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 - 硫脲嘧啶（PTU）蛋水。回到胚胎的孵化器。 </li><li>在24和96 HPF，胚胎筛选存在的荧光解剖范围所需的转基因。将所选择的胚胎在新鲜的的PTU蛋水和返回的孵化器。 </li><li>当幼虫已经达到了6天受精后（DPF）（或所需岁），他们从孵化器中删除选择几个幼虫安装，和他们转移到一个较小的菜。 </li><li>去除水中的从盘，并立即更换PTU约0.02％Tricane在蛋水。允许幼虫坐在麻醉约5分钟。 </li><li>的等分试样放置在烧杯中，用自来水和微波炉的0.8％低熔融琼脂糖，持续30秒，或直到琼脂糖熔化。允许冷却烧杯，直到琼脂糖文化管的触摸感觉不冷不热（不烫手）。 </li><li>选择麻醉幼虫，并将其转移到单层或多层以及35毫米的玻璃底菜。取出所有的水被转移的幼虫，然后立即盖上的幼虫有足够的温暖琼脂糖填补了玻璃底部分的菜，但不能创建一个大圆顶。 </li><li>正如琼脂糖变硬，用解剖针的位置在其一侧的幼虫在底部的菜，然后在它的后面稍稍倾斜的幼虫。当务之急是受伤和随后的成像，安装的幼虫接触玻璃底部的菜。用解剖针直到琼脂糖固化，固定幼虫，幼虫保持在这个位置。 </li><li>一旦琼脂糖完全硬化，慢慢吸取足够的Tricane水（可以是同一Tricane用于麻醉）入菜，完全覆盖琼脂糖和幼虫。 </li></ol> 2。激光校准和测试<ol><li>打开所有共聚焦显微镜仪器仪表，相应的二极管激光器，用于激发斑马鱼的转基因，和氮泵浦的染料激光。务必到位"空白"分光镜，激光衰减器是完全开放的，和435 nm的香豆素染料细胞很到位。在计算机上，打开图像处理软件。 </li><li>移动位置和一个63X 1.2NA水的浸泡的目的，适用于一小滴浸泡介质对水目标到目标。一个63X的目标将允许良好的激光消融精度和水，我mmersion允许获得较大的工作距离，这是必要的，当使用6-7单丝旦斑马鱼幼虫。 </li><li>获取一个载玻片与镜像侧用于校准的激光。将滑动，镜面朝下，到显微镜阶段。 </li><li>使用目镜在光照下，找到并专注于镜中的刮痕或腐蚀。明视场光将照耀下的载玻片上，但只能通过镜子的地方已被划伤或腐蚀掉的客观，会导致反光镜通过目镜看黑，版画看起来像斑点或光行。 </li><li>查看和版画集中在电脑屏幕上，使用成像软件。打开控制激光校准和功率设置窗口。设置的脉冲数为"1"，"3"％传输的衰减板。的脉冲数表示激光器将在每个感兴趣区域（RO触发的次数I）和％透光率表示的激光功率。请务必正确的校准设置的63X 1.2NA水浸泡的客观选择。 </li><li>从主工具栏中选择椭圆工具，然后单击计算机屏幕上的图像创建一个单一的圆形ROI。做3次以上，直到有4个圆形的ROI图像随机间隔。 </li><li>有些系统可能包括一个安全功能，不会使激光火，如果目镜的光路是开放的。如果是这样的话，在这个时候，手动关闭光路目镜。 </li><li>消防激光。这将创建4个小现货铜版画，1各通函内的投资回报率。如果激光不火，检查，以确保激光开启并正确连接，关闭光路目镜。如果激光火灾，但没有蚀刻看到，检查，以确保"空白"分光镜到位。如果一切都在发生，提高激光功率和"FRAP"的，直到蚀刻ARê可见。如果大于10的激光功率设置是必要的蚀刻玻璃，这可能表明激光等离子体盒需要更换。 </li><li>如果蚀刻的斑点出现在选定的ROI为中心是必要的，没有校准（请至2.12）。如果这些点不在中心位置，校准设置需要被更新。 </li><li>要校准，点击更新校准设置。激光会启动，会出现影像，包含一个单一的蚀刻点。如果点不出现，取消校准，提高激光功率，并开始重新校准。 </li><li>点击在现场的中心。激光将再次触发，会出现另一个点。点击这一点，并重复这个过程，直到校准完成9点出现在一格的时装。重复步骤2.6-2.9检查校准成功。 </li><li>取出载玻片从显微镜阶段，清洁的目的。打开光路OCULARS，并更换"空白"与"100％馆际互借"分光镜分光镜。 </li></ol> 3。使用激光消融时间推移共聚焦成像胶质细胞行为的神经切断术<ol><li>拆除阶段用来装载玻片和更换适合举办35毫米的玻璃底菜的舞台。继续要使用63X 1.2NA水的浸泡的目的。 </li><li>水浸泡中取一小颗的目标，并在舞台上安装的幼虫，将菜。稳定的剪辑菜。 </li><li>使用目镜和广角照明，重点的幼虫和定位运动神经。扫描的神经在10-20 hemisegments和选择运动神经横断。 </li><li>使用成像软件在电脑屏幕上实时取景的神经。选择Z型飞机和未受伤的神经轴突和神经胶质细胞的采集图像。 </li><li>准备时间推移成像设置在成像软件捕捉Z-突起在5-30分钟的间隔中的所有类型的细胞中，根据实验。这是最好的创建时间推移进行消融之前的所有必要设置，所以在启动时间推移，一旦受伤后有没有延迟。 </li><li>删除"100％病"的分光镜和更换的"空白"。关闭光路目镜。 </li><li>返回实时取景的神经。使用适当的荧光通道将横断查看的轴突。 </li><li>使用椭圆工具，创建一个薄的椭圆形消融在该地区的投资回报率，然后再创建所选区域内的投资回报率较小。 </li><li>在控制激光设置的窗口中，设定的脉冲数为"2"，"18"％传输的衰减板。消防所选的感兴趣区域内的激光。如果荧光遗体内的投资回报率，增加激光功率，等待约10秒，并再次发射激光。这样做，直到你达到一个设定，导致fluorescenCE消失内的投资回报率。 </li><li>等待10秒或更长时间，并再次检查烧蚀面积的荧光。当心，投资回报率最初可能出现烧蚀，它实际上是光漂白。如果荧光回报，增加激光功率，并再次发射激光。重复该步骤直至花期内消失的投资回报率，并在10秒内不返回。这是最好的开始与一个较小的激光功率，并增加必要的权力。所需的激光功率可能根据个人显微镜系统，香豆素染料的年龄，样品安装，幼虫的年龄，以及组织的厚度。一旦已建立一种理想的激光功率，此功率设置可能被再次使用，在同一实验中在随后的神经。 </li><li>一旦完成后，开始消融时间推移成像。 </li><li>当时间推移完成后，使用成像软件编译的数据，并创建Z-推算，每个时间点的彩色复合。创建一个QuickTime影片的行为进行分析轴突和神经胶质细胞的同时。 </li></ol>

<ol>
	<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>

什么都没有透露。

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。这些设置通...

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运?...

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 ...

motor-nerve-transection-time-lapse-imaging-glial-cell-behaviors-live

Research

JoVE Journal

Neuroscience

11.8K 次观看.  University of Virginia. 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。

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

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

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

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 ...

科研

神经科学

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

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

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

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

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

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

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

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

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 stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

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 ...

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis muscle of the garter snake. Raw data comprises time-lapse sequences of 3D z-stacks. Each stack contains 4-20 images acquired with epifluorescence optics at focal planes separated by 400-1,500 nm. Steps in the acquisition of image stacks, such as adjustment of focus, switching of excitation wavelengths, and operation of the digital camera, are automated as much as possible to maximize image rate and minimize tissue damage from light exposure. After acquisition, a set of image stacks is deconvolved to improve spatial resolution, converted to the desired 3D format, and used to create a 4D "movie" that is suitable for variety of computer-based analyses, depending upon the experimental data sought. One application is study of the dynamic behavior of two classes of endosomes found in nerve terminals-macroendosomes (MEs) and acidic endosomes (AEs)-whose sizes (200-800 nm for both types) are at or near the diffraction limit. Access to 3D information at each time point provides several advantages over conventional time-lapse imaging. In particular, size and velocity of movement of structures can be quantified over time without loss of sharp focus. Examples of data from 4D imaging reveal that MEs approach the plasma membrane and disappear, suggesting that they are exocytosed rather than simply moving vertically away from a single plane of focus. Also revealed is putative fusion of MEs and AEs, by visualization of overlap between the two dye-containing structures as viewed in each three orthogonal projections.

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals. Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

内吞体动力学在生活神经末梢与四维荧光成像可视化

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

Spinal Cord Transection in the Larval Zebrafish

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 to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

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

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

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

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon guidance, has not been fully elucidated. This is partly due to technical limitations of traditional axon guidance assays. To identify additional axon guidance cues influencing the oculomotor nerve, an ex vivo slice assay to image the oculomotor nerve in real-time as it grows towards the eye was developed. E10.5 IslMN-GFP embryos are used to generate ex vivo slices by embedding them in agarose, slicing on a vibratome, then growing them in a microscope stage-top incubator with time-lapse photomicroscopy for 24-72 h. Control slices recapitulate the in vivo timing of outgrowth of axons from the nucleus to the orbit. Small molecule inhibitors or recombinant proteins can be added to the culture media to assess the role of different axon guidance pathways. This method has the advantages of maintaining more of the local microenvironment through which axons traverse, not axotomizing the growing axons, and assessing the axons at multiple points along their trajectory. It can also identify effects on specific subsets of axons. For example, inhibition of CXCR4 causes axons still within the midbrain to grow dorsally rather than ventrally, but axons that have already exited ventrally are not affected.

An ex vivo slice assay allows oculomotor nerve outgrowth to be imaged in real time. Slices are generated by embedding E10.5 IslMN:GFP embryos in agarose, slicing on a vibratome, and growing in a stage-top incubator. The role of axon guidance pathways is assessed by adding inhibitors to the culture media.

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

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

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 ...