感谢Jacqueline Andratschke，Elena Werle，Sachi Takenaka和Matthias Toberer的出色技术帮助，以及Victor Tarabykin的有益讨论。这项工作得到了德意志银行授予SB（BR-2215）的支持。

所有实验程序均经动物福利委员会（RegierungspräsidiumTübingen）批准，并按照"德国动物福利法"和欧盟指令2010/63 / EU进行。 在Utero电穿孔<ol><li> 显微注射针 <ol><li>使用具有盒状细丝（2.5mm×2.5mm）的微量移液管拉拔器将硼硅酸盐玻璃毛细管（外径:1.0mm，内径:0.58mm，长度:100mm）拉入显微注射针，并且以下程序:HEAT:540，PULL :125，VELOCITY:20和DELAY:140.通过执行RAMP测试（HEAT值:RAMP + 25）确定每个单独灯丝的HEAT值。 </li><li>锥形针以38°的角度和中等至高的转速用微型研磨机获得，以获得在胚胎期（E）注射的20-30μm的尖端尺寸（E）13.5或更小和30至40μm用于在较老胚胎处注射阶段。为了储存，将针头固定在盒子或培养皿中，用造型粘土，以防止伤痕。 </li></ol></li><li> 质粒DNA溶液 <ol><li>根据制造商的说明书，使用不含内毒素的最大制剂试剂盒，制备含有荧光报告蛋白（ 如 GFP）的质粒DNA构建体。 </li><li>在含有Fast Green的无内毒素Tris-EDTA缓冲液中，终浓度为0.01％，将质粒DNA溶液稀释至终浓度为1-2μg/μL。等分溶液并储存在-20°C。 </li></ol></li><li> 抑菌氯化钠溶液 <ol><li>通过向等渗0.9％氯化钠溶液中加入苄醇至终浓度为0.9％来制备抑菌性氯化钠溶液。无菌过滤溶液即将使用。 </li></ol></li><li> 卡洛芬注射液 <ol><li>准备carprofen注射溶液将无毒等渗0.9％氯化钠溶液加入到最终浓度为0.5mg / mL的卡洛芬。在4°C储存长达28天。 </li></ol></li><li> 麻醉，DNA注射和电穿孔 <ol><li>称重E14.5定时怀孕的小鼠，并将其置于连接到蒸发器并用5％异氟烷饱和的透明麻醉室中。当动物无意识时，将其转移到连接到37°C加温板上的麻醉面罩上，并用1.8 - 2.2％异氟烷将其麻醉。 注意:通过尾部夹紧和脚踏反射（脚趾捏）的损失来评估手术麻醉的发展。 </li><li>对于镇痛药，每10 mg小鼠体重（5 mg / kg体重）皮下注射100μL卡洛芬注射液。转动鼠标背部，小心地用石油覆盖眼睛，以防止它们干燥。 </li><li>轻轻地散开四肢并修复它们用手术胶带加温板。用70％乙醇，然后使用纤维素拭子，用碘溶液（7.5mg / mL）灭菌腹部。重复此过程三次，以确保正确消毒。 </li><li>用无菌纱布覆盖腹部，在其中切开切口以排除腹部切口的一个场（直径为2 - 2.5厘米）。用抑菌氯化钠溶液润湿纱布。 注意:通过丧失脚踏反射来重新评估手术麻醉的发展。 </li><li>使用Micro Adson镊子（锯齿，长度:12厘米）和精细剪刀（与侧面成角度，长度为9厘米）沿着腹部中线切割皮肤约1.5厘米。沿着线条切下下面的肌肉。 </li><li>用环形镊子（长度:9厘米，外径:3毫米，内径:2.2毫米）取出一个子宫角，轻轻放在湿润的纱布上。 注意:只能在胚胎之间用环形镊子握住子宫角d不要扰乱胎盘或任何供应容器。使用抑菌性氯化钠溶液始终保持子宫潮湿。 </li><li>仔细定位胚胎与环镊子，并定位侧脑室，其呈现平行于矢状窦的新月形阴影。注射部位大致位于着色眼和鼻窦汇合处之间的一条线的中间，其中矢状窦分支到两个横向鼻窦。将显微注射针推过子宫壁并进入侧脑室。 注意:如有必要，注射深度可以通过针的缩回来矫正。 </li><li>使用以下设置使用微型注射器将1至2μLDNA溶液注入侧脑室，使用以下设置操作:25 psi，每脉冲10至20 ms，5至10个脉冲。成功注射可以通过彩色DNA溶液进行监测，该溶液应填充心室系统。 </li><li>放置镊子式电极（E13.5直径3 mm，E14.5以上直径5 mm），使"正"端与注射心室位于同一侧，"阴性"终端位于胚胎头部耳朵下方的注射心室的相对侧。 </li><li>用几滴抑菌氯化钠溶液润湿电穿孔部位，并以950 ms的间隔施加5次持续时间为50ms的5次电流脉冲（E13.5或更小的35 V，E14.5或更高的40 V）。 注意:60至90 mA的电流通常足以成功进行电穿孔。较低的电流不会有效地转染神经元，而较高的电流可能导致胚胎死亡。 </li><li>对子宫角的每个胚胎重复该程序（参见步骤1.5.7至1.5.10。），轻轻将其放回腹腔。 </li><li>从另一边取出子宫角并重复进行在步骤1.5.7中描述。至1.5.11。 </li><li>使用Micro Adson镊子，Mathieu针头支架（碳化钨，长度:14厘米）和不可吸收的外科缝合线（3/8圆形，13毫米，锥形点）缝合肌肉层，缝合肌肉层，关闭腹腔。 注意:缝合时要小心不要抓住子宫或其他器官。 </li><li>用碘溶液（7.5 mg / mL）仔细消毒皮肤缝线，并使用纤维素拭子轻轻除去眼周过多的凡士林。将鼠标放在红外线灯下，直到它醒来。在接下来的两天内密切监视鼠标。在12至24小时后重复步骤1.5.2所述的镇痛治疗。 </li></ol></li></ol> 2.器官切片培养<ol><li> 层粘连蛋白溶液 <ol><li>将1mg冻干层粘连蛋白溶解在无菌水中，得到1mg / mL层粘连蛋白储备溶液。准备50μL等分试样and存储在-80°C。 </li></ol></li><li> 聚-L-赖氨酸储备溶液 <ol><li>将50mg聚-L-赖氨酸溶解在无菌水中，得到1mg / mL聚-L-赖氨酸储液。准备0.5 mL等分试样并储存于-20°C。 </li></ol></li><li> 完成汉克平衡盐溶液（完成HBSS） <ol><li>通过将50毫升10倍HBSS溶液，1.25毫升1M HEPES缓冲液（pH 7.4），15毫升1M D-葡萄糖溶液，5毫升100毫克氯化钙溶液，5毫升100毫摩尔硫酸镁溶液和2mL 1M碳酸氢钠溶液。加入无菌水至0.5升，无菌过滤，并在4℃下储存。 </li></ol></li><li> 切片培养基 <ol><li>将35mL的Basal Medium Eagle（BME），12.9mL的完全HBSS（参见2.3.1。），1.35mL的1M D-葡萄糖，0.25mL的200mM L-谷氨酰胺和0.5ml的青霉素 - 链霉素取50 mL切片培养中。无菌过滤器并加入马血清，最终浓度为5％。在4℃下储存长达4周。 </li></ol></li><li> 低熔点（LMP）琼脂糖溶液 <ol><li>通过在中等功率下在微波炉中加热1至2分钟将1.5g LMP琼脂糖溶解在50mL的完全HBSS中来制备3％LMP琼脂糖溶液。 注意:仔细监测加热，以防止沸腾期间溢出。 </li><li>将溶液放在38°C的水浴中，直到准备使用。储存于4°C并重新使用一次。 </li></ol></li><li> 膜片的涂层 <ol><li>将一层层粘连蛋白储备溶液（参见步骤2.1.1。）和一份等分的聚-L-赖氨酸储备溶液（参见步骤2.2.1。）加入6 mL无菌水中，得到涂层溶液（足够用于6个插入物） 。 </li><li>将膜插入物放入含有每孔2毫升无菌水的6孔板中。加入1 mL涂层溶液（参见步骤2.6.1），并在37℃和5％二氧化碳气氛下孵育过夜。 注意:只能使用带有透明膜的插件，如聚四氟乙烯（PTFE）膜。 </li><li>用1 mL无菌水清洗膜三次，然后干燥。在同一天使用涂膜，或在4℃干燥的6孔板中储存长达四周。 </li></ol></li><li> 脑解剖和嵌入 <ol><li>电穿孔两天后，将小鼠放入与蒸发器连接并用5％异氟烷饱和的透明室。当动物无意识时，将其从室中取出并通过颈椎脱位进行牺牲。 </li><li>取出含有胚胎的子宫，并将其放入带有冰冷的HBSS的10厘米培养皿中。 注意:从此以后，将所有解决方案，胚胎和大脑保留在冰上。 </li><li>使用立体显微镜，精尖镊子（直，11厘米）和一对VannasTübingen弹簧剪刀（有角度，长度为9.5厘米），以将每个胚胎与子宫分离，并将其转移到另一个含冰冷的完整HBSS的培养皿中。 </li><li>在脑干水平进行切口，沿矢状中线切开。剥离皮肤和覆盖大脑的软骨。然后，切断半球后面的脑干，并从头骨中取出大脑。将大脑用微匙铲（185毫米×5mm）转移到含有冰冷的完整HBSS的12孔板中。从12孔板中的垃圾收集所有的大脑。 </li><li>使用荧光立体显微镜来筛选12孔板中的脑的荧光亮度以及电穿孔区域的大小。在推测体感皮层中选择2到4个具有明亮荧光信号的大脑进行进一步处理。 </li><li>将保持在38℃的3％LMP琼脂糖溶液倒入剥离的一次性嵌入模具中。祛瘀e使用微勺铲子从12孔板中取出大脑，并使用精细的纸巾小心地排出大脑周围的多余的完整HBSS。 </li><li>将大脑轻轻放入琼脂糖溶液中，将其推入模具底部，并使用20号针仔细调整其位置。对于冠状切片，使嗅球向上指向大脑。将模具保持在冰上，直到琼脂糖溶液固化并继续切片。 </li></ol></li><li> 振动切片和切片培养 <ol><li>从模具中取出琼脂糖块，并将其放入无菌培养皿的盖子上。用干净的剃须刀剪掉多余的琼脂糖，离开嗅球下方约2至3毫米，另一侧留下1毫米的琼脂糖。修复修剪的琼脂糖块，嗅球向下指向使用氰基丙烯酸酯胶的样品台。 注意:用70％乙醇灭菌仪器和设备在分割之前。 </li><li>将样品台转移到含有冰冷的完整HBSS的振动刀片切片机的切片室中，并将其背侧的脑部朝向叶片。切割含有电穿孔区域的250μm厚的脑切片，低至中等幅度为0.9mm，非常慢的切片速度为0.09-0.12mm / s。通常从每个成功电穿孔的脑中获得具有明亮荧光信号的4至6个切片。 </li><li>用弯曲的微型勺子铲和细小的镊子，仔细地将脑切片与周围的琼脂糖转移到含有冰冷的完整HBSS的6孔板中。 注意:小心不要在转移过程中用镊子在周围的周围触摸琼脂糖边缘来损伤脑组织。 </li><li>按照步骤2.8.1中所述处理所有选定的大脑。至2.8.3。 </li><li>在放置脑sl之前，用100μL完整的HBSS滋润膜片插入物在薄膜上，以便于切片的定位。最多可以在1片膜片上放置5片。 </li><li>使用弯曲的微型勺子刮刀和精细的镊子将切片小心地转移到膜上。只要用镊子接触琼脂糖边缘，轻轻地将切片拉到刮刀上，轻轻将切片从刮刀推到膜上。使用镊子定位切片，并继续传输剩余的切片。用移液器清除多余的完整HBSS。 注意:不要将切片与彼此重叠。 </li><li>在镊子的帮助下，小心地将带有切片的膜片插入到含有1.8mL切片培养基的6孔板中（参见步骤2.4.1。），并在37℃和5％二氧化碳气氛下孵育直至延时成像。 </li></ol></li></ol>时间延迟成像<ol><li> 显微镜设置 <ol><li>设置一个放置在舞台上的气候室倒置共焦显微镜至37℃和5％二氧化碳气氛。使用混合检测器来减少激光强度并改善细胞存活力。要详细分析，请使用数字孔径为0.6或更高的超长工作距离40X干物镜。 注意:显微镜的所有组件需要预热至37°C 2 - 3 h，然后进行成像，以在成像过程中提供稳定的对焦。 </li></ol></li><li> 切片成像 <ol><li>使用倒置荧光显微镜从包含膜插入片的6孔板中选择一个脑切片进行成像。 注意:避免长时间接触紫外线，因为这可能会导致细胞光损伤。 注意:选择上部SVZ中具有明亮单个神经元的切片，径向迁移到切片的侧面。跨越整个CP的径向胶质细胞的精细荧光过程表明使用的是完整的放射状胶质支架d通过径向迁移皮质神经元。 </li><li>将用于延时成像的切片转移到具有镊子帮助的含有2ml切片培养基的50mm直径的玻璃底盘中。将盘放入共焦显微镜的气候室。 </li><li>将分辨率设置为512×512像素，并将扫描速度从400Hz提高到700Hz，以便将帧速率分别从约1.4增加到约2.5帧。平均使用不超过两次。通过电穿孔区域（通常为400-100μm）定义z叠层，步长为1.5μm。总的来说，这些设置允许图像的足够的分辨率和亮度，同时在拍摄期间保持光损伤。每30分钟采取一个z-stack开始延时系列。 注意:长时间将切片暴露于激光将引起光损伤，从而降低细胞的活力。调整exp相应地激光照射时间。 </li><li>使用ImageJ软件（https://imagej.nih.gov/iij/）或等同物分析延时系列。 </li></ol></li></ol>

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(136), pl9 (2002).</li><li>Greig, L. C., Woodworth, M. B., Galazo, M. J., Padmanabhan, H., Macklis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+logic+of+neocortical+projection+neuron+specification,+development+and+diversity.">Molecular logic of neocortical projection neuron specification, development and diversity.</a> Nat Rev Neurosci. 14 (11), 755-769 (2013).</li><li>De Marco Garcia, N. V., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subtype-selective+electroporation+of+cortical+interneurons.">Subtype-selective electroporation of cortical interneurons.</a> J Vis Exp. (90), e51518 (2014).</li><li>Holubowska, A., Mukherjee, C., Vadhvani, M., Stegmuller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulation+of+cerebellar+granule+neurons+in+vitro+and+in+vivo+to+study+neuronal+morphology+and+migration.">Genetic manipulation of cerebellar granule neurons in vitro and in vivo to study neuronal morphology and migration.</a> J Vis Exp. (85), (2014).</li><li>Venkataramanappa, S., Simon, R., Britsch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+utero+electroporation+and+organotypic+slice+culture+of+mouse+hippocampal+tissue.">Ex utero electroporation and organotypic slice culture of mouse hippocampal tissue.</a> J Vis Exp. (97), (2015).</li><li>Simon, 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=A+dual+function+of+Bcl11b/Ctip2+in+hippocampal+neurogenesis.">A dual function of Bcl11b/Ctip2 in hippocampal neurogenesis.</a> EMBO J. 31 (13), 2922-2936 (2012).</li><li>Youn, Y. H., Pramparo, T., Hirotsune, S., Wynshaw-Boris, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+dose-dependent+cortical+neuronal+migration+and+neurite+extension+defects+in+Lis1+and+Ndel1+mutant+mice.">Distinct dose-dependent cortical neuronal migration and neurite extension defects in Lis1 and Ndel1 mutant mice.</a> J Neurosci. 29 (49), 15520-15530 (2009).</li><li>Nadarajah, B., Brunstrom, J. E., Grutzendler, J., Wong, R. O., Pearlman, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+modes+of+radial+migration+in+early+development+of+the+cerebral+cortex.">Two modes of radial migration in early development of the cerebral cortex.</a> Nat Neurosci. 4 (2), 143-150 (2001).</li><li>Higginbotham, H., Yokota, Y., Anton, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+analyzing+neuronal+progenitor+development+and+neuronal+migration+in+the+developing+cerebral+cortex.">Strategies for analyzing neuronal progenitor development and neuronal migration in the developing cerebral cortex.</a> Cereb Cortex. 21 (7), 1465-1474 (2011).</li><li>Stubbs, D., 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=Neurovascular+congruence+during+cerebral+cortical+development.">Neurovascular congruence during cerebral cortical development.</a> Cereb Cortex. 19, i32-i41 (2009).</li><li>Ayala, R., Shu, T., Tsai, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trekking+across+the+brain:+the+journey+of+neuronal+migration.">Trekking across the brain: the journey of neuronal migration.</a> Cell. 128 (1), 29-43 (2007).</li><li>Humpel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+brain+slice+cultures:+A+review.">Organotypic brain slice cultures: A review.</a> Neuroscience. 305, 86-98 (2015).</li></ol>

作者没有什么可以披露的。

径向迁移是新皮质发育的关键过程。在影响该过程的不同步骤基因中的突变可以引起严重的皮质畸形，包括无脑回畸形和白质异位1,2。我们最近表明，在年轻的迁移性皮层投影神经元中表达的Bcl11a在径向迁移中起作用。我们使用电穿孔大脑急性皮质切片中迁移神经元的延时共聚焦成像，直接证明迁移神经元中Bcl11a的遗传缺失会导致极化和迁移缺?...

新皮质是认知，情感和感觉运动功能的主要部位。它由六个水平层组成，平行于大脑的表面。在后期端脑侧壁发育的祖细胞期间，引起径向迁移到侧面的投影神经元并获得层型特异性神经元的身份。在心室/室下区（VZ / SVZ）产生后，这些神经元变得瞬时多极化，并减慢其迁移。在中间区（IZ）短暂停留后，它们切换到双极形态，附着于径向胶质支架，并继续径向定向迁移到皮质板（CP）中。在达到其最终目标投影神经元离开径向胶质细胞过程并获得层特异性身份时。影响不同步骤的神经元迁移的基因突变会引起严重的皮层畸形，如lissencephaly或白质异位1,2。 在子宫内的电是一个快速和强大的技术转染神经祖细胞在啮齿动物胚胎3,4的脑发育。使用这种技术，可以敲除和/或过度表达感兴趣的基因，以研究其在发育神经元中的功能。该方法已具体地有助于描述形态细节和表征径向迁移5，6，7，8，9的处理的分子机制。径向迁移的神经元经历细胞形态，迁移速度以及迁移方向的动态变化，这需要随时间直接和连续观察。器官切片培养电穿孔脑的再次和延时共焦成像允许随着时间的推移直接观察迁移神经元。使用这种组合方法，可以分析在电穿孔脑的固定组织切片中不能研究的迁移神经元的不同特征。 我们最近迁移应用在电穿孔的大脑切片培养的神经元皮质发育期间10来研究转录因子B细胞白血病/淋巴瘤11A（BCL11A）的作用时间推移共焦成像。 Bcl11a在年轻的迁移皮质神经元中表达，我们使用条件突变体Bcl11a等位基因（ Bcl11a flox ） 11来研究其功能。将Cre重组酶与绿色荧光蛋白（GFP）电穿孔到Bcl11a flox / flox脑的皮质祖细胞中，使得我们能够产生镶嵌突变体情况，其中只有少数细胞在否则野生型背景。以这种方式，可以在单细胞水平研究Bcl11a的细胞自主功能。我们发现Bcl11a突变体神经元显示出降低的速度，速度变化的变化，以及在其迁移期间的随机取向变化10 。在概述的协议中，我们描述了成功的电穿孔和切片培养制备12小鼠脑的工作流程，以及皮质切片培养物的延时共焦成像。

<table><tbody><tr><td>isoflurane</td><td>Abbott Laboratories&amp;nbsp;</td><td>506949</td><td>Forene</td></tr><tr><td>6-well plate</td><td>Corning</td><td>351146</td><td></td></tr><tr><td>12-well plate</td><td>Corning</td><td>351143</td><td></td></tr><tr><td>non-absorbable surgical suture</td><td>Ethicon</td><td>K890H</td><td>3/8 circle, 13 mm, taper point</td></tr><tr><td>Micro Adson Forceps</td><td>Fine Science Tools</td><td>11018-12</td><td>serrated, length: 12 cm</td></tr><tr><td>fine scissors</td><td>Fine Science Tools</td><td>14063-09</td><td>angled to side, length: 9 cm</td></tr><tr><td>Mathieu Needle Holder</td><td>Fine Science Tools</td><td>12510-14</td><td>tungsten carbide, length: 14 cm</td></tr><tr><td>fine tipped forceps</td><td>Fine Science Tools</td><td>11370-40</td><td>straight, 11 cm</td></tr><tr><td>Vannas T&amp;uuml;bingen Spring Scissors</td><td>Fine Science Tools</td><td>15005-08</td><td>angled up, 9.5 cm</td></tr><tr><td>ring forceps</td><td>Fine Science Tools</td><td>11103-09</td><td>OD: 3mm, ID, 2.2 mm, length: 9 cm</td></tr><tr><td>HBSS (10X)</td><td>Gibco</td><td>14180046</td><td></td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>25030081</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>horse serum</td><td>Gibco</td><td>26050088</td><td></td></tr><tr><td>BME</td><td>Gibco</td><td>41010026</td><td></td></tr><tr><td>borosilicate glass capillaries</td><td>Harvard Apparatus</td><td>30-0016</td><td>1.0 OD x 0.58 ID x 100 L mm</td></tr><tr><td>anesthsesia system</td><td>Harvard Apparaus</td><td>72-6471</td><td></td></tr><tr><td>anesthetizing chamber</td><td>Harvard Apparaus</td><td>34-0460</td><td></td></tr><tr><td>fluosorber filter canister</td><td>Harvard Apparaus</td><td>34-0415</td><td></td></tr><tr><td>low melting point agarose</td><td>Invitrogen</td><td>16520100</td><td></td></tr><tr><td>vibrating blade microtome</td><td>Leica</td><td>VT1200 S</td><td></td></tr><tr><td>fluorescence stereo microscope</td><td>Leica</td><td>M205 FA</td><td></td></tr><tr><td>stereo microscope</td><td>Leica</td><td>M125</td><td></td></tr><tr><td>inverted fluorescence tissue culture microscope</td><td>Leica</td><td>DM IL LED</td><td></td></tr><tr><td>confocal laser scanning microscope</td><td>Leica</td><td>TCS SP5II</td><td></td></tr><tr><td>hybrid detector</td><td>Leica</td><td>HyD</td><td></td></tr><tr><td>objective, 40x/0.60 NA</td><td>Leica</td><td>11506201</td><td></td></tr><tr><td>microscope temperature control system</td><td>Life Imaging Services</td><td>Cube, Brick &amp;amp; Box</td><td></td></tr><tr><td>cell culture insert</td><td>Millipore</td><td>PICM0RG50</td><td></td></tr><tr><td>microgrinder</td><td>Narishige</td><td>EG-45</td><td>use 38&amp;deg; angle for beveling</td></tr><tr><td>microinjector</td><td>Parker Hannifin&amp;nbsp;</td><td>052-0500-900</td><td>Picospritzer III</td></tr><tr><td>carprofen</td><td>Pfizer Animal Health</td><td>NDC 61106-8507</td><td>Rimadyl</td></tr><tr><td>emdedding mold</td><td>Polysciences</td><td>18986-1</td><td></td></tr><tr><td>endotoxin-free plasmid maxi kit</td><td>Qiagen</td><td>12362</td><td></td></tr><tr><td>fast green</td><td>Sigma</td><td>F7252</td><td></td></tr><tr><td>laminin</td><td>Sigma</td><td>L2020</td><td></td></tr><tr><td>poly-L-lysine</td><td>Sigma</td><td>P5899</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H4034</td><td></td></tr><tr><td>D-glucose</td><td>Sigma</td><td>G6152</td><td></td></tr><tr><td>calcium chloride</td><td>Sigma</td><td>C7902</td><td></td></tr><tr><td>magensium sulfate</td><td>Sigma</td><td>M2643</td><td></td></tr><tr><td>sodium bicarbonate</td><td>Sigma</td><td>S6297</td><td></td></tr><tr><td>square wave electroporator</td><td>Sonidel</td><td>CUY21EDIT</td><td></td></tr><tr><td>tweezers with 5 mm platinum disk electrodes</td><td>Sonidel</td><td>CUY650P5</td><td></td></tr><tr><td>micropipette puller</td><td>Sutter Instrument</td><td>P-97</td><td></td></tr><tr><td>box filament</td><td>Sutter Instrument</td><td>FB255B</td><td>2.5 mm x 2.5 mm</td></tr><tr><td>micro-spoon spatula</td><td>VWR</td><td>231-0191</td><td>185 mm x 5 mm</td></tr><tr><td>glass bottom dish, 50 mm</td><td>World Precision Instruments</td><td>FD5040-100</td><td></td></tr></tbody></table>

time-lapse confocal imaging of migrating neurons in organotypic slice culture of embryonic mouse brain using in utero electroporation

在子宫电穿孔是研究小鼠胚胎大脑皮层径向迁移过程的快速有力的方法。它有助于描述径向迁移的不同步骤，并表征控制该过程的分子机制。为了直接和动态地分析迁移神经元，他们必须随时间追踪。该协议描述了将子宫电穿孔与器官切片培养和延时共焦成像相结合的工作流程，其允许直接检查和动态分析径向迁移的皮质神经元。此外，迁移神经元的详细表征，例如迁移速度，速度分布以及径向取向变化是可能的。该方法可以容易地适应于通过损失和功能增益以及救援实验来进行径向迁移的皮层神经元感兴趣的基因的功能分析。时间推移迁移神经元的成像是一种最先进的技术，一旦建立，这是一种有效的工具，用于研究小鼠神经元迁移障碍模型中大脑皮质的发育。

以前，我们已经表明， 在子宫电穿孔中Bcl11a的遗传缺失会损害晚期出生的上层投影神经元的径向迁移10 。含有Cre-IRES-GFP的DNA质粒载体的电穿孔有效地在条件Bcl11a flox / flox脑中删除Bcl11a 11 。当我们在电穿孔后三天分析E14.5电穿孔大脑时，大多数控制神经元已经迁移到CP，而许多Bcl11a突变体?...

Watch this Scientific Journal Video about Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation at JoVE.com

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

该方案提供了直接观察径向迁移皮质神经元的指导。 在子宫电穿孔中，组织切片培养和延时共聚焦成像结合直接动态地研究过度表达或下调基因在迁移神经元中的作用并分析其在发育过程中的分化。

延期共聚焦显像移植神经元在器官性切片培养胚胎小鼠脑使用<em&gt;在Utero</em&gt;电穿孔

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

time-lapse-confocal-imaging-migrating-neurons-organotypic-slice

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

10.2K 次观看.  Ulm University. 该方案提供了直接观察径向迁移皮质神经元的指导。 在子宫电穿孔中，组织切片培养和延时共聚焦成像结合直接动态地研究过度表达或下调基因在迁移神经元中的作用并分析其在发育过程中的分化。 

视频: Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

器官型切片文化绿色荧光蛋白表达的实时成像周围神经生长的小鼠胚胎

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

科研

神经科学

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

对于高分辨率延时成像神经细胞迁移的一个器官型产后脑切片检测

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

神经元形态的研究方法：<em&gt;体外</em&gt; RNAi技术在胚胎小鼠大脑皮质的电穿孔

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which 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 ...

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 ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

在子宫内电途径研究神经元亚群的兴奋性和单细胞连接

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

前子宫内胚胎小鼠脑电穿孔和脊髓切片培养迁移 GABAergic 中间神经元的活体成像

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones -the sensory structures located at the distal tips of developing axons- execute this process. Studying the structure and dynamics of the growth cone is crucial to understanding axonal development and the interactions with the surrounding central nervous system (CNS) that enable it to form neural circuits. This is essential when devising methods to reintegrate axons into neural circuits following injury in fundamental research and pre-clinical contexts. Thus far, the general understanding of growth cone dynamics is primarily founded on studies of neurons cultured in two dimensions (2D). Although undoubtedly fundamental to the current knowledge of growth cone structural dynamics and response to stimuli, 2D studies misrepresent the physiological three-dimensional (3D) environment encountered by neuronal growth cones in intact CNS tissue. More recently, collagen gels were employed to overcome some of these limitations, enabling the investigation of neuronal development in 3D. However, both synthetic 2D and 3D environments lack signaling cues within CNS tissue, which direct the extension and pathfinding of developing axons. This protocol provides a method for studying axons and growth cones using organotypic brain slices, where developing axons encounter physiologically relevant physical and chemical cues. By combining fine-tuned in utero and ex utero electroporation to sparsely deliver fluorescent reporters along with super-resolution microscopy, this protocol presents a methodological pipeline for the visualization of axon and growth cone dynamics in situ. Furthermore, a detailed toolkit description of the analysis of long-term and live-cell imaging data is included.

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

This protocol demonstrates a straightforward and robust method to study in situ axon growth and growth cone dynamics. It describes how to prepare ex vivo physiologically relevant acute brain slices and provides a user-friendly analysis pipeline.

原位 急性 离体 胚胎脑切片培养物中轴突生长和生长锥动力学的可视化

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones ...

可视化发育中的小鼠大脑中的神经元和神经胶质细胞动力学

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

作者聚焦：探索发育中的大脑中的细胞迁移和基因作用

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

胚胎小鼠脑切片中迁移神经元和神经胶质祖细胞的延时成像

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain ...

延期共聚焦显像移植神经元在器官性切片培养胚胎小鼠脑使用

摘要

探索更多视频

此视频中的章节

器官型切片文化绿色荧光蛋白表达的实时成像周围神经生长的小鼠胚胎

对于高分辨率延时成像神经细胞迁移的一个器官型产后脑切片检测

神经元形态的研究方法：

急性片的成年小鼠前脑的神经细胞迁移的延时成像

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

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

在子宫内电途径研究神经元亚群的兴奋性和单细胞连接

前子宫内胚胎小鼠脑电穿孔和脊髓切片培养迁移 GABAergic 中间神经元的活体成像

原位急性离体胚胎脑切片培养物中轴突生长和生长锥动力学的可视化

胚胎小鼠脑切片中迁移神经元和神经胶质祖细胞的延时成像

延期共聚焦显像移植神经元在器官性切片培养胚胎小鼠脑使用

摘要

探索更多视频

此视频中的章节

器官型切片文化绿色荧光蛋白表达的实时成像周围神经生长的小鼠胚胎

对于高分辨率延时成像神经细胞迁移的一个器官型产后脑切片检测

神经元形态的研究方法：

急性片的成年小鼠前脑的神经细胞迁移的延时成像

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

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

在子宫内电途径研究神经元亚群的兴奋性和单细胞连接

前子宫内胚胎小鼠脑电穿孔和脊髓切片培养迁移 GABAergic 中间神经元的活体成像

原位 急性 离体 胚胎脑切片培养物中轴突生长和生长锥动力学的可视化

胚胎小鼠脑切片中迁移神经元和神经胶质祖细胞的延时成像

原位急性离体胚胎脑切片培养物中轴突生长和生长锥动力学的可视化