MT是由博士助学金[参考支持。 092839/Z/10/Z]和CR由新研究者奖[参考。从威康信托基金会095623/Z/11/Z]。

1。可选：准备阿菲 - 凝胶肝素珠（胶珠）为富时大马景点含量注：在开始植程序之前准备凝胶珠至少1天。 <ol><li> 20分钟的滚筒，在室温（RT）洗涤100μl的凝胶肝素珠悬浮液用无菌PBS。 </li><li>丸粒珠在台式离心机5分钟，在13000×g下。加入无菌PBS和重复洗涤过程4倍。 </li><li>最后一次洗涤后，除去PBS，浸泡珠子在小体积的含有精选的重组蛋白中的无菌溶液，小心地覆盖有孔玻璃珠与该溶液。该协议使用100纳克/微升重组人VEGF165在PBS重现以前发表的实验16。 </li><li>孵育凝胶肝素珠最少12小时的重组蛋白质溶液和最多1周上的辊子在4℃下</li></ol> 2。文化插入的涂层s 后脑外植体康宁文化培养插入一个8微米孔径的大小，或等效的插入。培养插入可以在协议完成后，可以重复使用，只要它们用蒸馏水洗涤，消毒用乙醇和直到需要保存在70％乙醇中。 注意：下面的步骤应在无菌条件下进行，在一个流罩。 <ol><li>制备外植体的培养基包括补充有B27（20微升/毫升）的Neurobasal培养基中，葡萄糖（6毫克/毫升）和青霉素/链霉素（5微克/微升）。 </li><li>洗涤培养插入用无菌PBS 5分钟，干燥的流罩下5-10分钟。 </li><li>将一种文化中插入操作12well板的每一个人好。注：刀片可能需要一个小推，以适应紧紧地里去了。 </li><li>覆盖培养插入10-20微克/毫升小鼠层粘连蛋白在Neurobasal培养基，并放置在一个组织培养箱（37℃，5％CO2 <sub&gt; 2）1小时。注意：涂层应进行上explanting的日子。 </li></ol> 3。从E11.5小鼠胚胎解剖Hindbrains的<ol><li>扑杀逾怀孕雌性小鼠与胚胎一天一个合乎伦理的程序（E）11.5，并把含有胚胎的子宫在一个100毫米的塑料培养皿用冰冷的L15培养基。 注意：所有的解剖步骤必须在冰冷L15来执行。 </li><li>用解剖显微镜和杜蒙钳钟表匠5号，撕裂子宫肌壁，暴露的胚胎，释放每个胚胎，割断脐带，并小心地取出卵黄囊。 </li><li>使用塑料巴斯德吸管用大口径开放，每个转移胚胎到一个干净的塑料盘用冰冷的L15。 </li><li>使用杜蒙钳，杀头胚胎刚刚前肢上方。如果实验需要的胚胎的基因分型，收集组织样本进行基因组DNA的提取（ 例如小PIECË卵黄囊或尾尖16,20）的。 </li><li>转动头部背侧，并确定4 个心室，其覆盖有薄的组织层（ 图2B）。小心刺破roofplate并开始尾端剥它拿走沿中线在后后脑和脊髓，并吻侧在中脑。后脑现在应该露出（ 图2C）。 </li><li>仔细逗相差其余头间质和连接到后脑（ 图2D）的软膜侧的任何脑膜。 </li><li>去除脑和脊髓组织，使后脑，随即渡过，并且可以在一个开放的书准备（ 图2E）平躺。 </li><li>使用宽口塑料巴斯德吸管中，每个解剖后脑转移到一个12孔板中含有冰冷L15，并将它们存储在冰上直到所有hindbrains已被切开。 </li><li>采用大口径塑料巴斯德吸管，转让罪GLE后脑到一个空盘子，保持一个开放的书准备，心室的一面朝上，在大约100微升L15液滴。 </li><li>转让的培养肝素凝胶珠几微升到同一个液滴。注意：因为珠子的大小是可变的，最好是转大约10珠，然后选择用于移植的最佳大小到后脑。 </li><li>做一个小撕裂在后脑组织并小心地插入1-3凝胶珠入后脑组织在R5 / 6的水平，对中线和后脑的横向边缘之间的半路上，降低他们进入组织，使胎圈正好位于后脑表面之下。 注：解剖过程可能需要5-20分钟/后脑之间，这取决于经验，可以舒展在较长时间内，如果胎大，在任何情况下，hindbrains应该在文化不长于3小时验尸好结果。 </li></ol> 4。后脑植文化<ol><li>删除包含从孵化器中培养插入板，吸层粘连蛋白涂层解决方案。 </li><li>放置1培养插入到一个单独的培养皿中充满了冰冷L15和，使用宽口塑料巴斯德吸管中，每个后脑腹侧转移到培养插入物（ 图2G）。后脑应该说谎完全平放在插入膜。 </li><li>小心地从培养皿解除培养插管并轻轻拍上干净的纸巾数次以除去过量的液体。这个过程确保了后脑坚持在平坦，开阔的书准备文化插入。如果后脑蜷缩起来，它的组织可能会不可逆转地共同成长。 </li><li>用500μl预热的培养基填充原来的12孔板，然后将插回这口井。仔细调整与另一400-600微升的媒体仅仅覆盖后脑的体积，确保后脑不浮离膜。如果它漂浮，则返回步骤4.4，并重复直到后脑仍然附着在膜上。 </li><li>在此阶段，有可能感兴趣的生物抑制剂添加到媒体研究其对FBM神经元的迁移效果。 注意：如果植入珠或给予其他治疗方法，建议保持在每个实验中正常生长条件下至少有2个控制外植体，以确保实验已经成功建立。 </li><li>培养外植体为24-30小时，在细胞培养箱（37℃，5％CO 2）。 </li></ol> 5。后脑植平片免疫荧光染色<ol><li>从每个孔中吸出介质，冲洗的PBS中并在4℃下，在冰冷却的4％多聚甲醛（新鲜制备的或刚解冻的4％多聚甲醛溶于PBS）温和搅拌定为2小时。注意：不要尝试从固定前文化插入后脑删除已完成。 </li><li>冲洗3次，用PBS。小心地从使用杜蒙镊子培养插入剥离hindbrains。有的外植体难以剥离，但是通常可以通过从移液管反复排出的PBS施加温和的压力被解除。 </li><li>转移hindbrains至2.0圆底管免疫荧光标记。在含有0.1％的TritonX-100（PBT），轻轻压延室温（RT）的PBS中透化hindbrains 30分钟。 </li><li>孵育1小时，在室温下在PBT中含有10％热灭活，轻轻压延正常山羊血清。 </li><li>培养外植体，轻轻压延，在4℃下进行5天，具体为ISL1初级抗体，1:100稀释在PBT中含有1％热灭活的正常山羊血清。 </li><li>在室温下用4倍的PBT，每次15分钟洗外植体。 </li><li>培养外植体用温和的轧制在室温搅拌3小时以荧光团偶联的山羊抗小鼠抗体（ 例如的Alexa Fluor 488羊抗小鼠，1:200稀释）在PBT中含有1％热灭活的正常山羊血清。 </li><li>洗植4倍于室温与PBT的15每个轻揉分钟。 </li><li> postfix的外植体在4％多聚甲醛在室温30分钟。 </li><li>盖上的黑色电工胶带3层和消费用手术刀分层磁带的小广场载玻片上创建一个口袋为外植体，或者，使用抑郁载玻片上。 </li><li>装载每个后脑在SlowFade试剂成一个口袋里，盖上盖玻片，小心避开陷阱气泡和图像使用激光扫描共聚焦显微镜。 注意：作为一个替代的免疫染色，与核糖核酸探针能识别FBMS（即ISL1或PHOX2B）原位杂交可用于可视化FBM神经元16,21。 </li></ol> 的步骤和时间安排概要 定时交配获得E11.25怀孕：〜14天 可选：珠制剂（ProtocoL-1）：〜2小时，对前胚胎隔离的日子 准备培养插入和媒体（协议2）：〜30分钟，前胚胎隔离 胚胎分离及后脑清扫术（步骤3.1-3.4）：〜10分钟/胚胎 后脑清扫术（步骤3.5-3.7）：〜5-10分钟/后脑 植过程（步骤3.8-3.9）：〜5-10分钟/后脑 可选：珠植入术（步骤3.10-3.11）：〜5-10分钟/后脑 外植体培养（步骤4.7）：24小时 固定的抗体染色（步骤5.1）：2小时 染色过程和成像（协议5）：12天

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没有作者都相互竞争的利益或利益冲突。

本协议描述了Transwell小系统来研究FBM神经元的迁移E11.5小鼠hindbrains的平片文化。该协议允许鼠标后脑运动神经元来维持生命和迁移，为期24小时，使体外操纵。这种方法有许多实验优势，为研究者试图找出神经细胞迁移的分子和细胞机制。而传统的迁移实验外植体小神经组织块放入矩阵的培养皿，使观察单个神经元，因为他们对外源性刺激作出反应，Transwell小法的主要优点是它的适宜宿主器官的环境中操纵神经元迁移，因此更生理环境。重要的是，物质可以容易地应用到体外后脑外植体，以测试它们对神经元迁移，避开与administe相关的可能副作用响这些物质的孕鼠。最后， 体外模型还允许不穿过胎盘屏障，如功能阻断抗体的物质进行测试。由于这些优点，在体外培养后脑提供使用斑马鱼胚胎中，可以用水溶性的小分子被视为在水族箱中的水，或在胚胎大脑的子宫内的电，这需要使用专门的替代和补充方法设备，并且更难以掌握比这里所描述的培养方法。这里描述的协议的另一个优点是它的顺从到植入珠浸泡在重组蛋白或其他试剂，因此允许开发操纵小鸡胚胎神经细胞迁移的小鼠模型中的标准胚胎学方法的应用。特别地， 在体外培养模型可应用于遗传工程小鼠hindbrains有缺陷略去中牵连的神经元迁移的特定分子，如生长或导向因子受体，和结合珠植入物来测试是否响应于配体的丢失。除了 ​​药理操纵，在离体培养的协议也可以适应于电穿孔，可以操纵感兴趣的基因的表达的表达载体，适当的方法电穿孔先前已描述的22,23。这个协议也可适于通过时间推移显微镜中从含有荧光标记的FBM神经元， 如ISL1-Cre重组酶的转基因小鼠后脑植可视化神经元的迁移; Rosa26Yfp 21。最后，该协议也可以用来研究其他类型的神经元迁移的后脑，如那些形成的下橄榄，虽然这将需要在较旧的胚胎阶段使用hindbrains的，并且可能需要培养长达48小时，根据神经元的存活率<em&gt;体外。 关键步骤和故障排除 对于本协议的成功，至关重要的是，胚胎在E11.5天，接近E11.25，当FBM神经元的迁移才刚刚开始早期收取。然而，它并不总是可能在该发育阶段的胚胎赶上由于小鼠自然交配变异性，因此，可能会出现一些变异性在不同实验之间FBM迁移的延伸。变异FBM如果迁移实验未分配的时间框架内完成，约3小时，如在图3E可以看出，也可以观察到。从E11.25小鼠胚胎后脑组织细腻。当解剖和整个外植体的过程，它不是撕裂后脑组织中的R4-R6，其中富时神经元所在的区域是很重要的。由于迪斯的微妙性主要树立的过程，因为这hindbrains被置于文化的速度影响的结果，该过程可能需要几个实践运行掌握，珍贵的样品或试剂在使用前特别。最后，重要的是后脑组织被放置到在培养插入打开的书的配置，因为在培养期间的后脑组织的折叠会阻止正常FBM迁移（参见图3F）。

胚胎神经元是出生在大脑的脑室区，但后来迁移到新的目的地到达了位于大的距离合适的目标区域。神经元细胞体沿大脑发育的背腹和前后轴的适当位置的正确定位是正确的接线，生存，而这些神经元的功能的迁移阶段1-4后必不可少的。类似于控制轴突导向5-7的分子机制，组合套具吸引力和排斥力的线索被认为是引导神经元迁移1,8。然而，由于多种细胞类型的相互作用，控制神经细胞迁移的信号已被减广泛研究比那些参与轴突导向，可以自主地研究细胞。脊椎动物的显影后脑已用于近来的一些研究，了解细胞和分子机制神经细胞迁移的，例如在鸡，小鼠，斑马鱼和1-4,9。这个器官包含了几种不同类型的神经元，其中包括前小脑的几个亚型和运动神经元5,7,10,11。 后脑motorneuron出生在脑室区靠近floorplate，他们分化 ​​为特定子集，根据产地1,12他们菱。在菱（R）4生成在后脑面部branchiomotor（FBM）神经元和背侧通过R4的出口点延伸它们的轴突到第二腮弓到支配面部肌肉2,9,13。斑马鱼和小鼠的神经元FBM提供优良的模型来研究神经细胞迁移的分子和细胞机制在于易于可视化的方法，因为这些神经元可再现易位其胞体在时空方式明确定义的过程。在小鼠中，富时大马神经元先迁移CAU玩弄通过R5，然后双方和尾端腹侧以达到他们在R6，在那里形成了第7届颅神经（VIIn）10,11,14的配对核境内后脑的软脑膜侧的最终位置。在斑马鱼中，FBM神经元最初迁移腹侧，然后在R4-R5边界改变方向继续在用层粘连蛋白依赖性4,12,15,16对软脑膜表面迁移。这种迁移进行了一段在发展的若干天，可分为切向和径向迁移的阶段，使调解这两个不同的过程的分子鉴定。相比之下，鸡胚后脑的神经元的FBM留在R4 3,13,17-19。 在他们的迁移，富时大马神经元可以被识别，像其他类型或运动神经元，通过他们的homoeodomain转录因子胰岛1（ISL1）14的表达。因此，wholemount免疫荧光染色或原位杂交对这个标记在不同的发育阶段揭示FBM胞体在斑马鱼和小鼠4,15,16从R4延伸至R6的明显迁移流。此外，荧光转基因记者如ISL1-GFP已经被用作合适的工具来可视化在斑马鱼3,17-19迁移FBM神经元。除了其适合用于成像，许多研究者已研究了FBM神经元在显影斑马鱼的迁移，因为它们的自由生活的胚胎可以很容易地与细胞移植技术和直接涂在水族馆的水药理化合物被操纵。相反，在小鼠胚胎发育封闭在子宫，排除的珠子进行指导线索或功能阻断性抗体不穿过胎盘屏障的给药植入。此外，给予孕妈妈药理化合物可能有未所需的副作用，可以间接地损害胚胎。绕过这个限制，我们已经开发了一个体外培养法在全鼠标后脑就是explanting 7,16后与FBM神经元的迁移和存活24小时兼容。这种方法可以方便的药理操纵，珠携带的指导线索或功能阻断抗体的行政植入，也可适应于不同的发育阶段，研究其他神经元亚型迁移的后脑。

<table border="0" cellpadding="0" cellspacing="0" width="575">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">Eppendorf round-bottomed reagent tube, 2.0 ml (Safe-Lock)</td>
			<td style="width:111px;">VWR</td>
			<td style="width:104px;">211-2120</td>
			<td style="width:147px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:215px;">Cell culture plates, 12 well</td>
			<td style="width:111px;">Thermo Scientific</td>
			<td style="width:104px;">150628</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">Plastic cell culture dish, 100 mm</td>
			<td style="width:111px;">Thermo Scientific</td>
			<td style="width:104px;">150288</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">15 mm Netwell insert with Mesh Polyester Membrane</td>
			<td style="width:111px;">Corning</td>
			<td style="width:104px;">3477</td>
			<td style="width:147px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Watchmaker forceps, no. 5</td>
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			<td style="width:104px;">91150-20</td>
			<td style="width:147px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Phosphate buffered saline</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:104px;">P4417</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">Laminin mouse protein</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:104px;">23017-015</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:215px;">Primary antibody, Isl1</td>
			<td style="width:111px;">Developmental Hybridoma Bank</td>
			<td style="width:104px;">39.4D5</td>
			<td style="width:147px;">Dilution 1/100</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:215px;">AlexaFluor 488-conjugated goat anti mouse secondary antibody</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:104px;">A11029</td>
			<td style="width:147px;">Dilution 1/200</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:215px;">Neurobasal medium</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:104px;">21103</td>
			<td style="width:147px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:215px;">B27 supplement (50x)&#160;</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:104px;">17504-044</td>
			<td style="width:147px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:215px;">Leibovitz&#8217;s L-15</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:104px;">21083-027</td>
			<td style="width:147px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:215px;">Penicillin/ streptomycin&#160;</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:104px;">15070</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Glucose</td>
			<td style="width:111px;">VWR</td>
			<td style="width:104px;">101174Y</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Heat-inactivated goat serum</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:104px;">G9023</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Triton X-100</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:104px;">T8787</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Paraformaldehyde</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:104px;">P6148</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">Slowfade Antifade Kit</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:104px;">S-2828</td>
			<td style="width:147px;">Alternative mounting solutions may be used</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Microscope slides</td>
			<td style="width:111px;">VWR</td>
			<td style="width:104px;">631-0912</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Cover glass</td>
			<td style="width:111px;">VWR</td>
			<td style="width:104px;">631-0137</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:215px;">Affi-Gel Heparin Beads</td>
			<td style="width:111px;">Biorad</td>
			<td style="width:104px;">153-6173</td>
			<td style="width:147px;">Glass, latex, or coloured beads may be used alternatively</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:215px;">Recombinant human VEGF165</td>
			<td style="width:111px;">R&#38;D systems</td>
			<td style="width:104px;">293-VE</td>
			<td style="width:147px;">Resuspend in PBS, store aliquots at -80oC</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Stereo Microscope, Leica MZ16</td>
			<td style="width:111px;">Leica</td>
			<td style="width:104px;"></td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">Confocal laser scanning microscope&#160; LSM710</td>
			<td style="width:111px;">Zeiss</td>
			<td style="width:104px;"></td>
			<td style="width:147px;"></td>
		</tr>
	</tbody>
</table>

mouse hindbrain ex vivo culture to study facial branchiomotor neuron migration

胚胎神经元是出生在大脑的脑室区，但后来迁移到新的目的地，以达到适当的目标。破译分子信号的共同指导神经细胞迁移在胚胎大脑因此重要的是理解复杂的神经网络是如何形成的，后来支持产后生活。在小鼠胚胎后脑面部branchiomotor（FBM）神经元免受菱（R）4尾侧迁移以在后脑的[6 - 衍生的区域配对的面神经核。在这里，我们为平片体外适宜的调查信号通路调节FBM迁移小鼠胚胎hindbrains文化的一个详细的协议。在该方法中，E11.5小鼠胚胎hindbrains在细胞培养插入物24小时开卷制备解剖并培养。在此期间，FBM神经尾侧移向r6和可暴露于功能阻断性抗体和小moleculES在装有重组蛋白研究的信号通路牵连指导神经细胞迁移的作用培养基或肝素珠。

本节说明了可以通过研究FBM神经元迁移，通过体外培养的小鼠后脑来获得结果的例子。我们表明，FBM神经元中从第11天小鼠胚胎中取出的hindbrains先经过切向迁移（ 图3A），然后开始以组装的面部运动核（ 图3B），类似于其在子宫内的行为（参见图1）。我们进一步证明，一个VEGF165浸泡过的珠子植入吸引FBM神经元（ 图3C和3D），如前所示16。重要的是，这个协议允许FBM学习迁移在没有血管或血管源性因素可能影响FBM移民在子宫内的，因为未注血管退化的文化16。因此，在使用上新鲜的ISL1小鼠抗体时的非特异性血细胞标记观察离体的小鼠hindbrains（ 图1）不再存在于后脑组织24小时培养（ 图3A-F）之后。最后，我们表明，没有正确地取出的，因此，含有氨酯神经元的异常分布hindbrains的两个例子中，这可能是因为后脑后胚胎隔离（ 图3E），或没有被取出的很快，因为后脑组织折叠起来，在transwell小（ 图3F）。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51397/51397fig1highres.jpg" src="/files/ftp_upload/51397/51397fig1.jpg" /> 图1。 FBM神经元迁移的共焦Z堆栈的ISL1平片免疫标记和flatmounting后野生型小鼠hindbrains;。在一个E11.5后脑的后脑正中表示，在所有面板带有星号（A）心室面<html>含ISL1阳性FBM神经元（箭头），显示了其切向迁移从R4至R6的面积;的一半相同后脑在含有原基的区域R4，R5和R6的位置被表示（A&#39;）的软脑膜表面。一个E12.5后脑含有氨酯神经元迁移切线方向（箭头所示）的一个配对FBM核（与VIIn表示），以及其他ISL1阳性的神经元群（B）的心室表面;箭头指示的示例含有循环细胞是由用于检测ISL1小鼠IgG抗体的抗小鼠第二抗体的交叉反应非特异性地标记的血管（B&#39;）。的一半相同E12.5后脑，其中包含的软脑膜表面一个配对FBM核。中线指示，在每个面板中的星号。比例尺（所有面板）：200微米。 V，腹，P，软脑膜。/ 51397fig1highres.jpg“目标=”_blank“&gt;点击这里查看大图。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51397/51397fig2highres.jpg" src="/files/ftp_upload/51397/51397fig2.jpg" /> 图2。 E11.5小鼠后脑解剖和体外培养。 （AE）在E11.5后脑解剖协议的关键步骤;比例尺：胚胎1毫米（A）打印头也被切离的胚胎前肢级之后的其余部分（B）的延髓部分。头被除去，头部组织的剩余部分定位，使得第 4心室（箭头）被向上定向（C） 第 4脑室的屋顶被剥离，并且后脑被剥离的组织的后脑下方远离暴露吻侧和尾侧（D）的软膜的膜除去（请注意，在这个前充足的，一些脊髓型颈椎病（SC）组织一直连接到后脑）（E）中脑过剩（MB）和脊髓（SC）的组织已被移除，保留只是后脑。（六）文化刀片涂有层粘连蛋白和放入12孔细胞培养板。（G）每个后脑被放置到一个插入和覆盖介质。（H）所采取的文化在24小时FBM迁移神经元（蓝色）路径的示意图。 <a href="https://www.jove.com/files/ftp_upload/51397/51397fig2highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图3" fo:content-width="4in" fo:src="/files/ftp_upload/51397/51397fig3highres.jpg" src="/files/ftp_upload/51397/51397fig3.jpg" /> 图3。鼠标后脑体外培养。 （A，B）的E11.5后脑为24小时和immunofluoresc培养<html> ently标示ISL1说明FBM神经元迁移的外植体;两个心室（A）和软脑膜后脑（二）双方都显示（C，D）E11.5同窝hindbrains在植入肝素珠浸泡的存在进行培养。在PBS中的（C）或VEGF 165（D），注意，FBM神经元迁移的朝向和到VEGF165胎圈，因此，迁移流扩展进一步尾端相比，在相同后脑或含有该控制珠后脑的未处理侧（E。 ，不尽人意E11.5后脑植体，其中FBMS没有从R4移民（E）或F）的例子中，后脑组织培养（F）的过程中被折叠。中线指示，在每个面板中的星号。比例尺（适用于所有面板）：200微米。 V，腹，P，软脑膜。“&gt;点击这里查看大图。

Watch this Scientific Journal Video about Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration at JoVE.com

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

胚胎神经元是出生在神经管的脑室区，但迁移到达到适当的目标。面部branchiomotor（FBM）的神经元，研究神​​经细胞迁移的有用模型。本协议描述的小鼠胚胎hindbrains平片的体外培养研究，规范FBM迁移机制。

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 ...

mouse-hindbrain-ex-vivo-culture-to-study-facial-branchiomotor-neuron

Research

JoVE Journal

Neuroscience

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

11.1K 次观看.  University College London. 胚胎神经元是出生在神经管的脑室区，但迁移到达到适当的目标。面部branchiomotor（FBM）的神经元，研究神​​经细胞迁移的有用模型。本协议描述的小鼠胚胎hindbrains平片的体外培养研究，规范FBM迁移机制。 

视频: Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

体外实时成像在小鼠神经上皮单细胞分裂

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

科研

神经科学

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted area of the brain, neural stem cells proliferate and constantly generate neuroblasts that migrate tangentially in chains along the rostral migratory stream (RMS) to reach the olfactory bulb (OB). Once in the OB, neuroblasts switch to radial migration and then differentiate into mature neurons able to incorporate into the preexisting neuronal network. Proper neuroblast migration is a fundamental step in neurogenesis, ensuring the correct functional maturation of newborn neurons. Given the ability of SVZ-derived neuroblasts to target injured areas in the brain, investigating the intracellular mechanisms underlying their motility will not only enhance the understanding of neurogenesis but may also promote the development of neuroregenerative strategies.
This manuscript describes a detailed protocol for the transfection of primary rodent RMS postnatal neuroblasts and the analysis of their motility using a 3D in vitro migration assay recapitulating their mode of migration observed in vivo. Both rat and mouse neuroblasts can be quickly and efficiently transfected via nucleofection with either plasmid DNA, small hairpin (sh)RNA or short interfering (si)RNA oligos targeting genes of interest. To analyze migration, nucleofected cells are reaggregated in &#39;hanging drops&#39; and subsequently embedded in a three-dimensional matrix. Nucleofection per se does not significantly impair the migration of neuroblasts. Pharmacological treatment of nucleofected and reaggregated neuroblasts can also be performed to study the role of signaling pathways involved in neuroblast migration.

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

Neuroblast migration is a crucial step in postnatal neurogenesis. The protocol described here can be used to investigate the role of candidate regulators of neuroblast migration by employing DNA/small hairpin RNA (shRNA) nucleofection and a 3D migration assay with neuroblasts isolated from the rodent postnatal rostral migratory stream.

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 ...

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 ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

产后的小脑颗粒细胞迁移利用共聚焦肉眼离体成像

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

低密度原海马神经元文化

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

The Mouse Hindbrain As a Model for Studying Embryonic Neurogenesis

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded forebrain neuroepithelium is not amenable to wholemount analysis to examine organ-wide neurogenesis patterns. Moreover, defining the mechanisms of forebrain neurogenesis is not necessarily predictive of neurogenesis in other parts of the brain; for example, due to the presence of forebrain-specific progenitor subtypes. The mouse hindbrain provides an alternative model for studying embryonic neurogenesis that is amenable to wholemount analysis, as well as tissue sections to observe the spatiotemporal distribution and behavior of neural progenitors. Moreover, it is easily dissected for other downstream applications, such as cell isolation or molecular biology analysis. As the mouse hindbrain can be readily analyzed in the vast number of cell lineage reporter and mutant mouse strains that have become available, it offers a powerful model for studying the cellular and molecular mechanisms of developmental neurogenesis in a mammalian organism. Here, we present a simple and quick method to use the mouse embryo hindbrain for analyzing mammalian neural progenitor cell (NPC) behavior in wholemount preparations and tissue sections.

This article demonstrates how the mouse embryonic hindbrain can be used as a model for studying developmental neurogenesis in both whole organ and tissue section preparations.

小鼠后脑作为研究胚胎神经再生的模型

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

利用体外大鼠脑切片研究变性主要事件的一种替代方法

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Facial Nerve Surgery in the Rat Model to Study Axonal Inhibition and Regeneration

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial nerve can be manipulated along its entire length, from its intracranial segment to its extratemporal course. There are three primary types of nerve injury used for the experimental study of regenerative properties: nerve crush, transection, and nerve gap. The range of possible interventions is vast, including surgical manipulation of the nerve, delivery of neuroactive reagents or cells, and either central or end-organ manipulations. Advantages of this model for studying nerve regeneration include simplicity, reproducibility, interspecies consistency, reliable survival rates of the rat, and an increased anatomic size relative to murine models. Its limitations involve a more limited genetic manipulation versus the mouse model and the superlative regenerative capability of the rat, such that the facial nerve scientist must carefully assess time points for recovery and whether to translate results to higher animals and human studies. The rat model for facial nerve injury allows for functional, electrophysiological, and histomorphometric parameters for the interpretation and comparison of nerve regeneration. It thereby boasts tremendous potential toward furthering the understanding and treatment of the devastating consequences of facial nerve injury in human patients.

This protocol describes a reproducible approach to facial nerve surgery in the rat model, including descriptions of various inducible patterns of injury.

大鼠模型中的面部神经外科研究同氧抑制与再生

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial ...

Ex Vivo Pressurized Hippocampal Capillary-Parenchymal Arteriole Preparation for Functional Study

From subtle behavioral alterations to late-stage dementia, vascular cognitive impairment typically develops following cerebral ischemia. Stroke and cardiac arrest are remarkably sexually dimorphic diseases, and both induce cerebral ischemia. However, progress in understanding the vascular cognitive impairment, and then developing sex-specific treatments, has been partly limited by challenges in investigating the brain microcirculation from mouse models in functional studies. Here, we present an approach to examine the capillary-to-arteriole signaling in an ex vivo hippocampal capillary-parenchymal arteriole (HiCaPA) preparation from mouse brain. We describe how to isolate, cannulate, and pressurize the microcirculation to measure arteriolar diameter in response to capillary stimulation. We show which appropriate functional controls can be used to validate the HiCaPA preparation integrity and display typical results, including testing potassium as a neurovascular coupling agent and the effect of the recently characterized inhibitor of the Kir2 inward rectifying potassium channel family, ML133. Further, we compare the responses in preparations obtained from male and female mice. While these data reflect functional investigations, our approach can also be used in molecular biology, immunochemistry, and electrophysiology studies.

The present manuscript details how to isolate hippocampal arterioles and capillaries from the mouse brain and how to pressurize them for pressure myography, immunofluorescence, biochemistry, and molecular studies.

Ex Vivo 加压希马卡毛细管- 功能研究准备

From subtle behavioral alterations to late-stage dementia, vascular cognitive impairment typically develops following cerebral ischemia. Stroke and ...

In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using calcium as a proxy for neuronal activity, these techniques provide a way to monitor the responses of individual cells within large neuronal ensembles to a variety of stimuli in real time. We, and others, have applied these techniques to image the responses of individual geniculate ganglion neurons to taste stimuli applied to the tongues of live anesthetized mice. The geniculate ganglion is comprised of the cell bodies of gustatory neurons innervating the anterior tongue and palate as well as some somatosensory neurons innervating the pinna of the ear. Imaging the taste-evoked responses of individual geniculate ganglion neurons with GCaMP has provided important information about the tuning profiles of these neurons in wild-type mice as well as a way to detect peripheral taste miswiring phenotypes in genetically manipulated mice. Here we demonstrate the surgical procedure to expose the geniculate ganglion, GCaMP fluorescence image acquisition, initial steps for data analysis, and troubleshooting. This technique can be used with transgenically encoded GCaMP, or with AAV-mediated GCaMP expression, and can be modified to image particular genetic subsets of interest (i.e., Cre-mediated GCaMP expression). Overall, in vivo calcium imaging of geniculate ganglion neurons is a powerful technique for monitoring the activity of peripheral gustatory neurons and provides complementary information to more traditional whole-nerve chorda tympani recordings or taste behavior assays.

Here we present how to expose the geniculate ganglion of a live, anesthetized laboratory mouse and how to use calcium imaging to measure the responses of ensembles of these neurons to taste stimuli, allowing for multiple trials with different stimulants. This allows for in depth comparisons of which neurons respond to which tastants.

在活体钙成像小鼠致病性甘里昂神经元对味觉刺激的反应

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using ...