MS和YZ是由KCL和KCL-中国博士学位奖学金支持。 MO是由一个生物技术和生物科学研究理事会博士助学金。我们感谢冈部胜和淳一宫崎的PCX-EGFP质粒和Alain Chedotal和雅典娜伊普西兰蒂上电宝贵意见。

这个程序是按照英国内政部规定（动物科学程序法，1986）。科学家应该遵循建立并获当地机构和国家动物监管机构的指引。 1。产后电穿孔 1.1。玻璃毛细管，DNA解决方案和电穿孔仪的研制<ol><li>用于DNA注射准备拉玻璃毛细管（0.86 mm外径:1.5毫米，ID）。 （对于萨特的P-97毛细管拉马指示设置是:热火283;拉50，速度90，时间50）。使在对应于约2微升体积的毛细管的标记。 </li><li>设定电穿孔仪的电压至5平方脉冲，50毫秒/脉冲，在100伏，用850毫秒的时间间隔（100伏，脉冲接通50毫秒，脉冲关850毫秒，脉冲5）。 </li><li>稀释高纯度（OD 260/280&gt; 1.80），无内毒素质粒DNA为1-2微克/微升终浓度用无内毒素的Tris-EDTA缓冲液中或PBS。它建议添加0.1％的快速绿色的DNA溶液（染料应在心室蔓延时成功注入）。 </li><li>准备5-7毫米的电极，电极凝胶和热身的加热垫。 </li></ol> 1.2。电穿孔<ol><li>从笼子里取出生后2鼠标小狗和异氟醚吸入麻醉它（在〜0.6 L / min的流量）。 </li><li>后约1分钟，用足捏响应确定麻醉的状态。如果没有发生移动，进行脑室注射。 </li><li>使用吸气管连接到所述毛细管装入毛细管针用1-2微升的DNA。 </li><li>在冷光源，握住你的拇指和食指少你的惯用手之间的小狗的头。皮肤稍向后拉的头部，以帮助识别正确的注射点。 </li><li>考虑眼睛和t之间的虚拟线他颅骨标志性的lambda（ 图1A）。插入毛细管针在约三分之一这条线的从拉姆达（约1从线中点毫米）15的长度。将约2毫米深的毛细血管，并确保避免在大脑深层渗透。 </li><li>通过吹嘴慢慢注入质粒（连接到所述毛细管的注射器也可以使用）。在此过程中，请确保您的手指没有施加太多的压力对大脑，因为这可以防止成功的质粒注射。 </li><li>停止喷射时，DNA溶液的少量留在毛细管中。最好是注入小于1微升，以避免在颅内压有害的增加。 </li><li>涂层既具有凝胶电极，并将它们与所述正侧上，其中所述DNA被注入（ 图1A）的半球的侧面。对于DNA纳入喙SVZ，地方电极头端稍微向注入点。改变电极的位置可以实现电穿孔的区域特异性的SVZ 22,25的不同区域。 </li><li>按脉冲脚踏开关踏板启动电流传输。当电完成后，检查了电穿孔仪显示屏上的电压（电压值应不低于90 V，因为较低的电压值相关性较差的电效率）。 </li><li>复活的小狗下对氧气加热垫了几分钟，并返回到笼子里，把它放在远离母亲。确保母亲检索小狗，并与垃圾的休息团聚了。电穿孔后，让幼仔与母亲的下一步骤前4-7天。 </li></ol> 2。急性脑切片培养的准备 2.1。解决方案和工具的制备<ol><li>下面的解决方案是必需的（但也可以使用高葡萄糖DMEM中进行解剖和成像17）: 夹层介质（500毫升） 盖伊的平衡介质 - 500毫升 45％葡萄糖 - 5毫升 电影中（10毫升） 45％葡萄糖 - 0.110毫升 HEPES 1米 - 0.100毫升 笔/链球菌 - 0.100毫升 FCS - 0.500毫升 B27 - 0.100毫升 谷氨酰胺 - 0.200毫升 的Dulbecco改良的Eagle培养基（无酚红） - 8.89毫升</li><li>在37℃下预热电影媒介</li><li>放置Millicell小插入到含有1ml的电影中，代替在湿润的培养箱中在37℃/ 5％CO 2的35毫米的玻璃底培养皿中。 </li><li>准备vibratome配件（螺丝起子，室，刀片，胶水等）。 </li><li>准备清扫工具:剪刀，小铲，直钳。 </li><li>准备工具，用于处理片:小画笔或软接种环（尺寸:10微升），塑料巴斯德吸管，冰盒和几个6厘米塑料菜肴。 </li><li>预冷夹层介质在2-4°C。 </li></ol> 2.2。脑片的制备<ol><li>填写一个6cm菜预冷的夹层介质，将其放在冰（保持新鲜切薄片）。 </li><li>下面的颈椎脱位，用剪刀斩首鼠标小狗。用解剖刀除去头皮，切割沿从OB中间矢状缝颅骨小脑，轻轻地使用镊子取下颅盖舌。确保整个大脑暴露，仔细用抹刀，请特别注意不要损坏组织解剖出来。解剖，必须小心进行，但在同一时间很快（不到一分钟，如果可能的话）。颈椎脱位是我们的首选方法，因为终端麻醉药品/气体麻醉剂可以影响神经母细胞和大脑切片文化，这就需要进行成像相对较快下列动物牺牲健康状态的迁移特性。 </li><li> Hemisect大脑与拉兹或叶片（ 图2）。丢弃未注射的半球或将用于其它实验。 </li><li>放置一小片的胶带上的vibratome保持器，并使用胶水的最小必需量的附加 ​​在它上面的大脑半球（ 图2）。这可以防止有害的支架表面的因反复胶水的应用程序。 </li><li>待胶水半干几秒钟。 </li><li>占位在vibratome托盘装满预冷解剖的解决方案。指向叶片嗅球（ 图2）。 </li><li>开始使用适当的设置切断大脑半球。以下参数建议:切片厚度300微米;速度〜3-5;频率〜9。高频率和低转速被推荐，以防止损坏的片。 </li><li>使用小画笔或软接种环收集切片。只保留切片可见OB（通常2-3片/脑）。通常，含有大部分的RMS的切片的CAn在约300微米的底表面被发现。 </li><li>切片检查标准荧光显微镜的GFP信号（并确保它们翻转过来检查荧光两侧）下，并选择那些呈现沿大部分的RMS明亮的荧光。 </li></ol> 2.3。培养脑片<ol><li>在细胞培养罩，切掉大脑切片的最尾端第三，取出用细直镊子或显微切割手术刀任何胶水的痕迹。 </li><li>使用塑料巴斯德吸管吸微妙片（切吸管尖以创建一个更大的开放，这将避免损坏切片），并把它放在预热的Millicell小的中心刀片。 </li><li>确保具有最亮的荧光信号的一侧被放置在与Millicell小插入（用于成像用倒置显微镜）相接触。 </li><li>卸下刀片的顶端多余的解剖液用移液管。 </li><li>离开切片文化，以定居在37℃/ 5％CO 2培养箱成像前至少1小时。 </li></ol> 3。成神经细胞迁移的时间推移成像<ol><li>前至少2小时成像，打开珀金埃尔默UltraViewVoX共聚焦旋转盘系统，倒尼康的Ti-E显微镜，滨松C10600-10B（ORCA-R2），冷却数字CCD相机，以及加热系统（索伦特科学）设定在固定导37℃的温度</li><li>打开Volocity软件采集模块，然后单击"即"按钮，选择激光（s）的成像。 </li><li>在2小时内脑片的准备​​，将包含在显微镜舞台上成像室大脑切片的玻璃底菜。 </li><li>使用尼康CFI超计划氟ELWD 20X/0.45目标在适当的荧光来选择和聚焦的切片的面积将被成像（ 如刚注射部位后在RMS的第一下降部分，或者ELBO瓦特的RMS的区域，或神经母细胞进入OB前刚过肘）。 </li><li>设置延时成像与以下操作: <ol><li>在显微镜上，单击L100按钮，以允许样品的激光扫描。 </li><li>在Volocity，通过选择合适的激光（绿色荧光蛋白如 488纳米激光）打开的Ultraview激光转换。 </li><li>设置曝光时间（通常为100-500毫秒）和激光强度（通常为20-30％），这取决于荧光信号的强度。 </li><li>调整图像的数字增益提高细胞可视化。 </li><li>选择z栈间隔图像的脑片内 （通常在100-120微米的间隔）。选择重叠尽可能（这将有利于后续的跟踪分析）的间隔与分离的细胞的一个合适的数，以避免可能的。 </li><li>选择每个Z堆叠图像（通常为2-4微米）之间的间距。 </li><li>选择时间INT每一Z堆叠捕获（ 例如 3分钟）和总成像时间（ 例如 3小时）之间erval。 </li><li>点击"保存"图标，以将更改保存到成像参数。 </li><li>按下录音按钮开始拍摄。 </li></ol></li><li>对照组和实验样品备用成像（例如车辆/药物治疗或不同的电穿孔质粒）全同一天。 </li></ol> 4。分析神经细胞迁移<ol><li>在Volocity定量模块，打开完成的时间推移实验之后创建期望库。从左上角的对话框（ 图4A，步骤1）选择"扩展对焦"。 </li><li>点击"测量"选项卡上显示测量窗口（ 图4A，步骤2）。 </li><li>任务列表是在屏幕的左下方可见。拖动"追踪"（下简称"杂项"目前在标题的底部上面的空间中的列表）。这将促使一个新的窗口被称为"轨道"在屏幕的左上部分的开口（ 图4A，步骤3）。 </li><li>请从往绩窗口中的"输入"选项卡（ 图4A，第4步）"点"。 </li><li>点击点工具（ 图4A，步骤5）。 </li><li>首先用鼠标点击到​​池体的中心区域跟踪迁移的神经细胞，并保持跟踪细胞运动，直到延时的最后时间点到达（例如点号61的3小时长的电影） 。 </li><li>要获取的数据选择从测量菜单（ 图4B，步骤6-7）"进行测量项目"。一个窗口会出现在屏幕的中央。 </li><li>在这个窗口中，选择"称为一种新的测量项目:"并键入一个名称（ 图4C，步骤8）。记住要选择公关之前，"所有时间点"选项咝声行（ 图4C，步骤9）。一个测量项目文件会出现延时的文件中，并将包含参数进行定量分析（ 如迁移距离，速度，位移，位移速率）。 </li><li>对测量项目文件，双击打开它作为一个窗口（ 图4D，步骤10）。 </li><li>可视化的单曲分析细胞，从"显示"选项（ 图4D，第11步）选择"跟踪点"。 </li><li>右键单击该测量项目文件，并将其导出为文本文件，然后可以在Excel等程式来分析迁移参数导入。 </li><li>为了测量连续帧和停顿在拍摄期间作出的每一个细胞中，测量项目文件从"显示"选项"点"或"人口"，选择并点击ID（每个轨道都有一个唯一的ID）之间的变动。通过导出着手生成的文件ing作为在步骤4.10解释。 </li></ol>

<ol>
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T., McConnell, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeletal+coordination+during+neuronal+migration.">Cytoskeletal coordination during neuronal migration.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 13652-13657 (2005).</li><li>Valiente, M., Marin, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+migration+mechanisms+in+development+and+disease.">Neuronal migration mechanisms in development and disease.</a> Curr.Opin. Neurobiol. 20, 68-78 (2010).</li><li>Comte, I., 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=Galectin-3+maintains+cell+motility+from+the+subventricular+zone+to+the+olfactory+bulb.">Galectin-3 maintains cell motility from the subventricular zone to the olfactory bulb.</a> J. Cell Sci. 124, 2438-2447 (2011).</li><li>Sonego, M., 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=Fascin+regulates+the+migration+of+subventricular+zone-derived+neuroblasts+in+the+postnatal+brain.">Fascin regulates the migration of subventricular zone-derived neuroblasts in the postnatal brain.</a> J. Neurosci. 33, 12171-12185 (2013).</li></ol>

作者什么都没有透露。

神经祖细胞沿RMS有效的迁移，确保他们的后续成熟为功能性的神经元10。神经祖细胞的朝向突出的OB流是可见的人类婴儿期，并有可能在出生后早期人类大脑发育27起到重要的作用。此外，这些细胞能够靶向脑受损伤和神经退行性变4,28的位点。能够实时监控基因操作对成神经细胞动力学的影响就显得至关重要，充分了解神经细胞的运动引导和规范。 在这里，我们描述了一个协议，通过耦合在产后体内电穿孔急性脑切片培养时间推移共聚焦旋转盘显微镜监测SVZ来源的神经细胞迁移。具体来说，我们已经展示了如何迁移的神经祖细胞可以在SVZ电穿孔质粒ENC标记oding绿色荧光蛋白。取决于研究的目的，多种类型的质粒，可以使用（ 例如 ，允许其它荧光蛋白或共表达的野生型/利息，Cre重组酶，或shRNA的突变蛋白一起荧光蛋白的表达）。我们强烈建议您使用含有鸡β肌动蛋白CAG启动子29 在体内表达质粒。从电穿孔的动物获得的脑片成像，也可用于电30后监测OB的神经前体细胞的放射状迁移在更长的时间点（7-10天）。 经过实践的初期， 在产后体内电穿孔变成了一个非常可靠的方法，可以让强大的神经细胞标记为时间推移成像和免疫荧光分析。此外，该技术提供了在成神经细胞特异性启动子表达荧光蛋白转基因小鼠的根本优势，对于广大神经母细胞被标记。事实上，电穿孔使神经母细胞稀疏标签，从而使它们的形态和迁移动力学的详细分析。相比于立体定向递送病毒载体31的，这种技术是便宜，快速和有出色的小鼠幼仔的耐受性。其主要缺点在于这样的事实，它被限制在出生后早期阶段。事实上，我们强烈建议您使用产后第2天的乳鼠，因为我们观察到的标记效率大幅下降在稍后的时间，当病毒交付方式更加符合31。然而，也可以使用类似的合适的小鼠遗传模型CRE-表达质粒电穿孔策略学习神经发生在成年阶段22。 双光子，标准共焦，旋转盘共聚焦和宽视场荧光显微镜都可以用于可视化神经细胞迁移17,23,24。旋转盘CON焦显微镜是一种便宜的替代双光子显微镜。它允许三维成像在更高的速度通过多z平面，限制了光漂白的标准相比，共聚焦显微镜，并提供了更高的分辨率比宽视场荧光成像31-33。多数神经细胞迁移的有一个清晰可见胞体和尖与生长锥一个高度动态的领先工艺。 所描述的其他17，有一个灌注室将允许直接评估控制和药物治疗的相同的大脑切片上的影响。虽然我们认为这是一个重要的优点，这里所描述的协议表明，它是不适合的细胞活力绝对必要灌注切片与充氧人工脑脊液（ACSF）或高糖DMEM 17,33。此外，使用倒置显微镜允许更容易操纵目标相比，装有水浸泡目标直立显微镜。我们发现，使用20X远距离的目标是一个最佳的妥协，使细胞的一个好数字（通常为每片25-40）跟踪和产生良好的分辨率的图像（ 视频1）。自动跟踪也是可以的，但它并不总是可靠的。为此我们建议视觉和，如果需要，自动跟踪数据手册验证。可使用尼康氟DIC M/N2 40X/0.80W目标进行单神经母细胞的高倍放大成像。免费提供的ImageJ插件MTrackJ也可以用来测量基本轨33的统计数据，但一些原始数据采集的文件可能会与本软件不兼容，可能需要转换为适用格式的分析，这在某些情况下可能是费时。由Volocity软件提供的图像采集和分析模块的组合可从图像捕获/处理洄游参数的定量分析立即转换TERS（速度，位移等 ），它可以在各种图形很容易地可视化（ 如显示迁徙模式/距离/方向性/速度/持续性/花费的时间不动， 等 ）。 神经母细胞显示一个跳跃式运动，交替迁徙到固定相34,35（ 图3C）。在光的事实，固定相可以是4-10分钟32之间，我们认为捕捉图像，每3分钟是一个很好的妥协，跟随迁移的神经母细胞与最低照明和光损伤。我们很少看到细胞停止时间超过6分钟，并发现，在3小时内，他们一般平均花30分钟不动。根据之前的报道，神经母细胞有60-100微米/小时的平均速度和平均蜿蜒指数（实际排量在迁移距离），略高于0.6 23,36，这与我们的观察一致。表型盟友，大约60％的细胞显示出一个"迁徙"的行为（ 如下面几乎是线性的迁移路径，用0.6-1之间蜿蜒指数）和20-25％是"试探性"（与0-0.4之间蜿蜒指数），而其余的约20％可以被归类为"中间"（交替迁徙和探索阶段，以0.4-0.6之间的曲折指数）。 我们认识到，仍然有限制这一技术，因为我们不能没有观察实质性改变他​​们的运动神经母细胞拍摄超过3-4小时。拍摄持续时间可以提高通过降低成像频率（不过，这将影响迁移分析的精度），以及通过采用灌注腔，以优化用于成像的环境条件。然而，这里描述的协议，包括一个3-4小时拍摄期间表示适当的妥协使足够的数据收集进行定量分析迁移。事实上，到目前为止，我们还设法通过蛋白过度表达/下调37或药理学操控与适当的控制（Sonego周，未公布结果）比较后发现对迁移产生的影响。 总之， 在体内电穿孔产后脑切片培养的旋转磁盘镜像的组合代表一个功能强大的工具来监测神经细胞迁移的动态系统中极为相似的体内环境，提供的可能性，以深入控制的分子机制研究成神经细胞活力。

在哺乳动物的大脑，新的神经元（神经）的一代出生后主要发生在两个区域，海马1的齿状回侧脑室和颗粒下层区的脑室下区（SVZ）。聚集在近年来相当多的证据支持对出生后神经发生在海马和嗅球记忆功能1-3的关键作用。重要的是，出生后的神经也持治疗的潜力，因为它与神经退行性疾病的关系，以及神经母细胞迁移到损伤部位在大脑4-6的能力。 脑室下区（SVZ）最近成为一个重要的神经利基。 SVZ来源的神经母细胞对嗅球（OB）通过喙迁移流（RMS）的迁移，使这个在产后大脑1,7,8最长的迁移过程。哺乳动物SVZ / RMS / OB系统已成为一个研究在神经发生不同的步骤，如增殖，迁移和分化1,8有用的模型。许多生长因子和细胞外调节线索SVZ神经发生和迁移沿RMS，但细胞内的分子机制还远远没有得到充分的理解1,9。沿着正确的RMS迁移是新生神经元10的后续成熟至关重要。此外，一些研究表明，SVZ来源的神经母细胞能出的RMS迁移到脑损伤部位4-6,11-13。因此，调查的信令机制，调节神经细胞迁移的根本不仅要了解神经同时也为潜在的治疗应用。 在这里，我们描述了一个详细的协议通过体内电穿孔产后标记SVZ神经祖细胞和使用时间推移旋转盘共聚焦microsco监控他们沿着急性脑切片培养的RMS迁移PY。电穿孔技术被广泛应用于从胚胎到成人阶段14-18发育研究。它是一个强大的工具，目标和操作SVZ神经祖细胞，代表了更便宜和相当快的替代立体定向注射病毒载体转基因模型1,15,19,20或生成。这是一个相对简单的过程，这并不需要手术，并具有高的存活率。电穿孔或shRNA的小鼠遗传模型采用的LoxP系统可用于靶向感兴趣的基因或实现SVZ祖细胞的永久标记，从而表示对成年神经发生的研究21,22的有用工具Cre重组酶表达质粒的构建。 在完整的脑成像RMS的神经细胞迁移仍是由于目前的技术限制的挑战。然而，这个过程可以使用急性脑切片，这提供了合适的共聚焦系统·旋转盘时间推移显微镜进行监测EM相像的体内条件也适合进行药物处理23,24。耦合在产后体内电穿孔随着时间推移成像将有助于控制神经细胞活力的分子机制的理解，并有助于为新的方法来促进大脑修复的发展。

<table border="0" cellpadding="0" cellspacing="0" width="854">
	<colgroup>
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:344px;">Millicell</td>
			<td style="width:195px;">Millipore</td>
			<td style="width:149px;">PICM0RG50</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 mm Glass bottom culture dish</td>
			<td>MatTek</td>
			<td style="width:149px;">P35G-0-14-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gey&#39;s Balanced media</td>
			<td style="width:195px;">Sigma</td>
			<td>G9779-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glucose, 45%</td>
			<td style="width:195px;">Sigma</td>
			<td>G8769-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td style="width:195px;">Sigma</td>
			<td>H3375-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pen/Strep</td>
			<td style="width:195px;">GIBCO</td>
			<td style="width:149px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FCS</td>
			<td style="width:195px;">GIBCO</td>
			<td style="width:149px;">10109-163</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">B27 supplement</td>
			<td style="width:195px;"><a>Invitrogen Life Technologies</a></td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">L-Glutamine</td>
			<td style="width:195px;"><a>Invitrogen Life Technologies</a></td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM (phenol red-free)</td>
			<td style="width:195px;">GIBCO</td>
			<td style="width:149px;">31053-028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fast Green</td>
			<td style="width:195px;">Sigma</td>
			<td>F7252-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:344px;">Glass capillaries for injection</td>
			<td style="width:195px;">Harvard Apparatus</td>
			<td style="width:149px;">30-0057</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:344px;">Aspirator tube</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:149px;">A5177</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sutter P-97 capillary puller</td>
			<td style="width:195px;">Sutter Instrument</td>
			<td style="width:149px;">P-97</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:344px;">ECM830 Square Wave Electroporator</td>
			<td style="width:195px;">Harvard Apparatus</td>
			<td style="width:149px;">45-0052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:344px;">Platinum Tweezertrodes 7 mm</td>
			<td style="width:195px;">Harvard Apparatus</td>
			<td style="width:149px;">45-0488</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:344px;">Footswitch Model 1250F</td>
			<td style="width:195px;">Harvard Apparatus</td>
			<td style="width:149px;">45-0211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel for electrodes</td>
			<td style="width:195px;">Cefar Compex</td>
			<td style="width:149px;">6602048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane</td>
			<td style="width:195px;">Merial</td>
			<td style="width:149px;">AP/DRUGS/220/96</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome</td>
			<td style="width:195px;">Leica</td>
			<td style="width:149px;">VT1000S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glue</td>
			<td>Roti coll</td>
			<td>Roti coll 1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UltraViEW VoX spinning disk system</td>
			<td style="width:195px;">Perkin Elmer</td>
			<td style="width:149px;"></td>
			<td>Customized setup (multiple laser sources can be used) equipped with Hamamatsu ORCA R2 C10600-10B CCD camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:344px;">Volocity software</td>
			<td style="width:195px;">Perkin Elmer</td>
			<td style="width:149px;"></td>
			<td>Acquisition, Quantitation, Visualization Modules</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:344px;">Environmental chamber for microscopy</td>
			<td style="width:195px;">Solent Scientific</td>
			<td style="width:149px;"></td>
			<td>Custom-made</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:344px;">Ti-E inverted microscope</td>
			<td style="width:195px;">Nikon</td>
			<td style="width:149px;"></td>
			<td>CFI Super Plan Fluor ELWD 20X/0.45 NA objective is recommended for the application described in this paper</td>
		</tr>
	</tbody>
</table>

in vivo postnatal electroporation and time-lapse imaging of neuroblast migration in mouse acute brain slices

脑室下区（SVZ）是在出生后大脑中的主神经利基之一。在这里，神经祖细胞增殖和神经母细胞能够沿着喙迁移流（RMS）对嗅球（OB）的移动引起。这种长距离的迁移需要在OB的新生神经元的后续成熟，但调控这一过程的分子机制仍不清楚。调查信号通路控制神经细胞活动力可能不仅有助于了解神经发生的基本步骤，同时也有治疗再生潜能，给了这些神经母细胞的目标受到损伤，中风或脑退化站点的能力。 在这个手稿，我们描述了在体内产后电穿孔，并在小鼠RMS的成神经细胞迁移的随后的时间推移成像的详细协议。产后电穿孔能有效转染SVZ祖细胞，从而产生神经母细胞沿RMS迁移。利用共聚焦旋转盘时间推移显微镜对急性脑切片培养，成神经细胞迁移可以在非常类似于在体内条件下的环境进行监测。此外，成神经细胞活力可以跟踪和定量分析。作为一个例子，我们描述了如何使用表达GFP的质粒在体内电穿孔产后标记和可视化的神经母细胞沿RMS迁移。在条件性敲除小鼠用人的loxP系统或shRNA的Cre重组酶表达质粒电穿孔也可用于靶向感兴趣的基因。药理操纵急性脑片培养可以进行调查不同的信号分子在神经细胞迁移中的作用。通过耦合在体内电穿孔随着时间推移成像，我们希望了解控制神经细胞活力的分子机制，并有助于开发小说换货方法来促进大脑修复。

的SVZ来源的成神经细胞迁移性标签可以沿着RMS可以观察到，一个成功的电穿孔（ 图1B）之后，通常4-8天。更长的时间点，也可以选择，但较少的细胞会在RMS中找到，因为他们大多会已经进入了转播。成神经细胞开始采集成熟粒细胞中的OB的典型形态学特征和电穿孔后约2-3周（未示出）。 〜1小时培养后，从电穿孔的乳鼠脑切片能够可靠地拍摄长达3-4小时。据此前报道23,26，神经母细胞能显示复杂的迁移动力学（ 图3和视频1），它可以使用电池跟踪（ 图4）定量分析。 <img alt="图1" fo:content-width="6in" src="/files/ftp_upload/50905/50905fig1.jpg" width="500px" /> 图1。 Postna<html>河谷体内电穿孔。 （A） 在体内电穿孔日龄2鼠标小狗的示意图。虚线（红）的眼连接到craniometrical里程碑的λ作为位置标记物的毛细管插入。注射点被表示为绿色的点。灰色椭圆形指示电极位置在布廷等人描述的绿色荧光蛋白表达质粒电穿孔后5天15（B）矢状小鼠前脑切片免疫染色的GFP。 SVZ来源的神经细胞迁移是可见沿RMS和他们中的一些已经开始到达OB。 CTX:皮质; SVZ:脑室下区，有效值:喙迁徙流;转播:嗅球。酒吧，400微米。 <a href="https://www.jove.com/files/ftp_upload/50905/50905fig1highres.jpg" target="_blank">点击这里查看大图</a> 。 <html> N"SRC ="/ files/ftp_upload/50905/50905fig2.jpg"宽度="500px的"/&gt; 图2。急性脑切片文化成像的原理制备步骤。 （A）尾部和intrahemispheric切口（虚线）上的新鲜解剖大脑被执行（B）将电穿孔的大脑半球被放到一个vibratome支架（C）矢状切片，得到了vibratome和标准荧光显微镜下观察（D）切片用最好的GFP信号是培养至少1小时在Millicell小插入玻璃底培养皿内，以及（E）随后放置在一个环境室成像由一个倒置的共聚焦旋转盘系统。 <a href="https://www.jove.com/files/ftp_upload/50905/50905fig2highres.jpg" target="_blank">点击这里查看大图</a> 。 les/ftp_upload/50905/50905fig3.jpg"宽度="500px的"/&gt; 图3。成神经细胞迁移的时间推移成像。 （A）的绿色荧光蛋白表达质粒电穿孔后第5天从小鼠大脑矢状切片取神经母细胞的旋转盘时间推移图像。图像1小时分开。每个小组是连续28图像的Z-堆叠投影4微米分开。箭头指示4代表沿神经母细胞向嗅球（位于出来的图片在右下角）的RMS迁移。（二）从4成神经细胞的时间推移成像获得的代表迁徙路径（A）中突出显示。 （三）图表显示了2代的成神经细胞迁移与时间的距离。细胞显示出典型的跳跃式能动行为。酒吧，70微米。 <a href="https://www.jove.com/files/ftp_upload/50905/50905fig3highres.jpg" target="_blank">点击这里查看大图</a> 。 图4。成神经细胞迁移的跟踪分析。使用使用Volocity软件来跟踪神经细胞迁移的顺序步骤。请参见文中详细说明。 <a href="https://www.jove.com/files/ftp_upload/50905/50905fig4highres.jpg" target="_blank">点击这里查看大图</a> 。 视频1:迁移的成神经细胞的时间推移成像的影片显示鼠标RMS用表达GFP的质粒电穿孔后得到5天GFP标记迁移的成神经细胞的一个部分。该OB位于拿出来看向右下角。共焦Z-堆栈被捕获在一个旋转盘共聚焦用20X物镜每3分钟3小时超过120微米的间隔。播放速度:10帧/秒。

Watch this Scientific Journal Video about In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices at JoVE.com

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

成神经细胞迁移是在出生后的神经发生一个根本性的事件。我们描述了一个协议，用于使用急性脑片时间推移成像在产后体内电穿孔和随后的迁移可视化的神经母细胞的有效标记。我们包括用于通过视频跟踪神经细胞动力学的定量分析的描述。

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 ...

in-vivo-postnatal-electroporation-time-lapse-imaging-neuroblast

Nanyang Technological University

Research

JoVE Journal

Neuroscience

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

13.1K 次观看.  King's College London. 成神经细胞迁移是在出生后的神经发生一个根本性的事件。我们描述了一个协议，用于使用急性脑片时间推移成像在产后体内电穿孔和随后的迁移可视化的神经母细胞的有效标记。我们包括用于通过视频跟踪神经细胞动力学的定量分析的描述。 

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

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

对于高分辨率延时成像神经细胞迁移的一个器官型产后脑切片检测

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

科研

神经科学

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 ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

在发展中国家小鼠视网膜体内电穿孔

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

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 ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

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 Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

产后的小脑颗粒细胞迁移利用共聚焦肉眼离体成像

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

延期共聚焦显像移植神经元在器官性切片培养胚胎小鼠脑使用<em&gt;在Utero</em&gt;电穿孔

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...