Name: motor nerve transection and time-lapse imaging of glial cell behaviors in live zebrafish
Uploaded: 2013-06-20T13:00:00
Description: Watch this Scientific Journal Video about Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish at JoVE.com

笔者想感谢Kucenas实验室有价值的讨论和仲裁技术，精湛的技术支持。这项工作得到了基金科学与技术卓越的UVA（巨星）（SK）。

1。斑马鱼胚胎的制备和安装消融和实时成像<ol><li>准备股票0.8％的低熔融琼脂糖鸡蛋水。分装成13X 100毫米的一次性文化管和存储在4°C，直到需要。 </li><li>交叉成年斑马鱼含有稳定整合的转基因荧光标记的运动神经元和神经胶质细胞类型的景点。收集斑马鱼胚胎的的蛋水和地方在28.5℃培养箱中培养正确的分期以后3。 </li><li>在受精后约24小时（HPF），去除蛋水，加0.002％，1 - 苯基-2 - ?...

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	<li>Geuna, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+3:+Histology+of+the+peripheral+nerve+and+changes+occurring+during+nerve+regeneration.">Chapter 3: Histology of the peripheral nerve and changes occurring during nerve regeneration.</a> Int. Rev. Neurobiol. 87, 27-46 (2009).</li><li>Hirata, K., Kawabuchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelin+phagocytosis+by+macrophages+and+nonmacrophages+during+Wallerian+degeneration.">Myelin phagocytosis by macrophages and nonmacrophages during Wallerian degeneration.</a> Microsc. Res. Tech. 57, 541-547 (2002).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+Embryonic+Development+of+the+Zebrafish.">Stages of Embryonic Development of the Zebrafish.</a> Developmental Dynamics. 203, 253-310 (1995).</li><li>Martin, S. M., O'Brien, G. S., Portera-Cailliau, C., Sagasti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wallerian+degeneration+of+zebrafish+trigeminal+axons+in+the+skin+is+required+for+regeneration+and+developmental+pruning.">Wallerian degeneration of zebrafish trigeminal axons in the skin is required for regeneration and developmental pruning.</a> Development. 137, 3985-3994 (2010).</li><li>O'Brien, G. S., Rieger, S., Martin, S. M., Cavanaugh, A. M., Portera-Cailliau, C., Sagasti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+axotomy+and+time-lapse+confocal+imaging+in+live+zebrafish+embryos.">Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos.</a> J. Vis. Exp. (24), e1129 (2009).</li><li>Parrinello, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EphB+signaling+directs+peripheral+nerve+regeneration+through+Sox2-dependent+Schwann+cell+sorting.">EphB signaling directs peripheral nerve regeneration through Sox2-dependent Schwann cell sorting.</a> Cell. 143, 145-155 (2010).</li><li>Rosenberg, A. F., Wolman, M. A., Franzini-Armstrong, C., Granato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+nerve-macrophage+interactions+following+peripheral+nerve+injury.">In vivo nerve-macrophage interactions following peripheral nerve injury.</a> J. Neurosci. 32, 3898-3909 (2012).</li><li>Stoll, G., Muller, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+injury,+axonal+degeneration+and+neural+regeneration:+basic+insights.">Nerve injury, axonal degeneration and neural regeneration: basic insights.</a> Brain Pathol. 9, 313-325 (1999).</li><li>Villegas, R., Martin, S. M., O'Donnell, K., Carrillo, S., Sagasti, A., Allende, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+degeneration+and+regeneration+in+developing+zebrafish+peripheral+axons+reveals+a+requirement+for+extrinsic+cell+types.">Dynamics of degeneration and regeneration in developing zebrafish peripheral axons reveals a requirement for extrinsic cell types.</a> Neural Development. 7, 19 (2012).</li><li>Waller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experiments+on+the+section+of+the+glossopharyngeal+and+hypoglossal+nerves+of+the+frog,+and+observations+of+the+alterations+pro-+duced+thereby+in+the+structure+of+their+primitive+fibres.">Experiments on the section of the glossopharyngeal and hypoglossal nerves of the frog, and observations of the alterations pro- duced thereby in the structure of their primitive fibres.</a> Philos. Trans. R. Soc. Lond. , 423-429 (1849).</li></ol>

这个实验的设计最关键的步骤是：1）正确安装幼虫损伤和随后的体内成像和2）激光校准和选择正确的电源设置，以创造一个干净的神经切断，结果最小的额外组织损伤。为了帮助确保成功切断后， 在体内成像和随后的分析，无论是单独的玻璃底菜，或在玻璃底菜分隔多个幼虫安装。激光校准后，我们​​建议测试幼虫上测试不同的电源设置，以确定最佳的参数神经切断。这些设置?...

斑马鱼已被广泛用于研究发展的神经系统，因为它们的光学透明的，易于转基因，耦合时，允许在活胚胎的动态细胞行为的壮观成像。此外，由于斑马鱼和哺乳动物共享几乎所有的神经系统的形成，在这个模型有机体的细胞和分子信息收集所需的基因直接听上去很像其他脊椎动物物种。虽然令人难以置信的强大的神经发育研究，斑马鱼和其独特的属性有潜力也阐明机制，维护和重建神经系统损伤后。斑马鱼幼虫保持到后期幼体阶段和色素沉着，可以有效地阻止黑色素生成抑制剂或缺乏色素细胞的遗传突变体，无论是使用半透明。因此，使用这种模式生物来研究伤害和regenerat离子在老年动物是可能的，并提供独特的机会，直接调查，重建神经系统的细胞和分子机制。在这篇稿件中，我们将介绍如何以高效率和重复性伤害PNS的斑马鱼幼虫的神经。这伤范式本身的研究不仅变性，而且外周神经胶质细胞和免疫细胞的反应，以及这些种群之间的相互作用，在再生过程中。 PNS的是一个复杂的网络是必要的中枢神经系统（CNS）和皮肤，器官和身体肌肉之间传递信息的运动和感觉神经，使生物体与它的环境交互，和生存。沿着这些神经，末梢神经胶质细胞，包括髓鞘化和非髓鞘的雪旺氏细胞和神经胶质细胞神经​​束膜，以及结缔组织，包住的轴突，并最终形成成熟的神经。这些神经损伤启动一个过程known Wallerian变性10。这种机制的轴突碎片，免疫招聘，杂物清理和再生是非常刻板和基因调控1。在哺乳动物系统中的先前的研究已经描述的作用的雪旺氏细胞在神经退化和再生的1，2，6，8。固定组织或细胞培养在这些研究中，雪旺氏细胞不仅招募巨噬细胞的损伤部位，以帮助在废墟的间隙，但也有助于髓鞘细胞吞噬自己。虽然这些研究已经非常翔实，我们有前所未有的可视化实时外围轴突损伤体内的胶质反应，并在这些事件中，没有其他的研究调查了不同类之间的关系外周神经胶质细胞。 最近，几个实验室研究Wallerian变性，利用斑马鱼和类似激光介导的轴突损伤，我们在这里描述的是什么<sup&gt; 4，5，7，9。在一些研究中，浅感觉轴突视神经切断在低龄幼虫使用一个定制的内置，双光子共聚焦显微镜4，5，9。在另一项研究中，这是我们自己非常相似，更深层次内腹侧运动神经轴突横断在5日龄幼虫使用市售的激光消融系统7。在这些实验装置，重点是Wallerian变性和轴突和免疫细胞进行成像。为了扩大在这些研究中，我们描述运动神经轴突受伤的老幼虫更加成熟，有髓神经和检测所有神经相关的外围神经胶质细胞变性和再生过程中的响应。 要做到这一点，我们横切6和7天受精后（DPF）幼虫的运动神经和可视化个别胶质人群的反应以及调查这些人群之间的相互作用，一起受伤的轴突。使用双重和三重TRA标签周围的神经胶质细胞，包括雪旺氏细胞和神经胶质细胞神经​​束膜，以及一个标记为轴突的nsgenic线，可使用市售的激光烧蚀系统组成的氮抽运染料激光器（波长435纳米）连接到一个旋转的圆盘共聚焦系统创建轴突横断。这实验装置，使我们能够想象现场，斑马鱼幼体轴突损伤和他们的关系，伤害特定的外设轴突束和时间推移图像的响应不同的胶质人口彼此。这个协议可以进一步用于建立神经损伤在斑马鱼的不同年龄段，不同的转基因株系或基因突变，以满足不同的科学问题。

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Phenylthiourea</td>
 <td>Sigma</td>
 <td>P7629-100G</td>
 </tr>
 <tr>
 <td>Finquel Tricaine Methanesulfonate MS-222</td>
 <td>Argent Chemical</td>
 <td>C-FINQ-UE-100G</td>
 </tr>
 <tr>
 <td>Low melting point agarose</td>
 <td>Sigma</td>
 <td>A9414-10G</td>
 </tr>
 <tr>
 <td>Quad CELLview Cell Culture Dishes, Glass Bottom, Sterile, Greiner Bio One</td>
 <td>VWR/Greiner</td>
 <td>89125-444</td>
 </tr>
 <tr>
 <td>Single well glass bottom Petri dishes 35 x 10 mm, 12 mm thick</td>
 <td>Willco Wells</td>
 <td><a href="http://www.willcowells.com/en/products/willco-dish-glass-bottom-dishes/gwst-3512/" target="_blank">GWSt-3512</a></td>
 </tr>
 <tr>
 <td>MicroPoint Laser System with all components</td>
 <td>Andor Technology - purchased through Quorum Technologies, Inc.</td>
 <td>2203-SYS</td>
 </tr>
 <tr>
 <td>MicroPoint Laser Courmarin dye (435 nm)</td>
 <td>Andor Technology </td>
 <td>MP-27-435-DYE</td>
 </tr>
 <tr> </tr>
 </tbody>
 </table>

motor nerve transection and time-lapse imaging of glial cell behaviors in live zebrafish

神经系统经常被描述为硬连线的身体组成部分，即使它是相当一致的，刻板的方式对外界刺激的反应的流体器官系统，同时保持令人难以置信的灵活性和可塑性。不同的是中枢神经系统（CNS），周围神经系统（PNS）是能够显著维修，但我们才刚刚开始了解细胞和分子机制，治理这种现象。利用斑马鱼作为模型系统，我们有前所未有的机会，夫妇再生研究在体内成像和基因操纵。是由周围神经轴突周围神经胶质细胞和结缔组织层。髓鞘或无髓鞘的雪旺氏细胞，这是由蜂窝鞘称为神经束膜包裹成一个分册依次的轴突ensheathed通过。受伤之后，成人末梢神经有非凡的能力，雷莫已经受损的轴突碎片和重新受神经支配的目标。要调查PNS中所有的外围神经胶质细胞再生的作用，我们在这里描述使用市售的氮泵浦染料激光axotomize在现场转基因斑马鱼的运动神经轴突横断检测。我们进一步描述的方法对夫妇这些实验时间推移成像的受伤和控制神经。此实验范式可以用来不仅评估的作用，神经胶质细胞发挥神经再生，但也可以是支配神经系统修复的分子机制的阐明平台。

这里所描述的测定法可用于评估在体内性轴索损伤的神经胶质细胞和其他神经相关的细胞群的响应。 电影1显示了一个例子使用该方法和响应周围的神经胶质细胞的神经损伤。进行该实验的Tg（nkx2.2a：megfp）中，Tg的（OLIG2：红色荧光蛋白），斑马鱼，束膜神经胶质细胞表达EGFP和运动神经元表达胞浆中红色荧光蛋白，对象的膜。沿着延髓投影在一个6 DPF直播的斑马鱼的躯干...

Watch this Scientific Journal Video about Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish at JoVE.com

Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish

虽然周围神经系统（PNS）能显着损伤后的修复，治理这种现象的细胞和分子机制知之甚少。使用现场，转基因斑马鱼和一个可重复的神经切断术实验，我们可以研究动态神经胶质细胞在神经变性和再生行为。

电机神经切断和时间推移成像胶质细胞行为在现场斑马鱼

The  nervous system is often described as a hard-wired component of the body even  though it is a considerably fluid organ system that reacts to external ...

Research

JoVE Journal

Neuroscience

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

在哺乳动物的中枢神经系统视神经横断后的实验操作方法

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be ...

科研

神经科学

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

在发展中国家斑马鱼的前脑的神经祖细胞克隆相关的定时实时成像

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Live Imaging of Dorsal Root Axons after Rhizotomy

实时成像背根轴突后根切断术

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

急性片的成年小鼠前脑的神经细胞迁移的延时成像

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

成神经细胞迁移的小鼠急性脑片在体内电穿孔产后和时间推移成像

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Spinal Cord Transection in the Larval Zebrafish

在斑马鱼幼虫脊髓横断

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

老鼠耳蜗外植体的长期时间推移成像

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth

来自胚胎GFP-表达小鼠的Ex Vivo Oculo运动切片培养,用于对Oculo运动神经外生长进行时移成像

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

坐骨神经粉碎损伤大鼠模型中功能评估的3D运动学分析

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...