作者感谢NINDS /美国国立卫生研究院，R00-NS064197和NINDS /美国国立卫生研究院，R01NS083897（既DLS）的资金。

1，制备媒体（图1，步骤1） <ol><li>片培养基<ol><li> 25毫升切片培养基足以制备5玻璃底培养皿中，每孔2片。 </li><li>在50ml锥形管中，加入250微升的100×N2溶液及500微升的50倍溶液B27无维生素A的DMEM/F12添加到22.5毫升的体积。 </li><li>过滤灭菌的溶液，随后加入1.25热灭活的马血清中的溶液和1.25ml胎牛血清。 </li><li>孵育在水浴溶液在37℃下进行切片的制备的持续时间。 </li><li>添加生长因子（FGF和EGF，10终浓度 纳克/毫升和20纳克/毫升，分别）之前立即培养的开始。该培养基的组成进行了优化，以促进培​​养细胞的存活，并增加神经祖细胞的增殖率。这将从而最大化的概率观察切片中的有丝分裂细胞。 </li></ol></li><li>全HBSS解决方案剖析<ol><li>制备500mL全HBSS中加入50毫升10×HBSS，1.25毫升1M HEPES（pH值7.4，FC 2.5毫米），6毫升2.5 M D-葡萄糖（FC 30毫摩尔），2.2毫升0.9M的碳酸氢钠 （FC 4毫米）和蒸压DIH 2 O至500ml的最终体积。 </li><li>过滤消毒溶液中，并储存于4°C，直到子宫角，从孕鼠的集合。此溶液可保持在4℃下进行周。 </li><li>整个解剖过程中保持在冰上的HBSS。 </li></ol></li><li>嵌入解决方案<ol><li>对于4胚胎大脑的嵌入，准备加入30ml 3％低熔点琼脂糖中的全部的HBSS溶液。在50ml锥形管中，加入0.9克低熔点琼脂糖和充分的HBSS〜30毫升。 </li><li>混合解决方案，旋涡混合器，并融化在微波与管站立和瓶盖打开。 </li><li>而在微波中，监测该溶液为pr事件溢出由于过度沸腾。当沸腾的开始，停止微波和涡流的解决方案。 </li><li>重复这些步骤，直到所有的琼脂糖融化。 </li><li>存储该管在水浴中于42℃ </li></ol></li></ol>的胚2。夹层（图1，步骤2） <ol><li>经过仔细安乐死孕鼠，收集子宫角，并将它们转移到装有冷全1X HBSS中有10 平方厘米的培养皿。胚胎的年龄可以根据研究有所不同。此协议已被成功地用于从胚胎E12.5天培养脑切片以E17.5。由于它们的大小，更年轻的大脑往往更难以处理。 </li><li>收集胚胎和解剖出如在大鼠和小鼠26,27先前所描述的胚胎的大脑，直到获得充分的胚胎脑包括后脑和前脑的两个大脑半球。这些可以是保持在室温，直到所有的大脑已经被解剖。 </li><li>收集胚胎和解剖出如在大鼠和小鼠26,27先前所描述的胚胎的大脑，直到获得充分的胚胎脑包括后脑和前脑的两个大脑半球。这些可以保持在室温，直至所有的大脑已被切开。 </li></ol>胚胎脑的3。嵌入（图1，步骤3） <ol><li>在含有冰水桶中，创建一个冰孔插入嵌入模具成。 </li><li>倒入3％琼脂糖介质进入一个塑料模具，并将该模具放入冰准备的孔。 </li><li>搅拌琼脂糖介质用数字温度计的尖端，直至温度达到35℃。 </li><li>大脑迅速转移到包埋剂。 </li><li>关键步骤：要去除多余的HBSS在大脑和琼脂糖之间的接口，仔细，轻轻下泻/旋转大脑反复在用钳子嵌入溶液。 </li><li>凝胶化琼脂糖的垫将形成在模具的底部与冰接触。一旦垫可以感觉到与钳子的前端，同时搅拌大脑，与背侧定位大脑。大脑不应该沉到模具的最底部。 </li></ol> 4，制备琼脂糖块（图1，步骤4） <ol><li>让琼脂糖硬化在冰上至少5分钟。 </li><li>用剃刀刀片，部分在模具的角部。 </li><li>用剃刀刀片仔细地刻在琼脂糖块周围的脑。尽量减少裁员的数量，以避免扰动嵌入大脑。 </li><li>确保该块具有更大的表面积在大脑中的尾区（相对于延髓区域，参见图1，步骤4），以增加对切片基座块的稳定性。 </li><li>最后切应该是一个在大脑的尾侧;这种切割将确定正确的切片平面与vibratome。 </li><li>通过对准垂直于大脑的rostro-尾轴和琼脂糖块刀片这样做。向下滑动叶片尾端沿rostro，尾椎轴，使晋级决赛长约5毫米远离大脑的最尾端区域。 </li><li>抛开琼脂糖/脑块，并重复其他大脑的嵌入。 </li></ol> 5，传输的嵌入式脑入Vibratome的托盘（图1，步骤5） <ol><li>加入一滴胶水到vibratome托盘的底部。放置胶，以使琼脂糖块将被定位在所述振动叶片的范围。 </li><li>将琼脂糖/脑块尾侧倒在刮刀的边缘，大约有50％以上的刮刀边缘的块小费。 </li><li>应用琼脂糖块的尾端面的胶水和滑动铲走，保持琼脂糖块的托盘的底部有s海达镊子的。不要让锅铲直接接触胶水。 </li><li>让胶在室温下固化5分钟。 </li></ol> 6，切片嵌入式脑的（图1，步骤6） <ol><li>填写vibratome托盘HBSS。 </li><li>定义vibratome刀片的起点和终点的位置。 </li><li>产生200 - 250微米的切片。随着VT1000s vibratome，请使用以下参数：速度2 - 4，频率8。 </li><li>小心地从vibratome取出切片，因为它们会被切除。传输用刮刀和镊子的后端成12孔培养皿中填充有充分的HBSS的切片。另外一些研究人员代替镊子用画笔关键点：在传送过程中，注意使大脑切片仍附着在周围的琼脂糖。 </li></ol> 7，Syto11的切片的染色（图1，步骤7） <ol><li>稀Syto11至终浓度0.5 - 1微米的切片培养培养基中的生长因子。 </li><li>在12孔板的孔，在37℃下孵育切片从一个脑于2.5ml 1小时的染色溶液</li><li>无染色20分钟的解决方案于2.5毫升切片培养基洗净切片。 </li></ol> 8，安装的片在玻璃底菜（图1，步骤8,9） <ol><li>准备15微升嵌入每片胶原溶液中。通过混合375微升的3毫克/毫升的I型胶原溶液以75微升的10×DMEM，9.4微升的1M NaOH和290微升H 2 O的制备1.5毫克/毫升的胶原溶液储存在冰。 </li><li>放置在35 mm的玻璃底微孔培养皿的底部的胶原溶液的15微升下降。用移液管尖端传播它相匹配的片段的大小。 </li><li>切片转移到胶原蛋白的下降与镊子锅铲和对关键点：安装在不同的多个切片耳鼻喉科菜肴增加收购适于成像片的概率。屏幕上通过不同的切片，并选择最能满足“代表结果”一节中下面的标准的人。 </li><li>让切片他们转移到37℃培养箱中培养，用5％的CO 2之前，在室温下孵育10分钟。 </li><li> 20分钟后，加入1.2毫升片培养基放入玻璃底微孔盘。添加600微升培养基中的在一个时间和一个枪头传播它。 </li><li>捕获的脑切片的低倍图片来记录片的解剖水平和完整性。 </li></ol>的片9。实时成像（图1，步骤10,11） <ol><li>让切片恢复在培养箱中培养至少1小时和30分钟，在37℃，5％CO 2的前实时成像。 </li><li>图像与选择的显微镜切片，牢记Syto11很容易漂白。这里的反演特德旋转盘共聚焦显微镜是用来（安道尔XD革命旋转盘共聚焦显微镜）。可替换地，激光扫描共聚焦显微镜都可以使用。下面的参数是旋转磁盘显微镜设置。 </li><li>在整个实时成像会话，保持在37℃，5％CO 2中附连到显微镜的加湿孵育室切片。 </li><li>与300μm的工作距离的60X硅油客观和1.3的数值孔径图像的细胞（例如参见图3B，3C，3E，3F，4A，4B）。为130微米的工作距离和1.4的数值孔径甲100X油浸物镜（例如参见图4C）也可以使用。相机的分辨率是512×512。 </li><li>图像中的单元格为30μm的z栈，与z堆栈位于约40μm的切片的表面下方的中心。试图像更深的组织可导致目标添加机械压力切片上。这可以影响脑切片的完整性。如果规划，使细胞的三维重建，使用不超过2微米的z间隔。 </li><li>调整激光功率和照射时间限制漂白。使用的曝光时间为30至200毫秒，这取决于Syto11信号的强度。 </li><li>随着机动阶段，安装在1盘在多个切片图像的多个位置。例如，图像达20个职位分布在4个不同的切片1张盘。 </li><li>实时成像，可使用少于5分钟的时间分辨率，以使有丝分裂的不同阶段的鉴定。使用Syto11和/或组蛋白H2B-EGFP，4 - 5小时的实时成像足以观察到显著数量有丝分裂的细胞。然而，如果成像疏标记的有丝分裂的细胞（例如由子宫内电穿孔来实现），那么较长的成像参数（过夜）是适当的，工作我们LL。 注意：使用此协议，我们通常观察每个位置平均10有丝分裂细胞。如上所述，成像多装片增加了一个成功的试验的可能性。通过这种方法，我们通常识别几乎所有与Syto11或组蛋白H2B-EGFP进行的实验有丝分裂细胞。 </li></ol> 10，有丝分裂的收购后分析（图2） 所有在本节的程序进行了优化，在斐济（ImageJ的），这是有利的，因为它是一个免费的开源软件。其他软件解决方案可用来执行相同的任务，如的Metamorph，Imaris里，和阿米拉。 <ol><li>有丝分裂的鉴定图在斐济<ol><li>打开数据集的一个位置作为“hyperstack”后，选择一个z平面（ 见图2A滚动条的位置）所在的组织和细胞看起来很健康（见标准在讨论部分）。跨越时间维度滚动，以确定细胞经历分裂后期，因为它是最简单的有丝分裂相鉴别。 </li><li>滚动时光倒流，以确定时间点的细胞有丝分裂进入（DNA凝聚）。该电池可行驶着的z飞机，但时间分辨率和前面描述的Z堆栈的参数进行了优化，使这个过程。 </li><li>在电子表格中，记录单元格中的hyperstack四维坐标（X，Y，Z和时间， 见图2A想像这些数字的位置，在斐济接口），因为它进入有丝分裂。了解这些坐标将防止计数相同的细胞数次。 </li><li>向前滚动，在时间和记录时间，当细胞的进行从一个阶段到另一个。记录每个有丝分裂阶段的持续时间为1给定的小区。 </li><li>继续与其他细胞，在整个数据集（内部和跨多个位置的细胞）。 </li></ol></li><li>三维可视化细胞的离子和旋转定量分裂中期期间（从盖达尔等人 ，2003改编）（ 图2B）。 <ol><li>在确定多个有丝分裂细胞后，用单元格的坐标和时间向前滚动，直到中期的开始。 </li><li>旋转整个“hyperstack”，使心室边界平面（使用“图像/变换/旋转...”命令）。使用“角度”工具来确定的角度来申请这个旋转。 </li><li>确定z平面，其中感兴趣的细胞是最明显的。在该平面上，使用“矩形选择”工具绘制一个选区，它包括整个小区。 </li><li>确定的z平面，其中包括所关注的细胞。使用“图像/复制”命令来创建一个“子堆”。在该对话框中，保留“重复hyperstack”检查。的“切片（z）的”参数对应于z平面包括整个CELL，和“帧（t）的”参数对应于当前时间点。 </li><li>如果分辨率差，增加了“分堆”使用“图像/调整/尺寸...”命令的大小。 </li><li>产生的细胞在使用“Image/Stacks/3D项目...”命令的当前时间点的三维重建。该参数为这个指令将显示在图2B中 。 “切片间距”对应于每个z平面图像（微米）之间的间隔。重要提示：请确保子堆的物理尺寸（微米）已被保存。如果不是这样，在z维度中的像素的插值将不正确，并且该三维重建将不准确。 </li><li>使用滚动，旋转所产生的三维重建，使得中期板的边缘是可见的。该帧的数目对应于图3B中描述的β角，记录该号码。运用的“角度”的工具和“分析/测量...”命令，测量水平和垂直线等分的中期板（ 图2B）之间的夹角。这是α角。 </li><li>记录这些角度针对感兴趣的单元的所有的中期时间点。旋转的时间点（t）和（T-1）之间的夹角等于在（T-1）的角处（t）和当前角度之间的差的绝对值。 </li></ol></li><li>测量解理面的方向<ol><li>后期在下面的三维重建中期板里的说明三维重建感兴趣的细胞在一个时间点。 </li><li>旋转重建，直到由分离的染色体所形成的两个板的边缘是可见的。 </li><li>使用角度工具来测量水平之间的角度（脑室边缘），并平行于由染色体分离所形成的两板的线。这是解理面（ 图2C）的角度。 </li></ol></li><li>的电影一代<ol><li>作为细胞通常在不同的z轴的平面移动，确定实时成像会话期间由细胞所占用的z平面上。生成使用“图像/堆栈/ Z轴项目...”命令这些Z-平面的“最大强度投影”。所得到的叠层另存为“TIF”文件。 </li><li>在1.45版本的ImageJ中打开此文件，斐济不包括稳定插件的“MOV”或“AVI”文件的生成。 </li><li>使用“文件/另存为/ ...”的ImageJ的命令来生成一个电影与你的下游应用兼容的格式。 </li></ol></li></ol>

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作者宣称他们没有竞争的经济利益。

我们已经描述了协议的主要优点是，它提供了神经祖细胞的有丝分裂的动态时间分辨率。通常情况下，检测到有丝分裂可视化在脑发育所使用的固定的组织切片进行免疫。但这种方法只提供了有丝分裂的快照在一个时间点。 有几个步骤是在大脑切片成像有丝分裂最关键的：1）脑片应保持附着在琼脂糖，转让和安装过程中保留的脑切片的完整性。 2）产生和切片成像，rostro-尾?...

本协议的总体目标是描述如何在胚胎大脑切片进行神经祖细胞有丝分裂的实时成像。用文化的脑片实时成像，该协议提供了一种简单的方法来测定在一个环境非常相似， 在体内环境的神经祖细胞有丝分裂的多个方面。它可以应用到已经被操纵以在子宫内电穿孔）1-5突变的动物和/或大脑的大脑。该技术也适合用于测试药剂对神经前体“有丝分裂的作用，通过简单地增加一个代理到培养基中。总而言之，本文将做一个技术上的挑战协议访问到那些学习神经。 在神经发生，不同的神经祖细胞群进行精确分割，产生神经元，最终有助于成年人的六个皮质层大脑皮层6-8。早在皮质发育，神经前体池扩大为神经上皮（NE）细胞分裂对称地自我更新。东北细胞，然后转换成放射状胶质细胞（RGC的）。最初，视网膜神经节细胞对称分裂产生两个新的视网膜神经节细胞，但大部分神经发生过程中，视网膜神经节细胞的主要分工模式是不对称的。在不对称分裂，1 RGC产生了一个新的RGC以及一个有丝分裂后的神经元，或更专门的祖细胞（无论是短期的神经前体（SNP），外放射状胶质（ORG），或一个中间祖（INP） 2,3,7,9。综合邻舍计划，单核苷酸多态性，以及ORGS然后可以在该子心室，左心室产生的神经元，和皮层的基底区域，分别，因此，祖细胞的细胞分裂是产生的神经元的基本处理新皮层。 许多研究指出，视网膜神经节细胞的有丝分裂的具体特征和子细胞的命运之间的相关性。海达尔等和Takahashi 等。已经表明，研资局有丝分裂期和细胞周期的长度增加为神经收益，这一发现回荡在后续研究10-13。大量的研究表明，有丝分裂纺锤体相对于心室取向影响神经发生和corticogenesis的各方面，包括在大脑中，生成的类型的神经元和后代的位置分别3,10,14-16。无论是解理面取向直接影响着细胞的命运是有争议的，但结论仍然是，这种有丝分裂参数影响神经发生。进一步强调了有丝分裂的重要性是观察，涉及有丝分裂的机制的许多基因是至关重要的神经发生和进行适当的大脑发育17-20。 有丝分裂是一个动态的过程，但迄今最为详细的研究神经祖有丝分裂利用的固定的组织切片或神经祖细胞成像通过体外细胞培养分析。因此，主流的方法来评价有丝分裂只提供这一过程的快照，并不能揭示细胞如何表现在组织。神经祖细胞有丝分裂实时成像日益成为了解神经祖细胞功能的重要工具。有关示例，请参阅这些引用4,8,10,21-25。几个突出的协议已经出版的准备和脑切片26,27的成像。然而迄今为止，全面协议用于成像和有丝分裂的分析还没有被描述，也不在视频演示。 这种技术提供了一些显著优势的脑切片固定的分析。脑切片的时间推移分析使之能够以一种灵活的方式来分析显著更多的数据点产生。首先，数据被聚集在单个时间点在数分钟或数小时的过程。我们可以分析各个时间点（创建一个静态蒙太奇）或可以结合不同的时间点成电影。其次，切片共焦成像能够产生数据在脑片不同的Z部分。其结果是，各个部分可以被分析。或者，堆叠各个部分可以被组合成一个最大强度投影。第三，分析是在一个组织的背景下完成的，揭示细胞如何分裂相对于邻近的细胞和结构。第四，它是非常适合的，显示有丝分裂缺陷的一些证据突变体的分析。总之这个协议将有助于澄清，以帮助调查谁愿意开展神经祖有丝分裂实时成像在自己的实验室关键步骤。

<table border="0" cellpadding="0" cellspacing="0" style="width:739px;" width="738">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:143px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:261px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100X N2</td>
			<td>Life technologies</td>
			<td>17502048</td>
			<td style="width:261px;">for culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50X B27 without vitamin A</td>
			<td>Life technologies</td>
			<td>12587010</td>
			<td style="width:261px;">for culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F12</td>
			<td>Life technologies</td>
			<td>11320033</td>
			<td style="width:261px;">for culture medium</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Heat-inactivated horse serum</td>
			<td>Sigma Aldrich</td>
			<td>H1138-500ml</td>
			<td style="width:261px;">for culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heat-inactivated calf serum</td>
			<td>Sigma Aldrich</td>
			<td>F4135-500ML</td>
			<td style="width:261px;">for culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FGF</td>
			<td>R&#38;D Sytems</td>
			<td>3139-FB-025</td>
			<td style="width:261px;">for culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGF</td>
			<td></td>
			<td></td>
			<td style="width:261px;">for culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10X HBSS</td>
			<td>Life technologies</td>
			<td>14065-056</td>
			<td style="width:261px;">for the dissection of the embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hepes Free Acid</td>
			<td>Sigma Aldrich</td>
			<td>&#160;H4034</td>
			<td style="width:261px;">dilute to 1M (pH7.4)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2.5M D-Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G8769</td>
			<td style="width:261px;">for the dissection of the embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.9M NaHC03</td>
			<td>Life technologies</td>
			<td>25080-094</td>
			<td style="width:261px;">for the dissection of the embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">low-melting agarose</td>
			<td>Fisher&#160;</td>
			<td>BP165-25</td>
			<td style="width:261px;">for generating slices</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Loctite 404 glue</td>
			<td>Loctite 404</td>
			<td>46551</td>
			<td style="width:261px;">keep at 4&#730;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">syto11</td>
			<td>Life technologies</td>
			<td>S7573</td>
			<td style="width:261px;">Make 5&#181;l aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3 mg/ml collagen type I&#160;</td>
			<td>Life technologies</td>
			<td>A1048301</td>
			<td style="width:261px;">for culturing slices</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glass bottom dish</td>
			<td>MatTek</td>
			<td>P35G-1.5-14-C</td>
			<td style="width:261px;">for culturing slices</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">petri dishes</td>
			<td></td>
			<td></td>
			<td style="width:261px;">for dissection of the embryos</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">digital thermometer</td>
			<td></td>
			<td></td>
			<td style="width:261px;">to measure the temperature of the agarose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">spatula</td>
			<td></td>
			<td></td>
			<td style="width:261px;">to transfer brains</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">paintbrush</td>
			<td></td>
			<td></td>
			<td style="width:261px;">alternative to transfer brains</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">vibratome</td>
			<td>Leica</td>
			<td>VT1000s</td>
			<td style="width:261px;">for generating slices</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dissecting microscope</td>
			<td></td>
			<td></td>
			<td style="width:261px;">dissecting out embryos</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">imaging microscope</td>
			<td></td>
			<td></td>
			<td style="width:261px;">A confocal microscope is required, it needs to be equipped with an incubation chamber</td>
		</tr>
	</tbody>
</table>

live imaging of mitosis in the developing mouse embryonic cortex

虽然时间短，有丝分裂是一个复杂和动态的多步骤过程，基本为器官，包括脑的发育。在发展中大脑皮层，神经祖细胞的有丝分裂异常可引起脑容量和功能的缺陷。因此，有迫切需要的工具来理解的神经祖有丝分裂的机制。在啮齿类动物皮质发育是研究这一过程的杰出典范。神经祖细胞有丝分裂通常在检查固定脑切片。该协议将详细描述用于在有丝分裂体外胚胎脑片实时成像的方法。我们将描述的关键步骤，此步骤，其中包括：脑提取，嵌入脑，脑切片vibratome切片，染色和切片的培养，和时间推移成像。然后，我们将演示并详细说明如何执行有丝分裂的收购后分析。我们有代表性的结果神父嗡使用活体染料Syto11，转基因小鼠（组蛋白H2B-EGFP和中心蛋白-EGFP），并在子宫内的电穿孔（的mCherry-α-微管蛋白），此测定法。我们将讨论如何此程序可以最优化，以及它如何可以修改为有丝分裂的基因调控的研究。有丝分裂中的脑切片的实时成像是一种灵活的方法，以评估年龄，解剖，和遗传扰动在一个受控制的环境的影响，并产生大量的具有高时间和空间分辨率的数据。因此，该协议将补充为神经前体有丝分裂分析现有的工具。

这个实验的成功，多有丝分裂细胞中的一个实时成像会议期间的观察很大程度上取决于双方的诚信和地点进行采集，切片解剖层次。如下面讨论的，切片的解剖水平是一个重要因素。 图3A示rostro-尾和中间-侧部，其中我们最成功的位置。对于这个问题的更多讨论，请参阅Noctor 26。切片的完整性可在该切片的不同区域而变化。这可以先于时间推移（使用整个切片的成像与步骤7.6?...

Watch this Scientific Journal Video about Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex at JoVE.com

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

神经祖细胞有丝分裂是神经发生的一个关键参数。很多我们的神经祖有丝分裂的理解是基于固定的组织分析。在胚胎大脑切片实时成像是一种通用的技术，以评估有丝分裂与在受控环境中高时空分辨率。

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

live-imaging-of-mitosis-in-the-developing-mouse-embryonic-cortex

Research

JoVE Journal

Neuroscience

15.2K 次观看.  Duke University Medical Center. 神经祖细胞有丝分裂是神经发生的一个关键参数。很多我们的神经祖有丝分裂的理解是基于固定的组织分析。在胚胎大脑切片实时成像是一种通用的技术，以评估有丝分裂与在受控环境中高时空分辨率。 

视频: Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

慢性成像小鼠的视觉皮层使用减薄，颅骨的制备

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

科研

神经科学

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

多鼠神经解剖学磁共振成像

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

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 ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

高分辨率实时成像发展中的神经上皮细胞的行为

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

双光子<em&gt;在体内</em&gt;树突棘中的鼠标皮质用稀疏的头颅成像准备

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

室管膜纤毛在小鼠脑侧脑室实时成像

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

同时双光子<em&gt;在体内</em&gt;突触输入的影像和突触后目标中的鼠标Retrosplenial的Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

生活成像的脑皮层细胞使用二维培养系统

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

前子宫内胚胎小鼠脑电穿孔和脊髓切片培养迁移 GABAergic 中间神经元的活体成像

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

Non-Invasive Modulation and Robotic Mapping of Motor Cortex in the Developing Brain

Mapping the motor cortex with transcranial magnetic stimulation (TMS) has potential to interrogate motor cortex physiology and plasticity but carries unique challenges in children. Similarly, transcranial direct current stimulation (tDCS) can improve motor learning in adults but has only recently been applied to children. The use of tDCS and emerging techniques like high&#8211;definition tDCS (HD-tDCS) require special methodological considerations in the developing brain. Robotic TMS motor mapping may confer unique advantages for mapping, particularly in the developing brain. Here, we aim to provide a practical, standardized approach for two integrated methods capable of simultaneously exploring motor cortex modulation and motor maps in children. First, we describe a protocol for robotic TMS motor mapping. Individualized, MRI-navigated 12x12 grids centered on the motor cortex guide a robot to administer single-pulse TMS. Mean motor evoked potential (MEP) amplitudes per grid point are used to generate 3D motor maps of individual hand muscles with outcomes including map area, volume, and center of gravity. Tools to measure safety and tolerability of both methods are also included. Second, we describe the application of both tDCS and HD-tDCS to modulate the motor cortex and motor learning. An experimental training paradigm and sample results are described. These methods will advance the application of non-invasive brain stimulation in children.

We demonstrate protocols for the modulation (tDCS, HD-tDCS) and mapping (robotic TMS) of the motor cortex in children.

发育中大脑运动皮质的非侵入性调制和机器人映射

Mapping the motor cortex with transcranial magnetic stimulation (TMS) has potential to interrogate motor cortex physiology and plasticity but carries ...