CYIY is supported by FAPESP (2012/14421-5) and FMV is supported by a fellowship from FAPESP (2009/53695-0). We thank the DNA Technologies Core at University of California, Davis for preparation and sequencing of the RNA-Seq libraries used in this work, the Galaxy team for providing an excellent and free interface for high-throughput sequencing data analysis tools and Dr. Marianne Bronner for allowing us to film in her laboratory space.

1.电穿孔兴趣的HH12-13鸡胚胎的神经管在大毛的构建用荧光报道，优选在双顺反子转录物或融合于目的蛋白质与嵌入病毒2a中的肽11，以使快速识别与转染的令人满意的水平的个体。注意：典型的孵育时间为这个阶段是围绕48小时，在37.8℃。 <ol><li>除去3毫升白蛋白与耦合到18G的X 1 1/2针头的注射器后，打开一个小窗口中的鸡蛋。以提高胚胎的可视化，注入少量的墨汁在无菌林格氏胚胎1-4下方溶液以1:50稀释的。 </li><li>覆盖用无菌林格氏溶液中的胚胎后，注入含有2.5微克/微升通过神经管的后端的DNA直到填满一个的10mM的Tris-HCl，pH值8.2和0.1％食品染料（F，D＆amp; C）溶液到后脑区域。电穿孔，通过4毫米1沿侧翼区域分离位置的铂电极（直径0.5mm），并提供5个脉冲的50毫秒和20 V与他们4之间100毫秒的间隔。 </li><li>电穿孔后，孵化操纵胚胎48小时以上，直到他们达到的阶段HH23，并确定个人在日光灯立体呈现转高层次的侧翼区域。 注：孵育电穿孔后的持续时间取决于实验的目的。电穿孔质粒可以在2小时后2产生显著的表达水平。因此，如果兴趣在于发病早期基因的电穿孔后潜伏期可以减小。 </li></ol> 2.收获成功转染胚胎解剖和电穿孔脊髓减半注意：此过程持续2-3个小时，每10胚胎和产量1-2每个胚胎微克总RNA。神经管的解剖，需要精细动作，并把它应用到实验样品前，一些练习是必要的。 <ol><li>净化用RNase去污溶液或通过在150℃下烘烤4小时12金属清扫工具和制备DEPC处理的PBS中。只使用新的认证无RNA酶的塑料制品。 注：收获后，按照标准的护理，防止清扫时RNase污染是很重要的。 </li><li>收获用一双精致的解剖剪刀和过筛收集勺胚胎。保持在100毫米的培养皿中冷DEPC处理过的PBS胚胎。 </li><li>用一双细镊子，切出整个侧翼区（前肢芽和后肢芽的前极限的后限之间），这部分转移到含冷DEPC处理过的PBS一道新菜。 </li><li>用两个细玻璃针穿孔在三个不同的点于神经管和近轴中胚层之间的胚胎各背外侧边。 </li><li>引入两个玻璃针，在中央​​穿孔，慢慢移动中的一个远离沿着神经管前后轴。重复此步骤不是由所述第一运动达到该穿孔。用一双细镊子取出分离旁轴中胚层。 </li><li>把胚胎侧身，插入神经管和脊索之间的镊子的提示和他们在沿前后轴分开的两个结构相反的方向滑动。 注意：这也应该除去大部分的中胚层的仍然附着在神经管。 </li><li>如有必要，删除仍然受到拉动用镊子，同时轻轻握住神经管与另一镊子附加任何大块mesodermic组织。完成后，轻轻的神经管含冷DEPC处理过的PBS采用转移到另一道菜耦合无RNase玻璃巴斯德吸管向手动移液管泵，并保持在冰上，直到所有的管子都解剖。 </li><li>分化神经管成两半。对于此，插入一个封闭镊子在管腔的尖端和通过移动镊子出通过它打开屋顶板。在此之后，通过切割底板与镊子尖端分开两半。 </li><li>来自非电穿孔半部排序电下的荧光解剖显微镜和转移到1.5ml管中填充用冰冷的DEPC处理过的PBS中。如果需要的话，轻弹的微管轻轻地释放附着在塑料壁的任何组织，并促进管的沉淀。 </li><li>旋转100 XG为3秒，取出非常缓慢尽可能多的PBS尽可能与1毫升微量。做到这一点，而在寻找一个光源照射，通过微管监测如有胚胎物质被吸入到枪头;如果发生这种情况，慢慢地排出，并重复centrifuga化。 </li><li>加入70微升的每个神经管半RNA提取裂解液和轻弹试管混合内容。在室温下孵育5分钟，并大力旋涡。重复5分钟温育和涡旋两次以上。储存在-80℃。 </li><li>收集每个样品至少八个神经管半部，以确保足够的产量为下一步骤。池小样本，如果几个电/夹层会话都需要积累的神经管的足够数量。储存足够的样本进行重复或一式三份每个条件。 </li></ol> 3.净化RNA和图书馆准备了Illumina的测序<ol><li>取下-80℃储存样品置于冰上15-30分钟。继续解冻在室温下，直到完成。 </li><li>涡流井，并在室温离开5分钟。旋涡再次离心以15,000×g离心1分钟以沉淀不溶的物质。 </li><li>将上清转移到新管中并根据该总RNA分离试剂盒手册说明书进行RNA提取。在洗脱建议的最低量，以增加浓度。 注：后清理DNase处理建议，以避免在RNA测序数据集的潜在基因组DNA序列的污染。 </li><li>轻弹管混合内容并删除5微升的质量控制分析。剩余的样品储存于-80℃直至文库制备;这些样品应解冻一次，以避免RNA降解。 </li><li>运行在生物分析仪器纯化的RNA用RNA 6000纳米试剂盒; RIN值必须至少为7以上进行。 </li><li>生成使用Illumina公司兼容的RNA测序文库制备试剂库和复用多达4个图书馆的HiSeq仪器设置为产生100 bp的单一读取一条车道。如果可能的话，包括在相同的运行，以尽量减少潜在的技术工件的所有样本。 注：本应产生高达50万转每个样品，这是绰绰有余的，以获得范围为大多数基因13 EADS。 </li></ol> 4.银河公共服务器10比较RNA测序数据集。 注：服务器的存储空间是有限的，并比较多个样品实验，可能需要删除在滤波步骤中生成的中间文件。 <ol><li>在银河公共服务器（创建用户帐户<a href="https://usegalaxy.org/">https://usegalaxy.org</a> ）和FTP上传Illumina的数据集FASTQ格式。 </li><li>准备与FastqGroomer 14岁以下的NGS工具上传的文件：质量控制和操作菜单;如果质量得分已经编码PHRED + 33（桑格），跳过此步骤只需更改文件类型下的属性为每个数据集fastqsanger。 </li><li>通过分析在NGS的FastQC工具，每个数据集的总体质量：质量控制和操纵我NU。 注：应特别注意给予对每个碱基序列的质量和每个碱基序列的内容所得到的图形，因为这些将提供估计用于修剪的读出端的参数。 </li><li>修剪读取放弃3“使用的工具FASTQ质量微调 14岁以下的NGS低质量的数据结束：质量控制和操作菜单。设置该工具只处理3&#39;端的质量分数低于20。 </li><li>卸下接头序列读取从使用裁剪工具 （ <a href="http://hannonlab.cshl.edu/fastx_toolkit/">http://hannonlab.cshl.edu/fastx_toolkit/</a> ）在NGS：质量控制和操作菜单。设置为丢弃具有小于45 bp的左，输入所使用的适配器的顺序读取，并选择输出到包括剪切和非截取序列的工具。此外，选择不丢弃具有未知碱基序列，因为大多数的低质量的碱中的最后一个步骤中已经除去。 </l我&gt; <li>使用工具修剪序列 （ <a href="http://hannonlab.cshl.edu/fastx_toolkit/">http://hannonlab.cshl.edu/fastx_toolkit/</a> ）在NGS：质量控制和操作菜单，修剪第一基地的读取，以消除FastQC检测5&#39;序列偏置（通常为10）。 注意：在这一点上，它是重新运行FastQC并与前面的分析进行比较，以确定该数据集的质量得到了改善一个好主意。 </li><li>下载并解压缩最新的鸡基因组组装从Illumina公司iGenomes在UCSC基因组浏览器（ <a href="http://support.illumina.com/sequencing/sequencing_software/igenome.ilmn">http://support.illumina.com/sequencing/sequencing_software/igenome.ilmn</a> ），并上传了全基因组FASTA格式的文件和基因注释GTF格式文件使用下获取数据菜单中的工具上传文件银河系的历史。 </li><li>使用下对准TopHat2 15地图读出各样品的鸡基因组农工商超市：RNA分析菜单。选择使用从历史基因组（选择步骤4.7上传的FASTA文件）。 </li><li>建立预测转录使用袖扣 16岁以下的NGS每个样本：RNA分析菜单。选择要使用的参考注解为指导（选择步骤4.7上传的GTF文件），打开选项执行偏差校正 （从历史，从步骤4.7 FASTA文件），并使用多读正确 。 </li><li>合并预测转录使用Cuffmerge 16岁以下的NGS所有样本：RNA分析菜单。选择要使用的参考注释（GTF文件从步骤4.7）和序列数据（从历史，从步骤4.7 FASTA文件）。 </li><li>通过比较TopHat2使用Cuffdiff 16岁以下的NGS每个样品产生BAM文件：RNA分析菜单。在现场笔录中选择了我rged转录由Cuffmerge生成并打开选项执行偏差校正 （从历史，从步骤4.7 FASTA文件），并使用多读正确 。 </li><li>排序差异表达测试使用的工具排序的筛选下表中列13（Q值）数值上升和排序菜单。 注：基因/成绩单显示实验条件之间的显著差异表达将呈现最低的Q值。 </li><li>与覆盖图形直观地检查结果，使用该工具创建的基因组覆盖率 17 下BEDTools菜单BedGraph先转换BAM文件bedGraph格式。禁用报告地区无覆盖，选择将拼接的条目不同的时间间隔，并通过了标准化测序深度被比较的所有数据集的一个因素缩放范围。 </li><li>使用所产生的bedGraph文件转换为权贵格式工具假发/ BedGraph到大人物 （ <a href="http://www.genome.ucsc.edu/">http://www.genome.ucsc.edu/</a> ）在转换格式菜单。上传要人文件作为在UCSC基因组浏览器（自定义轨道<a href="http://www.genome.ucsc.edu/">http://www.genome.ucsc.edu/</a> ）为galGal4组件和搜索感兴趣的基因进行可视化的覆盖在所述的数据集。 </li></ol>

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The authors have nothing to disclose.

在这里，我们为鸡脊髓电后分析效果的准则。虽然DNA载体进行电穿孔是更频繁地用于过表达感兴趣的基因，还可以使用构建体编码显性阴性，quimeric蛋白或前体的siRNA，以产生基因功能敲低的条件19-21。事实上，从两个GAIN-和丧失功能的分析得到的转录概况比较可以指出差异表达的基因在相反的方式中的每个条件，因此具有潜在地发现更多的具体效果。 如任何方法下游?...

在活的生物体的基因研究经常使用的鸡胚为模型，因为在OVO电代表了一个快速和廉价的方式来部分地转染胚胎结构在体内与DNA构建1-5。编码含有荧光报告与感兴趣的基因，如pCIG 6或机动设备7在一起的一个双顺反子转录物表达载体，允许在所希望的区域转质量的快速验证处理的胚胎用于下游分析之前，在立体显微镜下。 在DNA测序的最新进展，现在有可能从使用RNA测序-8,9- RNA样本获得数字全转录组表达图谱。因此，代替费时方法，使只有几个基因产物平行，分析如原位杂交，免疫组织化学和qPCR，用于制备cDNA文库的高通量测序可以提供全转录组的信息。对每一个基因的表达水平的实验效果的观察又可以提供关于所使用的构建体改变了通路有力的见解。这是特别容易的，如果一个基因组装配所使用的生物体是可用的，从而鸡胚又是一个合适的模型。 如果用户有访问权限的可靠的基因组测序中心，主要问题是获得高品质的RNA样品。这项工作表明获得的RNA样品从转染的神经管适用于RNA测序，以及在下游步骤中所得到的数据集的计算机分析的方法。电在HH12-13其次是48小时培养后，转染树干脊髓半解剖的核糖核酸酶自由的条件下，立即溶解并进一步降解保护的超低冰点，直到RNA纯化和图书馆preparat离子。 银河公共服务器10可以访问免费资源，用于分析的高通量测序数据。的空间供注册用户的数量是足够的小实验，从而省去了本地的计算基础设施。 RNA测序数据分析包括过滤，比对和基因表达的定量/对比。虽然有用于RNA测序数据分析的一般准则，滤波参数将在很大程度上取决于序列的质量，并应后的原始数据的质量控制分析来确定。 这个协议描述的步骤，以获得与从鸡胚胎转染体内脊髓比较RNA片段轮廓。作为代表性的结果，从转染了空载体两个独立的对照样品获得的数据集的比较表明，该方法是可重复的，应该允许鉴定差异EXPRES的SED成绩单实验样本。因此，该方法的应用有可能大大增加的发现的数量之后，一个单独的试验，从而提供一个大的数据集进行后续调查。

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			<td height="21" style="height:21px;">F D &#38; C Blue 1</td>
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			<td>5.FC.0010P0</td>
			<td>Food Dye used to visualize DNA injection</td>
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			<td height="21" style="height:21px;">Ringer&#39;s solution (1 L)</td>
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			<td height="21" style="height:21px;">&#160;&#160;&#160;&#160; - 7.2 g NaCl</td>
			<td>Sigma-Aldrich</td>
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			<td height="21" style="height:21px;">&#160;&#160;&#160;&#160; - 0.37 g KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P-4504</td>
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			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - 0.225 g CaCl2.2H2O</td>
			<td>Fisher Scientific</td>
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			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - 0.217 g Na2HPO4.7H2O</td>
			<td>Sigma-Aldrich</td>
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			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - 0.02 g KH2PO4</td>
			<td>Sigma-Aldrich</td>
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			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - ddH2O to 800 ml and adjust pH to 7.4</td>
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			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - ddH2O to 1 L</td>
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			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - Filter and autoclave</td>
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			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - 8 g NaCl</td>
			<td>Sigma-Aldrich</td>
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		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - 0.2 g KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P-4504</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - 1.15 g Na2HPO4.7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>S-9390</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - 0.2 g KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P-5379</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - ddH2O to 800 ml and adjust pH to 7.2</td>
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		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - ddH2O to 1 L and filter</td>
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			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - 1 ml DEPC (Diethylpyrocarbonate)</td>
			<td>Sigma-Aldrich</td>
			<td>D-5758</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - Shake vigorously and let stand overnight at room temperature</td>
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			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - Autoclave</td>
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			<td height="20" style="height:20px;">Sieved spoon</td>
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			<td height="20" style="height:20px;">Sterile 60 x 15 mm polystirene Petri dishes</td>
			<td>Corning Life Sciences</td>
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			<td height="20" style="height:20px;">RNaseZap RNase decontamination solution</td>
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			<td height="20" style="height:20px;">Pulled glass needle made from 1.1 x 75 mm glass capillary tubes</td>
			<td>Kimble Chase</td>
			<td>40A502</td>
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			<td height="20" style="height:20px;">9&#39;&#39; glass Pasteur pipette</td>
			<td>Any supplier</td>
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			<td height="20" style="height:20px;">Clear 1.5 ml microcentrifuge polypropilene tubes</td>
			<td>Corning Life Sciences</td>
			<td>MCT-150-C</td>
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			<td height="20" style="height:20px;">Microcentrifuge</td>
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			<td height="20" style="height:20px;">RNAlater solution for RNA stabilization</td>
			<td>Life Technologies</td>
			<td>AM7020</td>
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			<td height="20" style="height:20px;">RNAqueous total RNA isolation kit</td>
			<td>Life Technologies</td>
			<td>AM1912</td>
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			<td height="20" style="height:20px;">RNA 6000 Nano Kit</td>
			<td>Agilent Technologies</td>
			<td>5067-1511</td>
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			<td height="20" style="height:20px;">2100 Electrophoresis Bioanalyzer Instrument</td>
			<td>Agilent Technologies</td>
			<td>G2939AA</td>
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			<td height="20" style="height:20px;">Truseq RNA Sample Preparation Kit v2</td>
			<td>Illumina</td>
			<td>RS-122-2001/RS-122-2002</td>
			<td></td>
		</tr>
	</tbody>
</table>

rna-seq analysis of differential gene expression in electroporated chick embryonic spinal cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

为了验证所描述的方法，从胚胎电穿孔用空载体机动设备7和从胚胎用含有插入物构造SCRT2-ZNF，其编码鸡SCRT2 18的锌指结构域的相同向量中的一个样本中生成两个控制样品。样品得到11-22微克总RNA，每个从8至12中的胚胎池获得。所有三个样品中的生物分析仪（ 图1）进行的质量检查得分的10最佳的RIN值，这表明高品质的RNA可以与此协议来获得。 测序?...

Watch this Scientific Journal Video about RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord at JoVE.com

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

对基因差异表达的RNA序列中电穿孔鸡胚脊髓分析

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

rna-seq-analysis-differential-gene-expression-electroporated-chick

Research

JoVE Journal

Neuroscience

14.4K 次观看.  Universidade de São Paulo. This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

视频: RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

脊髓电二：外吸电极的制作

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues1 and survival factors2 located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons3. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons4. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro5,6.
Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth. 
SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol7) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival8.

We demonstrate how to dissect and culture chick E4 statoacoustic ganglion and E6 spinal cord explants. Explants are cultured under serum-free conditions in 3D collagen gels for 24 hours. Neurite responsiveness is tested with growth factor-supplemented medium and with protein-coated beads.

小鸡Statoacoustic神经节和脊髓植在胶原凝胶夹层和文化突起生长实验

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation ...

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1).
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however, other options have been discussed as well5.
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.

DII的标签DRG神经元的研究轴突分支在小鼠胚胎脊髓整装准备

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial and temporal control of ectopic microRNA expression. However, ventral brain structures like ventral midbrain are rather difficult to reach for any manipulations. Here, we demonstrate an efficient way to electroporate miRNA into ventral midbrain using thin platinum electrodes. This method offers a reliable way to transfect specific areas of the midbrain and a useful tool for in vivo studies.

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Ectopic expression is one technique to elucidate the microRNAs role in brain development. However, targeting specific areas using in ovo electroporation is challenging. Here, we show an efficient way to selectively electroporate ventral and dorsal midbrain regions.

在微RNA在腹鸡中脑大毛表达

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

立体定向注射病毒载体的基因操作在小鼠脊髓条件

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

解剖和斑马鱼的胚胎横向安装：脊髓发展分析

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

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 ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

一个<em&gt;离体</em&gt;激光诱导脊髓损伤模型，以评估轴索变性的机制在实时

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Intraspinal injection of recombinase dependent recombinant adeno-associated virus (rAAV) can be used to manipulate any genetically labelled cell type in the spinal cord. Here we describe how to transduce neurons in the dorsal horn of the lumbar spinal cord. This technique enables functional interrogation of the manipulated neuron subtype.

腺相关病毒介导的脊髓基因神经元的转基因表达

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...