这项工作是由威康信托计划资助授予PD和GL（089236/Z/09/Z）。 SG是由生物技术和生物科学研究理事会博士助学金支持。我们感谢马修Vermeren的shRNA表达载体和谢珍就成神经细胞核转染宝贵意见的一种恩赐。

这个程序是按照英国内政部规定（动物科学程序法，1986）。科学家应该遵循建立并获当地机构和国家动物监管机构的指引。 1。解剖和大鼠的RMS神经母细胞解离<ol><li>准备所需的RMS解剖和分离的解决方案： </li></ol> 夹层介质 （100毫升）中 Hank平衡盐溶液（HBSS） - 98.5毫升 5个M HEPES pH值7.4 - 0.5毫升 青霉素，链霉素（10,000单位/ ml和10,000微克/毫升） - 1毫升 解离培养基（2ml）中 HBSS - 1.760毫升 10倍的胰蛋白酶（2.5％） - 200微升 DNAse1（1毫克/毫升） - 40μL 贝科改良Eagle培养基（DMEM）+ 10％胎牛血清（FCS）（40毫升） DMEM -36毫升 FCS - 4毫升 完全培养基 （12毫升）中 Neurobasal培养基 - ​​11.46毫升 B27的补充 - 250微升 L-谷氨酰胺（200毫米） - 125微升 葡萄糖（45％） - 165微升 2。过滤灭菌的DMEM +10％FCS中，并在完全培养基中并preequilibrate它们在37℃/ 5％CO 2的培养箱中培养。 <ol><li>解剖<ol><li>颈椎脱臼牺牲P6-P7鼠窝（约12幼崽），并斩首用剪刀。 </li><li>使前后切口沿正中矢状缝线从鼻子到用手术刀刀片小脑皮肤。剥了一层皮，重复沿着头骨同一切口。 </li><li>轻轻取下颅瓣用钳子，仔细用锅铲取出大脑，照顾到包括嗅球。 </li><li>切断大脑的最尾端第三和丢弃它。 </li><li>斩日Ë脑组织成使用组织菜刀1.4毫米厚的冠状切片。 </li><li>将切片在含冷夹层介质的菜肴，仔细用针把它们分开。 </li><li>有效值显示为一个三角形，半透明区域的OB部分的中心，作为一个小的，圆形的面积更尾大脑切片。切出的RMS每片用显微刀，同时注意避免包括周围组织。在P7的幼鼠，通常〜8最喙片（包括OB）包含有效值。 </li><li>收集RMS片段用塑料巴斯德吸管，并将其放置在包含在冰冷的夹层介质一小碟。 </li><li>当清扫结束后，转移RMS片段入15ml试管用塑料移液管。留下的碎片，收于管子的底部。 </li></ol></li><li>解离<ol><li>用2毫升分离培养基更换夹层介质。 </li><li>轻轻p磨碎的RMS片段ipetting片段悬挂上下左右10倍使用P1000的移液管。 </li><li>离开该管的组织片段在37℃水浴中2分钟。 </li><li>再次吸取溶液10倍，并确保片段已经分离（暂停应该变得浑浊）。 </li><li>通过加入5ml预热的DMEM +10％FCS中的灭活胰蛋白酶。 </li><li>离心细胞悬浮液在433×g离心5分钟。 </li><li>在此期间等分试样所需的的siRNA / DNA的量成Eppendorf管中（通常是3-5微克DNA / shRNA的或每核转染5-9微克的siRNA寡核苷酸，然而DNA / siRNA的量，可能需要优化）。 </li><li>去除多余的介质，并轻柔吹打在5毫升预热的DMEM +10％FCS的重悬细胞沉淀。 </li><li>进行细胞计数。期望〜1×10 6每只大鼠幼鼠的脑细胞。最小2.5×10 6个细胞都需要为每个核转染，而最佳的结果是使用核转染每3-4×10 6细胞来实现。 </li><li>离心细胞悬浮液在433×g离心5分钟。一定要去除尽可能多的媒介越好。 </li></ol></li></ol> 2。核转染<ol><li> 立即 （如果使用小鼠细胞或小鼠）先前在室温下培养神经元的核转染溶液重悬细胞沉淀在大鼠。使用核转染每100微升注 ：通常为12幼仔大鼠垫料足以执行4 nucleofections和鼠标窝（12幼仔）足以执行2 nucleofections。 </li><li>转移100微升的细胞悬液至含有的siRNA / DNA的每个Eppendorf管中，用P200移液管轻轻混匀2-3倍通过移液。 </li><li>样品（cell-DNA/siRNA悬浮液）加入到nucleofectioncuvette的底部，注意避免气泡。 </li><li>使用Nucleofect程序的G-013（用于大鼠细胞）或O-005（用于小鼠细胞）。一核转染大约需要5秒。 </li><li>快速加入1 ml预热的DMEM +10％FCS中的nucleofected样品。 </li><li>重复步骤2.4和2.5所有其他样本。注意：为达到最佳效果，在整个核转染过程应该持续时间不超过5分钟。 </li><li>每个样品转移到15ml试管含有5ml预热的DMEM +10％FCS中的使用通过核转染试剂盒提供的塑料吸管。避免任何细胞碎片转移到管中。 </li><li>离心样品在433×g离心5分钟。 </li><li>小心地取出所有多余的介质，并使用P20吸管悬浮颗粒在25-30微升预热的DMEM +10％FCS的。不要使用超过30微升培养基。 </li><li>移液器的悬浮液一滴到p35的培养皿盖的内侧。 </li><li>逆转盖子以上含2ml完全培养基中的p35的培养皿（也参见图1）。 </li><li>留在培养箱（37℃/ 5％CO 2）在至少5小时和最多7小时。较长的培育时间可以更好地再聚集的细胞簇。 </li><li>从转移的盖子吊滴成使用P1000的吸管切尖这道菜的完全培养基。 </li><li>在37℃/ 5％CO 2 24小时进行DNA nucleofections和48小时用于siRNA / shRNA的nucleofections。 </li></ol> 3。嵌入<ol><li>备的完全培养基（25毫升）中，并在37℃/ 5％CO 2的几个小时preequilibrate它。 </li><li>取出基底膜基质的冷冻等分试样在-80℃下冷冻并在冷室中在冰上融化。 </li><li>对于每个核转染制备含有多达813毫米​​无菌盖玻片一个6cm培养皿中。 </li><li>将菜在冰盒盖上保鲜膜。重要的是要保持盖玻片凉爽重要的是要防止在嵌入过程中，基体凝固。 </li><li>为了保持湿度，潮湿的地方组织的条15厘米的培养皿内，瓦特生病被用来撑起来包含嵌入神经母细胞36厘米菜肴。 </li><li>加入完全培养基在1:3的比例解冻矩阵。例如，通过移液混合40微升完全培养基中以120μl的基质。这一数额矩阵是足够的八个12毫米盖玻片嵌入聚合。 </li><li>转移reaggregated细胞簇到15毫升管中，离心 433×g离心5分钟。 </li><li>删除多余的介质和悬浮颗粒于10微升完全培养基。 </li><li>将2微升的细胞聚集体悬浮到每个无菌盖玻片，并加入18μl矩阵/完全培养基混合物。用枪头传播矩阵在整个盖玻片。 </li><li>立即将含有盖玻片的6cm培养皿中的15cm培养皿，并在培养箱（37℃/ 5％CO 2）15-20分钟离开。当矩阵已经凝固，轻轻地加入5 ml完全培养基，以每6cm培养皿照顾，以推下任何浮动盖玻片用枪头。 </li><li>孵育24小时后，在37℃/ 5％CO 2，让成神经细胞迁移出细胞聚集体。 </li></ol> 4。 3D迁移实验<ol><li>准备所需的免疫染色的解决方案。 <ol><li>准备块的解决方案： 山羊块溶液（50ml）中 磷酸盐缓冲盐水（PBS） - 5毫升 山羊血清 - 7.5毫升 10％的Triton X-100 - 1.5毫升 牛血清白蛋白 - 50毫克 H 2 O的- 36毫升</li><li>过滤器和储存在4°C。 </li><li>准备固定的解决方案： 定影溶液（100毫升）中 多聚甲醛（PFA） - 4克注意：始终处理PFA一个引擎盖下 蔗糖 - 20克（可选） PBS至100ml </li><li>在热板上，并在不断搅拌下溶解PFA在80毫升PBS中保持在65℃。 </li><li>一旦PFA溶解，加20克蔗糖。 </li><li>调节pH值至7.4（通常通过添加〜60微升的1M NaOH每100毫升溶液）。 </li><li>弹出至100ml，用PBS总体积。 </li></ol></li><li>免疫组化<ol><li>放置盖玻片的24孔板。 </li><li>冲洗盖玻片用PBS 2倍。 </li><li>修复RMS成神经细胞聚集体在室温下45分钟固定液。 </li><li>冲洗盖玻片用PBS 3X（5分钟/洗 - 摇摆平台上）。 </li><li>阻断30-60分钟，山羊块的解决方案。 </li><li>稀释一抗山羊块的解决方案，并孵育过夜，在4°C。 （如果需要的话，荧光鬼笔环肽（1:400）和Hoechst的染料（1:10,000）也可以加入到第一抗体溶液进行可视化的丝状肌动蛋白和核）。 </li><li>冲洗盖玻片用PBS 3X（5分钟/清洗）。 </li><li>稀释的二抗山羊块溶液孵育2小时，在室温下进行。 </li><li>冲洗coversliPS用PBS 3X（5分钟/洗涤） </li><li>贴装盖玻片用荧光封固剂和离开干燥过夜室温。 </li></ol></li><li>迁移分析<ol><li>捕捉固定有效值成神经细胞聚集的图像使用10X物镜的荧光显微镜。包括在样本图像中的比例尺。 </li><li>要设置刻度量化，通过选择ImageJ的工具栏上的“直线”工具测量图像中的比例尺。 </li><li>选择“分析”选项，并单击“设置比例”。 </li><li>在规模窗口中设置了“已知的距离”，并勾选“全球”框保留为所有测量相同的设置。 </li><li>使用ImageJ的工具栏上的“分段线”工具来测量距离从总的边缘，在整个集合（ 图3B）周围6个不同行业的最远迁移成神经细胞。只考虑隔离AGgregates进行分析。 </li><li>从计算每个聚合得到的6个值的平均移动距离。 </li><li>测量10-20集料为每个条件中的至少三个独立实验的每个独立的实验和游泳池结果。总是包括一个核转染的控制（ 例如 ，GFP或控制的sh /干扰）。 </li></ol></li></ol>

<ol>
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Neurosci. 33, 12171-12185 (2013).</li><li>Ward, M., Rao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigations+of+neuronal+migration+in+the+central+nervous+system.">Investigations of neuronal migration in the central nervous system.</a> Methods Mol. Biol. 294, 137-156 (2005).</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><li>Peretto, P., Giachino, C., Aimar, P., Fasolo, A., Bonfanti, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chain+formation+and+glial+tube+assembly+in+the+shift+from+neonatal+to+adult+subventricular+zone+of+the+rodent+forebrain.">Chain formation and glial tube assembly in the shift from neonatal to adult subventricular zone of the rodent forebrain.</a> J. Comp. Neurol. 487, 407-427 (2005).</li><li>Khlghatyan, J., Saghatelyan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+Imaging+of+Neuroblast+Migration+in+Acute+Slices+of+the+Adult+Mouse+Forebrain.">Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain.</a> J. Vis. Exp. , e4061 (2012).</li><li>Bozoyan, L., Khlghatyan, J., Saghatelyan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+Control+the+Development+of+the+Migration-Promoting+Vasculature+Scaffold+in+the+Postnatal+Brain+via+VEGF+Signaling.">Astrocytes Control the Development of the Migration-Promoting Vasculature Scaffold in the Postnatal Brain via VEGF Signaling.</a> J. Neurosci. 32, 1687-1704 (2012).</li><li>Whitman, M. C., Fan, W., Rela, L., Rodriguez-Gil, D. J., Greer, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+Vessels+Form+a+Migratory+Scaffold+in+the+Rostral+Migratory+Stream.">Blood Vessels Form a Migratory Scaffold in the Rostral Migratory Stream.</a> J. Comp. Neurol. 516, 94-104 (2009).</li><li>Sonego, M., Ya, Z., Oudin, M. J., Doherty, P., Lalli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+Postnatal+Electroporation+and+Time-lapse+Imaging+of+Neuroblast+Migration+in+Mouse+Acute+Brain.">In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain.</a> J. Vis. Exp. , (2013).</li><li>Feliciano, D. M., Lafourcade, C. A., Bordey, A., lique, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+Subventricular+Zone+Electroporation.">Neonatal Subventricular Zone Electroporation.</a> J. Vis. Exp. , e50197 (2013).</li></ol>

神经母细胞沿RMS在OB中的最终位置的迁移是在出生后的神经发生的一个基本步骤。然而，控制这个复杂的过程的分子机制还远远没有得到充分的理解。 这里描述的实验步骤，使得成神经细胞迁移的体外研究。我们已经适应了以前发表的协议，用于从出生后早期小鼠或大鼠25 RMS隔离神经细胞。以达到最佳效果，掌握剥离步骤是很重要的，因为它是非常重要的保持解剖和核转染到最低限度之间的时间间隔。核转染后，成神经细胞可以reaggregated，嵌入在一个三维矩阵和左迁移超过24小时的时期。另外，用于规定以外的迁移（ 如免疫荧光或免疫印迹分析）等，电池可以立即核转染对polyornithine/laminin-镀后涂盖玻片，在那里他们存活长达4-5天。小鼠和大鼠的神经母细胞在基底膜迁移到类似的程度，但是小鼠细胞似乎具有更强的倾向链比大鼠细胞迁移。 根据不同的研究目的，神经母细胞可以nucleofected不同质粒编码的荧光蛋白质或野生型/突变体蛋白。为了获得最佳的蛋白表达质粒与CAG启动子（β-肌动蛋白启动子与CMV增强子和β-珠蛋白多聚A尾巴）26强烈推荐。此外，寡核苷酸的siRNA或shRNA质粒可以nucleofected拦截的利益目标。有效消耗的蛋白质可以通过免疫荧光或免疫印迹（通常裂解嵌入式聚集从1大鼠小狗与50微升标准裂解缓冲液）进行可视化。 核转染是一种比较简单的方法转染的主要成神经细胞，提供了一个更容易和更快的替代方案为vi拉尔载体介导的转染，并能达到高（〜70-80％）的转染效率。这是至关重要的核转染过程中快速地工作，因为留下的成神经细胞中的核转染溶液为延长的时间大大减少了细胞活力。 从RMS夹层的平均细胞产量是相对低的P7小鼠（〜5×10 5个细胞/脑）相比，P7大鼠（〜1×10 6个细胞/脑）和每核转染至少3×10 6个细胞是必需的实现转染〜50％的效率。此外，大鼠的神经母细胞出现抵抗更好核转染相比，小鼠成神经细胞。因此，出生后早期（P6-P7）幼鼠可能代表一个方便的成神经细胞源，也考虑到大鼠和小鼠的RMS的组织都显着西米拉尔27， 在体外大鼠和小鼠成神经细胞迁移的程度也不相上下。最好不要让nucleofected神经母细胞的reaggregated集群停牌超过48小时更长的时间来避免对细胞形态和迁移（我们未发表意见）异常的影响。 这里所描述的三维测定可用于在基体（ 例如 ，24小时）后嵌入到量化成神经细胞迁移在固定的时间点。不同尺寸的聚集体可以在分析中使用，由于存在聚集体的大小和迁移距离（我们的未发表的观察结果）之间没有显著相关。可视化，并进一步探讨神经细胞迁移的动态，时间推移成像都可以使用。建议在执行迁移分析后嵌入一个24小时的时间间隔内，由于神经母细胞的速度出现大幅更长的时间点下降（我们未发表意见）。&lt;/ P&gt; 有一些限制，以该协议。首先，核转染可到目前为止，用于产后早期啮齿类动物的神经母细胞，而感染病毒载体仍然是最有效的转染方法对成人神经母细胞28。其次， 在体外迁移测定不能完全重现体内观察到的RMS的复杂结构。事实上，虽然神经母细胞维持到，迁移以类似的方式，以它们的体内对应于这里描述它们缺乏与其它RMS的组分，如星形胶质细胞和血管，这也有助于调节其蠕动9,29相互作用的实验装置的能力， 30。这个问题可能在未来通过三维共培养模型系统的优化来解决。 总括来说，在一个3D迁移实验相结合的核转染代表一个有价值的工具，以便更好地了解潜在的分子机制成神经细胞迁移。此实验过程提供了一个初始，快速和相对简单的方法来评价成神经细胞迁移的候选调节剂，其可以通过其他方法，如在体内产后电穿孔和脑切片培养的延时成像进一步证实的作用28,31,32 。

在产后哺乳动物大脑，产生新的神经元（神经）发生在整个生命和被限定为两个神经源性龛：侧脑室的脑室下区（SVZ）和海马1的齿状回颗粒下层区域。最近的一些研究表明，成年神经发生的促进学习和记忆任务2,3的重要作用。此外，增殖脑损伤后4-7神经祖细胞和招聘的证据提出了神经发生的神经修复药理活性的可能性。 产后神经发生有严格的规定在其各个阶段，包括神经祖细胞的增殖，迁移，分化，存活和新生的神经元8的最终突触融合。在SVZ来源于干细胞的神经祖细胞（神经细胞）有很大的距离在迁移穿过喙迁徙流（RMS）对嗅球（OB），发育为功能性的神经元9。神经母细胞迁徙主要是单极的，有延伸的单个领先的流程细长的细胞体。这些细胞移动进行集体链，滑过彼此10。迁移是用于随后的成熟SVZ来源的祖细胞为功能性神经元11的一个关键步骤，是由多种因素和指导分子，包括控制：多唾液酸神经细胞黏附分子（PSA-NCAM）12，肝配蛋白13，整14，狭缝15，生长因子16和神经递质17，但保留该过程的分子机制尚不完全清楚。调查的细胞内信号途径调节神经细胞迁移不仅将提供一个更好地了解成人神经，也将有助于新的治疗发展方法促进大脑修复。 这个手稿描述了一个详细的协议使用核转染和三维迁移实验来研究神经细胞迁移的候选监管机构在体外的作用。核转染是基于电穿孔的改良方法的细胞转染技术。细胞类型特异性的电流和核转染溶液使聚阴离子大分子如DNA和shRNA的载体和siRNA寡核苷酸的转移直接进入细胞核并慢慢地分割或有丝分裂无活性细胞如胚胎和哺乳动物的神经元18允许转染。这种方法是快速的，相对易于执行和结果在广泛的细胞类型，包括原发性神经母细胞和神经元19-21的高度重复性的转染。 RMS组织的离解允许迁移性的神经母细胞的分离，从而可以成功地nucleofected与DNA /的shRNA载体或寡核苷酸的siRNA靶向目的基因。下面核转染，成神经细胞被reaggregated在悬滴，随后包埋在三维基质胶基质。这些条件允许的成神经细胞迁移出细胞聚集扼要体内观察到的迁移模式，从而提供了一个很好的模型系统来研究参与神经细胞迁移的信号通路，并评估药物治疗对这些细胞的运动的影响。

<table border="0" cellpadding="0" cellspacing="0" width="1639">
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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:688px;">Hank&#8217;s Balanced Salt Solution (HBSS)</td>
			<td style="width:339px;">Invitrogen Life Technologies</td>
			<td style="width:255px;">14175129</td>
			<td style="width:359px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:688px;">HEPES</td>
			<td style="width:339px;">Sigma-Aldrich</td>
			<td>H3375-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:688px;">Penicillin-Streptomycin</td>
			<td style="width:339px;">Invitrogen Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:688px;">2.5% Trypsin-EDTA (10x)</td>
			<td style="width:339px;">Gibco</td>
			<td>15090-046</td>
			<td>store 200 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:688px;">DNAse I Vial (D2)</td>
			<td style="width:339px;">Worthington</td>
			<td style="width:255px;">LK003170</td>
			<td>&#8805;1,000 units per vial; store 50 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:688px;">Dulbecco Modified Eagle&#39;s Medium (DMEM)</td>
			<td style="width:339px;">Gibco</td>
			<td style="width:255px;">11960-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:688px;">Fetal Calf Serum (FCS)</td>
			<td style="width:339px;">Hyclone</td>
			<td style="width:255px;">SH3007902</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:688px;">Neurobasal medium</td>
			<td style="width:339px;">Gibco</td>
			<td style="width:255px;">21103-049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:688px;">B27 supplement</td>
			<td style="width:339px;">Invitrogen Life Technologies</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:688px;">L-Glutamine (200 mM)</td>
			<td>Invitrogen Life Technologies</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:688px;">D-(+)-Glucose solution (45%)</td>
			<td style="width:339px;">Sigma-Aldrich</td>
			<td style="width:255px;">G8769</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel Basement Membrane Matrix, Growth Factor Reduced (GFR), Phenol Red-free, 10 ml, LDEV-Free</td>
			<td style="width:339px;">BD Biosciences</td>
			<td>356231</td>
			<td>prepare 120 &#181;l aliquots at 4 &#176;C, then store at -80 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:688px;">PFA</td>
			<td style="width:339px;">Sigma-Aldrich</td>
			<td style="width:255px;">441242</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:688px;">Sucrose</td>
			<td style="width:339px;">BDH</td>
			<td style="width:255px;">102745C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:688px;">Goat serum</td>
			<td style="width:339px;">Sigma-Aldrich</td>
			<td style="width:255px;">69023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:688px;">Triton X-100</td>
			<td style="width:339px;">VWR International Ltd.</td>
			<td style="width:255px;">306324N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:688px;">BSA</td>
			<td style="width:339px;">Fisher Chemical</td>
			<td style="width:255px;">BPE9701-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:688px;">Dako fluorescence mounting medium</td>
			<td style="width:339px;">Dako</td>
			<td style="width:255px;">S3023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:688px;">Rat neuron nucleofection kit</td>
			<td style="width:339px;">Lonza</td>
			<td style="width:255px;">VPG-1003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:688px;">Mouse neuron nucleofection kit</td>
			<td style="width:339px;">Lonza</td>
			<td style="width:255px;">VPG-1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:688px;">Microsurgical knife</td>
			<td style="width:339px;">Angiotech</td>
			<td style="width:255px;">7516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:688px;">McIlwain tissue chopper</td>
			<td style="width:339px;">The Mickle Laboratory Engineering Company</td>
			<td style="width:255px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:688px;">Nucleofector II</td>
			<td style="width:339px;">Lonza</td>
			<td style="width:255px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

nucleofection of rodent neuroblasts to study neuroblast migration in vitro

位于侧脑​​室外侧壁脑室下区（SVZ）中扮演成年神经发生了根本性的作用。在大脑中的这一禁区，神经干细胞增殖和不断产生的切向链，迁移沿喙迁移流（RMS）达到嗅球（OB）的神经母细胞。一旦在OB，神经母细胞切换到径向迁移和再分化成能够纳入已经存在的神经元网络的成熟神经元。适当的神经细胞迁移是在神经发生的基本步骤，确保新生神经元的正确功能成熟。鉴于SVZ来源的神经母细胞为目标的脑部受伤的地方，调查相关的运动细胞内机制的能力，不但可以提高神经的理解，而且可促进神经再生策略的发展。 此手稿描述了详细的协议主要啮齿动物的转染RMS产后神经母细胞和它们的蠕动使用3D 体外迁移实验扼要重述它们的迁移模式在体内观察到的分析。大鼠和小鼠的神经母细胞可以快速而有效地转经核转染与任何质粒DNA，小发夹（SH）RNA或短干扰（SI）的RNA寡核苷酸靶向目的基因。分析迁移，nucleofected细胞reaggregated在“悬滴”，并随后包埋在三维矩阵。核转染本身并不显著损害神经母细胞的迁移。也可进行药物治疗nucleofected和reaggregated成神经细胞的研究信令涉及成神经细胞迁移的途径的作用。

神经母细胞可成功地分离自剖分RMS的组织（ 图1A）和嵌入在三维矩阵。细胞无论从大鼠或小鼠出生后的RMS隔离免疫阳性是候鸟成神经细胞标志物，如doublecortin（DCX），βIII微管蛋白或PSA-NCAM（ 图1B-C）。 离解成神经细胞可以有效地nucleofected与DNA（ 例如 ，GFP的质粒编码，图2）或shRNA的质粒（ 图4）来实现蛋白质消耗，这可以通过免疫印迹分析来评估（ 图4B）或免疫荧光（未示出） 。 细胞nucleofected与绿色荧光蛋白编码质粒径向迁移出reaggregated成神经细胞群（ 图3A）。相对迁移距离24小时后嵌入（ 图3B）的量化显示了迁移的GFP-断定没有区别<html>的ive细胞和GFP阴性，nonnucleofected细胞（ 图3C），表明核转染本身不会中断迁移。也有在nucleofected神经母细胞和神经母细胞直接迁移出的RMS植（数据未显示）之间迁移的程度无显著差异。 <img alt="图1" fo:content-width="6in" src="/files/ftp_upload/50989/50989fig1.jpg" width="500px" /> 图1。解剖有效值神经母细胞。有效值成神经细胞解剖（一）示意图。有关详细说明，请参阅文本。 （二）大鼠离体RMS细胞免疫阳性的成神经细胞迁徙者DCX和βlll微管蛋白。酒吧，20微米（C）细胞迁移出鼠标的RMS植表达迁徙神经细胞标志物DCX和PSA-NCAM。酒吧，20微米。0989/50989fig1highres.jpg“目标=”_blank“&gt;点击这里查看大图。 <img alt="图2" fo:content-width="6in" src="/files/ftp_upload/50989/50989fig2.jpg" width="500px" /> 图2。小鼠成神经核转染。游离鼠标有效值 神经母细胞被nucleofected用PMAX-GFP，reaggregated，嵌入在一个三维矩阵和允许迁移6小时。神经母细胞迁移出reaggregated细胞簇（顶部，相衬图像）显示转染效率高（底，绿色荧光蛋白通道的图片）。右边一栏面板显示对应于左栏板突出的插图更高的放大倍率的照片。酒吧，20微米。 <a href="https://www.jove.com/files/ftp_upload/50989/50989fig2highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" fo:c<html> ontent宽度=“6英寸”SRC =“/ files/ftp_upload/50989/50989fig3.jpg”宽度=“500px的”/&gt; 图3。 3D迁移实验。 （A）大鼠成神经细胞被nucleofected用PMAX-GFP（绿色荧光蛋白）或pCAG-IRES-EGFP 22（EV），reaggregated，嵌在基质和左迁移24小时。然后将细胞固定并进行免疫染色的GFP（绿色）和βIII微管蛋白（红色）。酒吧，50微米（B）使用ImageJ测量迁移距离。该reaggregated细胞群被分成6等份。集群（虚线）和最远的迁移细胞的边缘之间的距离的测量是对每个扇区。的相对距离（C）的定量通过nucleofected细胞迁移（GFP阳性）和对照，nonnucleofected细胞（GFP阴性） 。 <a href="https://www.jove.com/files/ftp_upload/50989/50989fig3highres.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="&lt;/ HTML">“图4”FO：内容宽度=“6英寸”SRC =“/ files/ftp_upload/50989/50989fig4.jpg”宽度=“500px的”/&gt; 图4。监测shRNA的核转染后的神经细胞迁移。 （一）大鼠神经母细胞进行nucleofected与对照shRNA表达载体（PCA-B-EGFPm5消音器3，这也表达了EGFP 23）或含有shRNA的中fascin目标，一个肌动蛋白束蛋白24相同的向量。细胞reaggregated超过48小时，嵌在基质和左迁移24小时。聚集体，然后固定和免疫染色的GFP（绿色）和βIII微管蛋白（红色）。酒吧，50微米（B）中fascin有效 损耗可以检测shRNA的核转染的Western blot分析后，50小时。肌动蛋白在此处显示为一个负载控制（C）相对迁移距离的显示，枯竭中fascin损害显著成神经细胞迁移的定量分析。（平均值±标准差; ** P &lt;0.01，n = 3的独立实验）。 <aHREF =“https://www.jove.com/files/ftp_upload/50989/50989fig4highres.jpg”目标=“_blank”&gt;点击这里查看大图。

Watch this Scientific Journal Video about Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro at JoVE.com

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

成神经细胞迁移是在出生后的神经发生了关键性的一步。在这里描述的协议可以被用于研究神经细胞迁移的候选调节器通过使用DNA /小发夹RNA（shRNA的）核转染，并与成神经细胞从啮齿动物出生后的头端迁移流中分离出来的三维迁移实验中的作用。

鼠神经母细胞核转染到研究神经细胞迁移<em&gt;在体外</em

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted ...

nucleofection-rodent-neuroblasts-to-study-neuroblast-migration

Research

JoVE Journal

Neuroscience

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

11.4K 次观看.  King's College London. 成神经细胞迁移是在出生后的神经发生了关键性的一步。在这里描述的协议可以被用于研究神经细胞迁移的候选调节器通过使用DNA /小发夹RNA（shRNA的）核转染，并与成神经细胞从啮齿动物出生后的头端迁移流中分离出来的三维迁移实验中的作用。 

视频: Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection和胚胎小鼠海马和皮层神经元的原代培养

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

科研

神经科学

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation从啮齿动物脑切片的神经元，研究突触传递和图像突触前的码头

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

胚胎腹侧中脑器官型片文化：一个系统来研究多巴胺能神经元发展在体外</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

急性片的成年小鼠前脑的神经细胞迁移的延时成像

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

啮齿动物的中枢神经系统药物直接脑室交货

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

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

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

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

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

成神经细胞迁移的小鼠急性脑片在体内电穿孔产后和时间推移成像

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this protocol focuses on how to image dividing neuroblasts, which are found underneath the epidermal cells and may be important for epidermal morphogenesis. Tissue formation is crucial for metazoan development&#160;and relies on external cues from neighboring tissues. C. elegans is an excellent model organism to study tissue morphogenesis in vivo due to its transparency and simple organization, making its tissues easy to study via microscopy. Ventral enclosure is the process where the ventral surface of the embryo is covered by a single layer of epithelial cells. This event is thought to be facilitated by the underlying neuroblasts, which provide chemical guidance cues to mediate migration of the overlying epithelial cells. However, the neuroblasts are highly proliferative and also may act as a mechanical substrate for the ventral epidermal cells. Studies using this experimental protocol could uncover the importance of intercellular communication during tissue formation, and could be used to reveal the roles of genes involved in cell division within developing tissues.

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes how to image dividing cells within a tissue in Caenorhabditis elegans embryos. While several protocols describe how to image cell division in the early embryo, this protocol describes how to image cell division within a developing tissue during mid late embryogenesis.

可视化的神经细胞胞质分裂过程中<em&gt; C。线虫</em&gt;胚胎

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this ...

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

Embryonic neurons are born in the ventricular zone of the brain, but subsequently migrate to new destinations to reach appropriate targets. Deciphering the molecular signals that cooperatively guide neuronal migration in the embryonic brain is therefore important to understand how the complex neural networks form which later support postnatal life. Facial branchiomotor (FBM) neurons in the mouse embryo hindbrain migrate from rhombomere (r) 4 caudally to form the paired facial nuclei in the r6-derived region of the hindbrain. Here we provide a detailed protocol for wholemount ex vivo culture of mouse embryo hindbrains suitable to investigate the signaling pathways that regulate FBM migration. In this method, hindbrains of E11.5 mouse embryos are dissected and cultured in an open book preparation on cell culture inserts for 24 hr. During this time, FBM neurons migrate caudally towards r6 and can be exposed to function-blocking antibodies and small molecules in the culture media or heparin beads loaded with recombinant proteins to examine roles for signaling pathways implicated in guiding neuronal migration.

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

Embryonic neurons are born in the ventricular zone of the neural tube, but migrate to reach appropriate targets. Facial branchiomotor (FBM) neurons are a useful model to study neuronal migration. This protocol describes the wholemount ex vivo culture of mouse embryo hindbrains to investigate mechanisms that regulate FBM migration.

鼠标后脑<em&gt;前体内</em&gt;企业文化来研究面部Branchiomotor神经元迁移

Embryonic neurons are born in the ventricular zone of the brain, but subsequently migrate to new destinations to reach appropriate targets. Deciphering ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

的实时成像<em&gt;果蝇</em&gt;幼虫神经母细胞

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...