斯基德莫尔学院发展基金资助的这个手稿的编写和出版。

1。安装胚胎（所选择可视化技术，如免疫细胞的完成， 原位杂交等 ） <ol><li>把胚胎在35毫米的培养皿中充满了选择的缓冲器（ 图1A）。缓冲液的选择是基于标记过程。通常PBS基缓冲剂是合适的。 </li><li>在解剖显微镜下，用钳，固定头与一对钳子的，而拉远蛋黄与另一对（ 图1B）。或者，使用昆虫针昆虫针固定器轻轻扯远了蛋黄。 </li><li>使用胚胎扑克（钓鱼线（0.41毫米直径）粘到任何一毛细管或巴斯德吸管）将胚的培养皿中不具有大量的卵黄碎片（ 图1C-D）的一部分。 </li><li>用玻璃巴氏吸管，吸在尽可能少的液体胚胎成为可能，并克ently吸取它们放到一个滑动（ 图1E）。 </li><li>轻轻地用实验室擦拭吸走多余的液体。避免与胚胎接触。 </li><li>加入一滴安装介质胚胎。对于荧光标记，使用选择的抗淬灭剂。对于色度信号，70％的甘油可以用作安装介质。 </li><li>利用胚胎扑克，定位于行（ 图1F），在其两侧的胚胎。 </li><li>加入凡士林或高真空硅脂之一“DAB”的盖玻片（ 图1G）的每个角落。这可以防止胚胎的过度压缩的伤害。 </li><li>放置盖玻片（凡士林面朝下）上的胚胎（ 图1H）的顶部。 </li><li>轻轻拍打在每个角落盖玻片，直到安装媒体（70％甘油或防褪色安装试剂）接触到盖玻片（ 图1H）。 </li><li>如果有必要，吹打旁边的CO​​V添加了更多的安装介质erslip，在20-200毫升的范围内使用移液器。这将灯芯下。 </li><li>用实验室擦拭，彻底干净周围的盖玻片。肯定不施加压力，以盖玻片，因为这样会损坏胚胎。这个区域必须是干燥的，没有凡士林或安装媒体上。 </li><li>用指甲油密封周围的盖玻片（ 图1I）的边缘。 </li></ol>

<ol>
	<li>Gribble, S. L., Nikolaus, O. B., Dorsky, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+function+of+Dbx+genes+in+the+zebrafish+spinal.">Regulation and function of Dbx genes in the zebrafish spinal.</a> Dev. Dyn. 236 (12), 3472-3483 (2007).</li><li>Bonner, J., 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=Midline+crossing+is+not+required+for+subsequent+pathfinding+decisions+in+commissural+neurons.">Midline crossing is not required for subsequent pathfinding decisions in commissural neurons.</a> Neural Dev. 7 (1), 18 (2012).</li><li>Ross, A. B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+Wnt+signaling+using+Lithium+Chloride:+Inquiry-Based+Undergraduate+Laboratory+Exercises.">Activation of Wnt signaling using Lithium Chloride: Inquiry-Based Undergraduate Laboratory Exercises.</a> Zebrafish. , (2012).</li><li>Bonner, J., 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=Proliferation+and+patterning+are+mediated+independently+in+the+dorsal+spinal+cord+downstream+of+canonical+Wnt+signaling.">Proliferation and patterning are mediated independently in the dorsal spinal cord downstream of canonical Wnt signaling.</a> Dev. Biol. 313 (1), 398-407 (2008).</li><li>Gribble, S. L., 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=Tcf3+inhibits+spinal+cord+neurogenesis+by+regulating+sox4a+expression.">Tcf3 inhibits spinal cord neurogenesis by regulating sox4a expression.</a> Development. 136 (5), 781-789 (2009).</li><li>England, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+Hedgehog+pathway+components+and+retinoic+acid+signalling+in+specifying+zebrafish+ventral+spinal+cord+neurons.">Roles of Hedgehog pathway components and retinoic acid signalling in specifying zebrafish ventral spinal cord neurons.</a> Development. 138 (23), 5121-5134 (2011).</li><li>Challa, A. K., Beattie, C. E., Seeger, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+roundabout+orthologs+in+zebrafish.">Identification and characterization of roundabout orthologs in zebrafish.</a> Mech Dev. (1-2), 101-101 (2001).</li><li>Lee, J. S., Ray, R., Chien, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+expression+of+three+zebrafish+roundabout+homologs+suggest+roles+in+axon+guidance+and+cell.">Cloning and expression of three zebrafish roundabout homologs suggest roles in axon guidance and cell.</a> 221 (2), 216-230 (2001).</li><li>Downes, G. B., Waterbury, J. A., Granato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+in+vivo+labeling+of+identified+zebrafish+neurons.">Rapid in vivo labeling of identified zebrafish neurons.</a> Genesis. 34 (3), 196-202 (2002).</li><li>Kim, H., 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=Notch-regulated+oligodendrocyte+specification+from+radial+glia+in+the+spinal+cord+of+zebrafish+embryos.">Notch-regulated oligodendrocyte specification from radial glia in the spinal cord of zebrafish embryos.</a> Dev. Dyn. 237 (8), 2081-2089 (2008).</li><li>Sylvain, N. J., Brewster, D. L., Ali, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+embryos+exposed+to+alcohol+undergo+abnormal+development+of+motor+neurons+and+muscle+fibers.">Zebrafish embryos exposed to alcohol undergo abnormal development of motor neurons and muscle fibers.</a> Neurotoxicol. Teratol. 32 (4), 472-480 (2010).</li><li>de Soysa, T. Y., 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=Macondo+crude+oil+from+the+Deepwater+Horizon+oil+spill+disrupts+specific+developmental+processes+during+zebrafish+embryogenesis.">Macondo crude oil from the Deepwater Horizon oil spill disrupts specific developmental processes during zebrafish embryogenesis.</a> BMC Biol. 10 (40), (2012).</li><li>Langenberg, T., Brand, M., Cooper, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+brain+development+and+organogenesis+in+zebrafish+using+immobilized+embryonic+explants.">Imaging brain development and organogenesis in zebrafish using immobilized embryonic explants.</a> Dev Dyn. 228 (3), 464-474 (2003).</li><li>Deschene, E. R., Barresi, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Targeted+Embryonic+Chimeras:+Zebrafish+Gastrula+Cell+Transplantation.">Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation.</a> J. Vis. Exp. (31), (2009).</li></ol>

没有竞争的经济利益存在任何作者。

脊髓发展的固定斑马鱼胚胎辅助可视化横向安装。通过除去卵黄球的，需要特殊的显微成像的工作距离被减小。我们的代表性的结果被限制到24个高倍视野的阶段，然而，这种技术可以早在18个高倍视野中使用，尽管早期胚胎是更难以解剖由于胚胎的大小和组织的脆弱性。这种技术也适用于年龄大于24个高倍视野胚胎。由胚胎斑马鱼的光学透明性，执行正向遗传筛选，反向遗传学的能力，并且转基因方法的帮助下，几种发育过程可能进行调查。这包括神经元祖细胞具有类似的BrdU和抗磷酸化组蛋白3月4日至6日 ，通过在PAX，NKX，DBX，并OLIG FAMILIE神经元和神经胶质祖细胞标记物的表达图案标记有丝分裂指数s 1,4-6。试剂用于报告胶质和神经元确定（如胶质纤维酸性蛋白（GFAP）和于克/ D）1,4,5,10都可以使用。神经元亚型的分化可以通过胰岛，VSX，engrailed基因和转录因子GATA基因1,4-6的表达进行分析。最后，轴突引导的分析有可能通过抗体诸如抗乙酰化的微管蛋白，3A10和ZNP-1 2,11,12。虽然其他脊椎动物模型系统（小鼠和鸡）被用来研究脊髓的发展，只有斑马鱼可以同时观看未受影响胚胎发育的所有轴。 最明显的限制，这种方法是，胚胎是固定的，这就排除了实时成像。事实上，除去（或损坏）的蛋黄结果中快速胚胎死亡，因此是不能够减少使用这种技术的工作距离在活的胚胎。然而，高倍率脊髓合作在活胚胎的RD成像是可能的。为了在实时成像以保持蛋黄，胚胎可以被放置在低气压的幻灯片，它提供的标本和盖玻片之间更大的空间。可选地，几个22毫米x 22毫米的盖玻片可以粘在×1一3在显微镜载玻片或幻灯片上。盖玻片作为一个“桥梁”，为覆盖玻片。如果胚胎是年龄超过18 HPF，三卡因用于麻醉胚胎，以防止移动。而与嵌入琼脂糖活胚胎中工作时，具有完整的蛋黄是必需的0.288毫米的工作距离为deyolked胚胎在24个高倍视野，一个显微镜头为3.3毫米的工作距离是足够的。或者，从deyolked胚来源的外植体培养可使用血浆凝块固定化技术13可视化。细胞的不同人群可以通过使用遗传编码荧光蛋白如GFP，mCherry的，或者photocon的被观察vertible染​​枫（仅举几例）。另外，嵌合体胚胎内荧光标记的细胞也可以用这种方法14观看。 一致的安装技术，可稳定几个月是发育和细胞生物学的重要工具。这个简单的技术是可重复的，允许不同的实验试验之间易于比较。此外，在胚胎行的对齐方式容易进行鉴定后分析具体的胚胎。

在斑马鱼中的脊髓的可视化是由若干因素的抑制。由于上覆体节的厚度和脊髓的内部位置，在相当长的工作距离是必需的高细胞分辨率。卵黄球（它仍然存在于神经发生的早期阶段），进一步增加了所需的工作距离，并且很容易通过从盖玻片压力损坏。此外，从受损的蛋黄碎屑禁止清晰的组织的可视化。虽然在背腹（DV）轴的横截面是可能的，它们不容易允许同时可视化中的前-后（AP）的轴1。 为了克服这些障碍，胚胎解剖和幻灯片上。此过程提供了一些好处。首先，斑马鱼胚胎容易出在侧面朝上，这有利于AP轴可视化。第二，去除蛋黄的BAL升降低要求的工作距离，并且限制碎屑。三，本次安装程序允许荧光和明场显微镜。四，安装的胚胎是稳定了几个月，在4°C，使长时间的标本可视化。最后，开发进展发生在眼前段比后段更成熟。为了确保分级匹配脊hemisegments胚胎之间进行比较时，上覆体节组织作为一个指南。例如，第十最后部体节覆盖在第十脊hemisegment，这是在阶段相匹配的胚胎发育当量。完整胚胎此安装程序可方便识别体节。 我们利用遗传学和基因敲除技术研究轴突导向的机制在发展中脊髓。特别是，我们已经确定ROBO2，ROBO3，和DCC所需的连合小学ASCEnding（COPA）轴突路径。使用此处描述的安装过程，我们能够审视腹增长，中线交叉，合缝宽度，背鳍和前增长2,3。该安装过程也可以应用到DV或脊髓的AP构图。我们已经用这种方法来确定在脊髓的DV图案Wnt信号的下游效应物不同的角色。使用这种安装，我们能够取得的DV标志的分辨率在单细胞水平，并确定分钟图案变化为改变的Wnt信号接收4,5的结果。此安装程序还允许有丝分裂指数的计算通过抗磷组3或BrdU标记（祖细胞增殖）分化5。

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dissection and lateral mounting of zebrafish embryos: analysis of spinal cord development

斑马鱼的脊髓是神经系统研究的一个有效的调查模式有以下几个原因。首先，基因，转基因和基因敲除的方法可以被用来检查相关的神经系统发育的分子机制。其次，同步发育胚胎的大型离合器提供大型实验的样本量。三，斑马鱼胚胎的光学清晰度允许研究人员能够可视祖，胶质和神经元群体。尽管斑马鱼的胚胎是透明的，试样厚度可以阻碍有效的微观可视化。其中一个原因是脊髓和覆体节组织的串联发展。另一个原因是大卵黄球，这仍然是在早期神经发生期间存在。在这篇文章中，我们展示了显微切割和拆除蛋黄固定胚胎，这使得微观可视化，同时保留周围的体节的组织。我们阿尔斯Ø证明斑马鱼胚胎的半永久性的安装。这就允许观察神经发育中的背腹和前后轴线，因为它保留了立体感的组织。

当阐明背后诸如蜂窝图案形成，分化和轴突导向各个发育事件的机制，它能够为他们的组织的范围内可视化的细胞是重要的。背腹图案缺陷的典型表现为腹部或背部转变的转录因子的表达域在PAX，NKX，或DBX家庭4。有时，变化可能是微妙的，包括只有少数细胞直径4。截面分析允许这种类型的分析。然而，试剂与较弱的信号，或者横截面是不垂直于轴的AP可以限制解释。此外，截面分析不容易考虑到的变化是否坚持在AP轴，并依靠低温恒温器的可用性。这个限制是由脊髓侧景规避。正如图2B所示 ，NKX6.1 mRNA表达为distributed中在24小时后受精（HPF）的脊髓大致腹侧一半，祖域之内。祖细胞是从由于底板，这是在脊髓的中间部分发现的存在下交的有丝分裂的细胞区分开来。与DIC的光学来看，单个细胞清晰可辨，并表达和nonexpressing细胞之间的边界定义是显而易见的。 祖细胞周期出口是伴随着基因表达的变化。例如，所有的有丝分裂后神经元表达HUC / D，它是检测与免疫细胞化学1,5。基因探针胰岛，GATA和VSX系列标记每个脊髓hemisegment 6中的一贯立场和一些具体的有丝分裂后神经元。此外，轴突导向受体，如那些在机器人的家庭表示在有丝分裂后神经元的受限人群（ 图2C） <suP&gt; 2,7,8。胚胎横向安装允许有丝分裂后神经元的准确计数。同样，该祖细胞继续分裂可以量化与抗磷酸化组蛋白3免疫组织化学以及BrdU标记。此外，细胞死亡可以与TUNEL标记4-6进行评估。 在24个高倍视野，下面的有丝分裂后神经元的轴突可以用各种方法来可视化，并且是可区分的在单细胞水平：背外侧递增（多拉），连合主升序（COPA），连合次要升序（COSA），腹侧纵降序（草原），Kolmer-Agdur（KA），连合分叉/纵向（COB / L），圆周升序（CIA），圆周递减（CID）和单极连合递减（UCOD），Rohon - 比尔德（RB），运动神经元（ M）9。轴突从这些神经元在背，腹，前和后方向上延伸。他们的分支，越过中线，并退出这两个背和脊髓腹侧。如果再加上激光共聚焦扫描显微镜，这些不同的细胞行为是明显的横向安装的胚胎，用免疫细胞化学和基因编码的荧光蛋白如GFP 2。在图2D中 ，ZNP-1免疫细胞化学法用于标记显影运动神经元。退出运动神经元脊髓腹侧以支配周围的肌肉发展。 <img alt="图1" fo:content-width="6in" fo:src="/files/ftp_upload/50703/50703fig1highres.jpg" src="/files/ftp_upload/50703/50703fig1.jpg" width="500px" /> 图1。解剖和横向装载斑马鱼胚胎（A）处理后，免疫荧光， 原位杂交等 ，胚胎放置在35毫米培养皿中填充的PBT（PBS含有0.5％的Triton X-100）或PTW（PBS与0.1％吐温20）。卵黄球（YB）可见一斑。 （B）中的蛋黄是由固定的胚胎随后小心剥离的头部去除。 （C）一种胚胎扑克（渔线粘到巴斯德吸管）用于单独的胚胎和蛋黄碎片（D）。清洗胚胎吸移到载玻片（E）和对齐（F）。 （G）凡士林加到盖玻片的角落。 （H）的盖玻片轻轻地放置在安装介质中的胚胎的顶部。 （ 一）指甲油是用来密封盖玻片的边缘。 <a href="https://www.jove.com/files/ftp_upload/50703/50703fig1highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" fo:content-width="3.5in" fo:src="/files/ftp_upload/50703/50703fig2highres.jpg" src="/files/ftp_upload/50703/50703fig2.jpg" width="500px" /> &lt;<html> STRONG&gt;图2。基因表达模式和轴突路径的分析横向安装的斑马鱼胚胎（A）在24 HPF绘制一个斑马鱼胚胎。在BD，上面卵黄囊延长大约4 hemisegments被可视化（盒装区）。 （B）NKX6.1表达于脊髓腹侧，包括底板（FP）。 （ 三）ROBO3表现在有丝分裂后神经元细胞（PMN）。底板和祖区不是体现在这个更横向焦平面。在B，C，脊髓是通过在图像的左侧的支架为界。 （D）的共焦显微镜检查用于图像ZNP-1免疫荧光法，其中标记运动神经元轴突腹侧离开脊髓。中的所有图像，前壁是向左和背到了。一个40X长工作距离，开水浸泡（NA 0.8）镜头使用（3.3毫米）。oad/50703/50703fig2highres.jpg“目标=”_blank“&gt;点击这里查看大图。

Watch this Scientific Journal Video about Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development at JoVE.com

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

发育过程，如增殖，图案，分化和轴突导向可以很容易地模拟在斑马鱼的脊髓。在这篇文章中，我们描述了一个安装程序，斑马鱼的胚胎，从而优化了这些事件的可视化。

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 ...

dissection-lateral-mounting-zebrafish-embryos-analysis-spinal-cord

Research

JoVE Journal

Neuroscience

13.7K 次观看.  Skidmore College. 发育过程，如增殖，图案，分化和轴突导向可以很容易地模拟在斑马鱼的脊髓。在这篇文章中，我们描述了一个安装程序，斑马鱼的胚胎，从而优化了这些事件的可视化。 

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

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

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

侧向液压冲击：创伤性脑损伤小鼠模型

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

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 ...

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 ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

完全性脊髓损伤和脑解剖协议后续Wholemount<em&gt;原位</em&gt;杂交苗种海七鳃鳗

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

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.

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 ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

斑马鱼<em&gt;原位</em&gt;脊髓制备用于脊髓感觉和运动神经元的电生理记录

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

成人脊髓细胞核在大规模平行单核 RNA 测序中的分离

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

幼虫斑马鱼外系头发细胞的活体钙成像

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...