandidate ProteinsControl Of Axonal BranchingWiring Of The Nervous SystemParallel Processing Of InformationIntermediate Target RegionsTerminal ArborizationAxonal GrowthGrowth ConeSplitting BranchingDelayed BranchingInterstitial BranchingCentral Projections Of NeuronsDorsal Root Entry Zone (DREZ)Stereotyped Pattern
Citazione di questo articolo
Schmidt, H., Rathjen, F. G. DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord. J. Vis. Exp. (58), e3667, doi:10.3791/3667 (2011).