<p class="jove_content"我们感谢博士尤瓦的戈特利布-德罗尔电插图。这项工作是由渠务署授予国家在以色列的心理生物学研究所和萨克森 - 以色列研究合作计划和补助AK从以色列科学基金会，以色列卫生部和优秀传统遗产中心的支持生物医学科学合作。</p&gt;

1。后脑电穿孔 1.1处理蛋<ol><li>将鸡蛋在培养箱（37-38.5°C）水平。胚胎电孵育65-70小时后，当他们到达16-17（HH）阶段（25-30体节）。 </li><li>从培养箱中取出鸡蛋，它们保持在水平位置。 </li></ol> 1.2准备工作<ol><li>上拉玻璃毛细管（直径0.5 mm）。 </li><li>与L形的弯曲的黄金Genetrodes电极（直径3 mm）连接到脉冲发生器的适配器保持。电参数包括25伏，5个脉冲数，45毫秒脉冲长度，300毫秒脉冲间隔。 </li><li>准备口吸管，10毫升的注射器针头，封口膜条，软毛刷，锋利的解剖剪刀和25毫升无菌PBS溶液。这个数额通常是足以应付一个鸡蛋托盘（鸡蛋18）。 </li><li>准备了充足的DNA质粒的混合物（5微克/微升）和增加约0.025％的快速绿色染料，以可视化DNA注射。一般是足够的一个托盘的注射体积为4微升。 </li></ol> 1.3窗口，注射和电<ol><li>用70％乙醇清洁鸡蛋壳。 </li><li>一次处理一个鸡蛋胚胎曝光时间，以尽量减少在空气中。 </li><li>用剪刀结束了一个洞的鸡蛋极白蛋白用注射器除去3-4毫升。不要取出白蛋白量较大，因为它降低了生存能力。 </li><li>打开一个椭圆形的小窗口（2.5×2厘米大小）用解剖剪刀将鸡蛋壳上的中心。 </li><li>的PBS淌下几滴之上的胚胎，在整个过程中，以防止干燥。 </li><li>加载少量的DNA混合物到玻璃毛细管口移液管使用。 </li><li>将胚胎后端向你。没有墨水喷射需要在此阶段的胚胎是清晰可见的。避免墨水注入增加胚胎存活。尾后脑穿透毛细管〜45°。不要删除任何来自各地的胚胎膜。仔细DNA溶液注入到后脑腔和呼气，而形成气泡。 DNA溶液向前传播。 </li><li> DNA注射后，立即将平行电极在精确的AP和DV后脑的位置，你的目标是目标。腹侧电极小幅推而不触及心脏，因为它可能会降低活力。脉冲电穿孔。电极应覆盖有气泡，表示导电性（ 图1A）。 </li><li>小心地取出电极和滴几滴的PBS，来冷却胚胎，并除去气泡。密封正确打开的鸡蛋在孵化过程中，以防止干燥用封口膜条。用软毛刷清洗电极在PBS。 </li><li>将蛋在孵化器中所需的时间长度。胚胎的生存期是大约90％后2-3天电穿孔，并减少70％的12天电穿孔。 </li></ol> 2。分析胚胎 2.1平面安装准备和免疫<ol><li>平板式筹备的后脑可以执行E6.5显微镜或立体显微镜下轻松地可视化轴突。 </li><li>通过仔细解剖胚胎从周围膜和血管分开。将胚胎在一个小的硅 - 涂层培养皿中含有PBS。钨针朝向背侧，将胚胎的菜。执行后脑屋顶板使用的是锋利的玻璃毛细管中缝背。 </li><li>使用尖锐的镊子和微型剪刀持有的头部和上部拉后脑组织非常仔细地从吻端到尾鳍。后脑分离后，​​除去剩余的腹侧组织，以达到清洁制剂。 </li><li>孵育后脑4％多聚甲醛（PFA）的溶液在室温下1-2小时（RT）。 PBT（PBS/0.1％的Triton X-100）后脑洗净去除PFA。 </li><li>对于在平面安装hindbrains免疫荧光，加500微升的封闭液（5％山羊血清在室温下孵育2小时，用第一抗体对在4℃下洗涤（15分钟×3）与PBT PBT和孵育孵育与第二抗体在RT下搅拌2小时。与PBT（15分钟×3）进行清洗。 </li><li>放置在玻片上，后脑朝向背侧。日光灯的安装介质添加到后脑。使用玻璃毛细管或钨针，后脑平坦的幻灯片上。 </li><li>添加少量润滑脂在每一个角落，以防止挤压的组织由盖玻片。小心地放置盖玻片后脑上，避免产生气泡。对于坚持补充指甲油覆盖玻璃边缘。 </li></ol> 2.2冷冻切片和免疫<ol><li>低温部分的脑干CAN是在任何阶段轴突轨迹可视化。然而，E6.5后，这应该是优选的方法，因为可视化的平面安装的胚胎，变得太厚的轴突的难度。此外，示范突触需要切片的组织和高倍率显微镜下检视。 </li><li>解剖胚胎，并删除所有周围的组织。用PBS冲洗。削减的胚胎尾部分的髓质使用微型剪刀和尖锐的镊子。小脑和脑之间的一个大切口，取出整个脑干。组织应包含延髓，脑桥和小脑。 </li><li>在4％PFA过夜（ON）在4℃，修复脑干，用PBS（10分钟×3）冲洗，并在4°C，直到那颗孵育30％的蔗糖。 </li><li>低温模具中嵌入后脑与华侨城（最佳切削温度）的化合物，将其放置在-20°C至冻结，如在其他地方11。 </li><li&gt;在OCT块放置在低温恒温器中所需的剖轴。横截面宽度为12μm。 </li><li>干燥的幻灯片。添加100微升封闭液在室温下2小时，每张幻灯片上。添加100μl的抗体在封闭溶液稀释至所需浓度。 1次和第2次抗体的潜伏期为ON时，在4℃下2小时，分别在RT，。对于每个温育时间，轻轻地添加封口膜条的顶部滑动，以防止干燥。用PBS洗涤（10分钟×3）抗体之间。如果需要，添加大埤1:1,000（4&#39;-6-二脒基-2 - 苯基吲哚），在PBS中稀释，在RT下20分钟。用PBS冲洗。 </li><li>安装幻灯片日光灯安装媒体。让干1小时前分析。 </li></ol>

<ol>
	<li>Altman, J., Bayer, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+precerebellar+nuclei+in+the+rat:+II.+The+intramural+olivary+migratory+stream+and+the+neurogenetic+organization+of+the+inferior+olive.">Development of the precerebellar nuclei in the rat: II. The intramural olivary migratory stream and the neurogenetic organization of the inferior olive.</a> J. Comp. Neurol. 257, 490-512 (1987).</li><li>Rose, M. F., Ahmad, K. A., Thaller, C., Zoghbi, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitatory+neurons+of+the+proprioceptive,+interoceptive,+and+arousal+hindbrain+networks+share+a+developmental+requirement+for+Math1.">Excitatory neurons of the proprioceptive, interoceptive, and arousal hindbrain networks share a developmental requirement for Math1.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 22462-22467 (2009).</li><li>Storm, R., 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=The+bHLH+transcription+factor+Olig3+marks+the+dorsal+neuroepithelium+of+the+hindbrain+and+is+essential+for+the+development+of+brainstem+nuclei.">The bHLH transcription factor Olig3 marks the dorsal neuroepithelium of the hindbrain and is essential for the development of brainstem nuclei.</a> Development. 136, 295-305 (2009).</li><li>Lumsden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cellular+basis+of+segmentation+in+the+developing+hindbrain.">The cellular basis of segmentation in the developing hindbrain.</a> Trends Neurosci. 13, 329-335 (1990).</li><li>Liu, Z., 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=Control+of+precerebellar+neuron+development+by+Olig3+bHLH+transcription+factor.">Control of precerebellar neuron development by Olig3 bHLH transcription factor.</a> J. Neurosci. 28, 10124-10133 (2008).</li><li>Muller, T., 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=The+bHLH+factor+Olig3+coordinates+the+specification+of+dorsal+neurons+in+the+spinal+cord.">The bHLH factor Olig3 coordinates the specification of dorsal neurons in the spinal cord.</a> Genes Dev. 19, 733-743 (2005).</li><li>Avraham, O., 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=Motor+and+dorsal+root+ganglion+axons+serve+as+choice+points+for+the+ipsilateral+turning+of+dI3+axons.">Motor and dorsal root ganglion axons serve as choice points for the ipsilateral turning of dI3 axons.</a> J. Neurosci. 30, 15546-15557 (2010).</li><li>Avraham, O., 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=Transcriptional+control+of+axonal+guidance+and+sorting+in+dorsal+interneurons+by+the+Lim-HD+proteins+Lhx9+and+Lhx1.">Transcriptional control of axonal guidance and sorting in dorsal interneurons by the Lim-HD proteins Lhx9 and Lhx1.</a> Neural Dev. 4, 21 (2009).</li><li>Avraham, O., Zisman, S., Hadas, Y., Vald, L., Klar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+axonal+pathways+of+genetically+defined+groups+of+neurons+in+the+chick+neural+tube+utilizing+in+ovo+electroporation.">Deciphering axonal pathways of genetically defined groups of neurons in the chick neural tube utilizing in ovo electroporation.</a> J. Vis. Exp. (39), e1792 (2010).</li><li>Kohl, A., Hadas, Y., Klar, A., Sela-Donenfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+Patterns+and+Targets+of+dA1+Interneurons+in+the+Chick+Hindbrain.">Axonal Patterns and Targets of dA1 Interneurons in the Chick Hindbrain.</a> J. Neurosci. 32, 5757-5771 (2012).</li><li>Vogel, J., Mobius, C., Kuschinsky, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+delineation+of+ischemic+tissue+in+rat+brain+cryosections+by+high-contrast+staining.">Early delineation of ischemic tissue in rat brain cryosections by high-contrast staining.</a> Stroke. 30, 1134-1141 (1999).</li><li>Helms, A. W., Abney, A. L., Ben-Arie, N., Zoghbi, H. Y., Johnson, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoregulation+and+multiple+enhancers+control+Math1+expression+in+the+developing+nervous+system.">Autoregulation and multiple enhancers control Math1 expression in the developing nervous system.</a> Development. 127, 1185-1196 (2000).</li><li>Lumpkin, E. A., 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=Math1-driven+GFP+expression+in+the+developing+nervous+system+of+transgenic+mice.">Math1-driven GFP expression in the developing nervous system of transgenic mice.</a> Gene Expr. Patterns. 3, 389-395 (2003).</li><li>Lu, Y., Lin, C., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PiggyBac+transgenic+strategies+in+the+developing+chicken+spinal+cord.">PiggyBac transgenic strategies in the developing chicken spinal cord.</a> Nucleic Acids Res. 37, e141 (2009).</li><li>Wang, 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=piggyBac-like+elements+in+the+pink+bollworm,+Pectinophora+gossypiella.">piggyBac-like elements in the pink bollworm, Pectinophora gossypiella.</a> Insect Mol. Biol. 19, 177-184 (2010).</li><li>Alsina, B., Vu, T., Cohen-Cory, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+synapse+formation+in+arborizing+optic+axons+in+vivo:+dynamics+and+modulation+by+BDNF.">Visualizing synapse formation in arborizing optic axons in vivo: dynamics and modulation by BDNF.</a> Nat. Neurosci. 4, 1093-1101 (2001).</li><li>Leal-Ortiz, 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=Piccolo+modulation+of+Synapsin1a+dynamics+regulates+synaptic+vesicle+exocytosis.">Piccolo modulation of Synapsin1a dynamics regulates synaptic vesicle exocytosis.</a> J. Cell Biol. 181, 831-846 (2008).</li><li>Gardzinski, P., 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=The+role+of+synaptotagmin+I+C2A+calcium-binding+domain+in+synaptic+vesicle+clustering+during+synapse+formation.">The role of synaptotagmin I C2A calcium-binding domain in synaptic vesicle clustering during synapse formation.</a> J. Physiol. 581, 75-90 (2007).</li><li>Nowack, A., Yao, J., Custer, K. L., Bajjalieh, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SV2+regulates+neurotransmitter+release+via+multiple+mechanisms.">SV2 regulates neurotransmitter release via multiple mechanisms.</a> Am. J. Physiol. Cell Physiol. 299, C960-C967 (2010).</li><li>Itasaki, N., Sharpe, J., Morrison, A., Krumlauf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+Hox+expression+in+the+vertebrate+hindbrain:+influence+of+paraxial+mesoderm+and+rhombomere+transposition.">Reprogramming Hox expression in the vertebrate hindbrain: influence of paraxial mesoderm and rhombomere transposition.</a> Neuron. 16, 487-500 (1996).</li><li>Clarke, J. D., Lumsden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segmental+repetition+of+neuronal+phenotype+sets+in+the+chick+embryo+hindbrain.">Segmental repetition of neuronal phenotype sets in the chick embryo hindbrain.</a> Development. 118, 151-162 (1993).</li><li>Diaz, C., Glover, J. C., Puelles, L., Bjaalie, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+hodological+and+cytoarchitectonic+organization+in+the+vestibular+complex+of+the+11-day+chicken+embryo.">The relationship between hodological and cytoarchitectonic organization in the vestibular complex of the 11-day chicken embryo.</a> J. Comp. Neurol. 457, 87-105 (2003).</li><li>Marin, F., Puelles, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+fate+of+rhombomeres+in+quail/chick+chimeras:+a+segmental+analysis+of+hindbrain+nuclei.">Morphological fate of rhombomeres in quail/chick chimeras: a segmental analysis of hindbrain nuclei.</a> Eur. J. Neurosci. 7, 1714-1738 (1995).</li><li>Niwa, H., Yamamura, K., Miyazaki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+selection+for+high-expression+transfectants+with+a+novel+eukaryotic+vector.">Efficient selection for high-expression transfectants with a novel eukaryotic vector.</a> Gene. 108, 193-199 (1991).</li></ol>

<p class="jove_content"&gt;没有利益冲突的声明。</p

OVO中电是一个可行的，可靠的，有效的工具来检查细胞规范和轴突指导在鸡胚神经系统的发展20。在这个协议中，我们描述了一个模式把小鸡后脑电E2.75使用增强子元件，​​使有条件的标签特定的interneurons。这一战略的结合换位系统PiggyBac转介导外源基因插入到鸡基因组，这使跟踪的的轴突航线，预测和突触网站在胚胎发育晚期。 以前的方法标签轴突/把小鸡?...

后脑是一个关键的中继枢纽神经系统之间的通信中枢和周围神经系统神经网络通过递增和递减。它调节的基本功能，包括呼吸，意识，听觉和运动协调1-3。在早期胚胎发育过程中，脊椎动物的后脑瞬时细分成重复菱沿其前后轴（AP），不同的神经细胞类型的形成，并产生多个脑干中心4。后脑也被划分成基底和鼻翼板，在指定离散神经祖细胞变得和区分不同的DV地点3,5,6沿背腹轴（DV）。怎样早期AP和DV特定的神经元模式的脑干功能设计电路的设立在很大程度上是未知。 为了获得这个基本知识的问题，工具都需要以特定亚群的神经元在早期后脑贴上标签，以跟踪它们的轴突轨迹和连接更高级的阶段。我们先前已经利用特异性增强子元件，和一个CRE / LoxP位条件的表达背脊髓在早期鸡胚7-9的轴突轨迹跟踪系统。在当前的手稿中，我们有针对性的后脑和标记后期胚胎后脑的interneurons，轴突和其突触目标，使用修改后的电战略和PiggyBac转 - 介导DNA换位的升级实验范式。我们的新战略，可以在一侧的后脑和他们的的轴突预测和突触位点不同的胚胎阶段的跟踪标记不同的神经元亚型，从2至12天电。基于这种方法，我们贴上了最背的分组后脑的interneurons（dA1/Atoh1 + </s&gt;细胞），并揭示了两个对侧上升轴突投射图案，分别来自不同AP的位置和拉长了一个独特的索。 DA1轴突被发现在听觉核，中脑，小脑10多层的项目，并形成突触。 小鸡电的结合，遗传神经元和先进得多的发展阶段分析预测网站跟踪研究在大脑中形成的神经网络，并阐明控制电路形成的分子机制，提供一个独特的平台。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Equipment</td>
			<td>Company</td>
			<td>Catalogue Number</td>
		 </tr>
		 <tr>
			<td>L-shaped gold Genetrodes 3 mm electrodes</td>
			<td>BTX, Harvard Apparatus</td>
			<td>45-0162 </td>
		 </tr>
		 <tr>
			<td>pulse generator, ECM 830</td>
			<td>BTX, Harvard Apparatus</td>
			<td>45-0002</td>
		 </tr>
		 <tr>
			<td>OCT (Optimal Cutting Temperature) Compound</td>
			<td>Tissue-Tek
				Sakura</td>
			<td>4583 O.C.T. Compound </td>
		 </tr>
		 <tr>
			<td>Nail Polish</td>
			<td>From Any Commercial Supplier </td>
			<td></td>
		 </tr>
		</tbody>
 </table>

electroporation of the hindbrain to trace axonal trajectories and synaptic targets in the chick embryo

鸡胚神经管电具有许多优点，如快速，高效的成神经细胞的外源基因表达。在这篇稿件中，我们提供了一种方法，展示了独特的电穿孔DNA禽流后脑E2.75为了明确标示神经祖细胞的一个子集，以及如何按照他们的轴突预测和突触的目标非常先进的发展阶段， E14.5。我们利用新的遗传工具，包括特异性增强子元件，​​酶Cre /液氧 - 基于质粒和PiggyBac转介导的DNA换位后脑细胞的一种亚型（最背的分组的interneurons，DA1）系统来驱动GFP表达。随后在早期和晚期胚胎阶段，在不同的脑干区域的轴突轨迹和DA1轴突的目标。这种策略有助于先进的技术，针对感兴趣的细胞在胚胎后脑TRA庆安电路形成多个发展阶段。

这个协议最近发现轴突的模式和投影网站的DA1群的interneurons在小鸡后脑10。要具体标明这些轴突，增强子元件（Atoh1的），先前已经被作为具体的脊柱DI1神经元8,12,13其特征在于，确认表达在后脑DA1单元10。该元件上游克隆到Cre重组和合作电在E2.75随着一个CRE依赖细胞质GFP记者质粒（pCAGG LoxP位停止LoxP位cGFP的图1BI）。由平坦的安装准备分析hindbrains 48小时以下的电?...

Watch this Scientific Journal Video about Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo at JoVE.com

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

神经网络是建立在胚胎大脑发育神经生物学的一个基本问题。在这里，我们结合电穿孔技术与新的遗传工具，如酶Cre / LOX-质粒和PiggyBac转介导的DNA换位系统在禽流后脑背中间贴上标签，并跟踪他们的轴突预测和突触在不同发育阶段的目标。

电穿孔轴突轨迹跟踪和突触目标的后脑在鸡胚

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

electroporation-hindbrain-to-trace-axonal-trajectories-synaptic

Research

JoVE Journal

Neuroscience

10.8K 次观看.  The Hebrew University of Jerusalem. 神经网络是建立在胚胎大脑发育神经生物学的一个基本问题。在这里，我们结合电穿孔技术与新的遗传工具，如酶Cre / LOX-质粒和PiggyBac转介导的DNA换位系统在禽流后脑背中间贴上标签，并跟踪他们的轴突预测和突触在不同发育阶段的目标。

视频: Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1 . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

在斑马鱼后脑细胞标记和成像

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

科研

神经科学

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent progenitor cells to be an integral part of the neural crest1. Phenotypically, neural crest stem cells (NCSC) are defined by simultaneously expressing p75 (low-affine nerve growth factor receptor, LNGFR) and SOX10 during their migration from the neural crest2,3,4,5. These progenitor cells can differentiate into smooth muscle cells, chromaffin cells, neurons and glial cells, as well as melanocytes, cartilage and bone6,7,8,9. To cultivate NCSC in vitro, a special neural crest stem cell medium (NCSCM) is required10. The most complex part of the NCSCM is the preparation of chick embryo extract (CEE) representing an essential source of growth factors for the NCSC as well as for other types of neural explants. Other NCSCM ingredients beside CEE are commercially available. Producing CCE using laboratory standard equipment it is of high importance to know about the challenging details as the isolation, maceration, centrifugation, and filtration processes. In this protocol we describe accurate techniques to produce a maximized amount of pure and high quality CEE.

To cultivate neural crest stem cells (NCSC) in vitro, a special medium (NCSCM) is required. Essential part of NCSCM is chick embryo extract (CEE). We here describe accurate techniques to produce a maximized amount of pure and high quality CEE, including details as the isolation, maceration, centrifugation, and filtration processes.

小鼠神经嵴干细胞的培养鸡胚提取物的生产

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.

We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface.&#160;The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.

使用普洛林来评估阿克森指导受体在细胞培养和小鸡胚胎中的动力学

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

嗅觉系统作为模型来研究轴突生长的模式和形态<em&gt;在体内</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

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 ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

使用的跟踪经典条件反射，评估胎儿酒精谱系障碍大鼠模型海马功能障碍

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions in the immature and adult brain. Here we describe a zinc histochemical staining procedure that reveals differences in staining patterns among different layers and brain regions. Others have utilized this procedure not only to reveal the distribution of zinc-containing neurons and circuits in the brain, but also to successfully delineate areal and laminar boundaries in the developing and adult brain in several species. Here we illustrate this staining procedure with images from developing and adult ferret brains. We reveal a zinc-staining pattern that serves as an anatomical marker of areas and layers, and can be reliably used to distinguish visual cortical areas in the developing and adult visual cortex. The main goal of this protocol is to present a histochemical method that allows the accurate identification of layers and regions in the developing and adult brain where other methods fail to do so. Secondarily, in conjunction with densitometric image analysis, this method allows one to assess the distribution of synaptic zinc to reveal potential changes throughout development. This protocol describes in detail the reagents, tools, and steps necessary to successively stain frozen brain sections. Although this protocol is described using ferret brain tissue, it can easily be adapted for use in rodents, cats, or monkeys as well as in other brain regions.

We describe a histochemical procedure that reveals characteristic laminar and areal zinc staining patterns in different brain regions. The zinc-staining pattern may be used in conjunction with other anatomical markers to reliably distinguish layers and regions in the developing and adult brain.

利用突触锌组织化学在发育和成人大脑中揭示不同的区域和叶片

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

光遗传学与人工 microRNAs 相结合对完整神经元回路中基因击倒对突触前功能的影响

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...