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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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. 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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陷。延时序列用于跟踪各个神经元的迁移路径，并计算迁移神经元的特定参数，包括速度曲线，平均迁移速度和迁移方向10 。 该方案描述了研究分子时的一个重要方法r径向迁移机制。通过使用短发夹RNA或DNA表达质粒，几乎任何感兴趣的基因可分别被敲低或过量表达。将电穿孔与Cre-LoxP系统结合在一起是一种强有力的方法。用Cre重组酶转染条件突变体（"floxed"）小鼠的大脑产生镶嵌突变情况并允许对由野生型细胞包围的单突变神经元的选择性分析。通过这种方式，可以研究迁移神经元中的细胞自主分子机制。此外，容易执行功能救援实验。准备延时系列电穿孔脑切片允许动态分析单个迁移神经元的迁移行为，其提供比从固定脑部分获得的信息更多的信息。 在子宫内的电3，4时需要进行初始训练，但-一旦掌握了-是快速和直接的技术在体内可重复地转染大脑皮质的神经元。在本文所述的方案中，我们在E14.5的子宫电穿孔中使用，当出生晚期新生的上皮新皮质投射神经元时。对于子宫电穿孔中早期出生的深层投影神经元的研究必须在早期发育阶段进行（ 即 E12.5） 13 。原则上，该方案还可用于研究可以用电穿孔转染的脑中其他神经元亚群的迁移，包括中间神经元14和小脑颗粒细胞的迁移15 。 我们描述了切片培养协议改编自Polleux和天哪12和先前，我们已经使用了前子宫内电16,17给STUdy海马发育。最重要的是，必须在整个手术过程中小心处理脑切片以保持其生存能力。我们使用倒置显微镜和光学质量的玻璃底层，将沉积在细胞培养插入物上的切片培养物成像。这种配置非常容易设置并允许无菌条件，因为目标不会与切片培养物或培养基接触。然而，需要具有足够高数值孔径（0.6或更高）的长工作距离物镜（&gt; 2 mm）。其它研究已经直接就把脑切片到玻璃底和中使用的胶原基质18,19固定在适当位置的切片，这使得用更短的自由工作距离可能使用的目标。然而，使用细胞培养插入物提供了易于处理，可重现的结果，并且允许研究人员在其制备后1至2天对成片进行成像，脑切片的生存能力（数据未显示）。另一个关键问题是通过激光损伤切片。我们使用了高灵敏度的混合检测器，这使得我们能够将激光功率降至最低，同时仍然获得足够的荧光信号用于延时成像。与常规共焦显微镜相比，使用多光子成像将进一步减少光损伤，并且允许更深层的组织穿透以及更有效的光检测。 尽管研究神经元迁移是有用的，但该方案具有局限性，并不能完全保留完整脑内迁移神经元的情况。细胞外基质，粘合剂细胞间接触，并迁移区内脉管系统可以动态地影响径向迁移神经元20,21。具体来说，上层神经元依赖于细长的径向胶质过程他们的迁徙22 。对于成功的成像，因此重要的是选择具有GFP电穿孔的放射状胶质细胞的脑切片，其扩展其整个皮层宽度的过程。放射状胶质支架在到达脑部表面之前已被切除的"斜切片"应进一步分析。有时，细胞死亡可能发生在脑片的空气表面，这限制了图像采集到样品内更深的区域。培养的脑切片中的电穿孔神经元可能与体内电穿孔神经元相比迁移的效率较低，这可能是由于准备恢复不足引起的。这需要对实验和控制情境的多个切片培养物进行分析，以及对代表性数据集的批判性解释。在某些情况下，与子宫电穿孔相关的侮辱可能会导致胚胎死亡或结构性改变脑的离子，包括扩大的脑室，不对称变薄的皮层，或由DNA注射引起的胶质瘢痕形成。在这些情况下，样品应进一步分析。 虽然来自胚胎供体的脑切片在膜插入物23上存活良好，但分析仅限于早期发育事件，包括神经元迁移，并且我们尚未将其用于研究后续事件，例如枝晶发育或突触形成。总之，我们提供了一个详细的方案来直接和动态地研究切片培养物中电穿孔神经元的神经元迁移，其可以容易地适应于分析参与神经发育障碍的基因的功能。

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 border="0" cellpadding="0" cellspacing="0" width="771">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">isoflurane</td>
			<td style="width:171px;">Abbott Laboratories&#160;</td>
			<td style="width:111px;">506949</td>
			<td style="width:209px;">Forene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">6-well plate</td>
			<td style="width:171px;">Corning</td>
			<td style="width:111px;">351146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">12-well plate</td>
			<td style="width:171px;">Corning</td>
			<td style="width:111px;">351143</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">non-absorbable surgical suture</td>
			<td style="width:171px;">Ethicon</td>
			<td style="width:111px;">K890H</td>
			<td>3/8 circle, 13 mm, taper point</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Micro Adson Forceps</td>
			<td style="width:171px;">Fine Science Tools</td>
			<td style="width:111px;">11018-12</td>
			<td>serrated, length: 12 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">fine scissors</td>
			<td style="width:171px;">Fine Science Tools</td>
			<td style="width:111px;">14063-09</td>
			<td>angled to side, length: 9 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Mathieu Needle Holder</td>
			<td style="width:171px;">Fine Science Tools</td>
			<td style="width:111px;">12510-14</td>
			<td>tungsten carbide, length: 14 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">fine tipped forceps</td>
			<td style="width:171px;">Fine Science Tools</td>
			<td style="width:111px;">11370-40</td>
			<td>straight, 11 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Vannas T&#252;bingen Spring Scissors</td>
			<td style="width:171px;">Fine Science Tools</td>
			<td style="width:111px;">15005-08</td>
			<td>angled up, 9.5 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">ring forceps</td>
			<td style="width:171px;">Fine Science Tools</td>
			<td style="width:111px;">11103-09</td>
			<td>OD: 3mm, ID, 2.2 mm, length: 9 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">HBSS (10X)</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:111px;">14180046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">L-Glutamine</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:111px;">25030081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Penicillin/Streptomycin</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:111px;">15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">horse serum</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:111px;">26050088</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">BME</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:111px;">41010026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">borosilicate glass capillaries</td>
			<td style="width:171px;">Harvard Apparatus</td>
			<td style="width:111px;">30-0016</td>
			<td>1.0 OD x 0.58 ID x 100 L mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">anesthsesia system</td>
			<td style="width:171px;">Harvard Apparaus</td>
			<td style="width:111px;">72-6471</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">anesthetizing chamber</td>
			<td style="width:171px;">Harvard Apparaus</td>
			<td style="width:111px;">34-0460</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">fluosorber filter canister</td>
			<td style="width:171px;">Harvard Apparaus</td>
			<td style="width:111px;">34-0415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">low melting point agarose</td>
			<td style="width:171px;">Invitrogen</td>
			<td style="width:111px;">16520100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">vibrating blade microtome</td>
			<td style="width:171px;">Leica</td>
			<td style="width:111px;">VT1200 S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">fluorescence stereo microscope</td>
			<td style="width:171px;">Leica</td>
			<td style="width:111px;">M205 FA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">stereo microscope</td>
			<td style="width:171px;">Leica</td>
			<td style="width:111px;">M125</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">inverted fluorescence tissue culture microscope</td>
			<td style="width:171px;">Leica</td>
			<td style="width:111px;">DM IL LED</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">confocal laser scanning microscope</td>
			<td style="width:171px;">Leica</td>
			<td style="width:111px;">TCS SP5II</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">hybrid detector</td>
			<td style="width:171px;">Leica</td>
			<td style="width:111px;">HyD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">objective, 40x/0.60 NA</td>
			<td style="width:171px;">Leica</td>
			<td style="width:111px;">11506201</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">microscope temperature control system</td>
			<td style="width:171px;">Life Imaging Services</td>
			<td style="width:111px;">Cube, Brick &#38; Box</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">cell culture insert</td>
			<td style="width:171px;">Millipore</td>
			<td style="width:111px;">PICM0RG50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">microgrinder</td>
			<td style="width:171px;">Narishige</td>
			<td style="width:111px;">EG-45</td>
			<td>use 38&#176; angle for beveling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">microinjector</td>
			<td style="width:171px;">Parker Hannifin&#160;</td>
			<td>052-0500-900</td>
			<td style="width:209px;">Picospritzer III</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">carprofen</td>
			<td style="width:171px;">Pfizer Animal Health</td>
			<td style="width:111px;">NDC 61106-8507</td>
			<td>Rimadyl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">emdedding mold</td>
			<td style="width:171px;">Polysciences</td>
			<td style="width:111px;">18986-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">endotoxin-free plasmid maxi kit</td>
			<td style="width:171px;">Qiagen</td>
			<td style="width:111px;">12362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">fast green</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:111px;">F7252</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">laminin</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:111px;">L2020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">poly-L-lysine</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:111px;">P5899</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">HEPES</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:111px;">H4034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">D-glucose</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:111px;">G6152</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">calcium chloride</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:111px;">C7902</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">magensium sulfate</td>
			<td>Sigma</td>
			<td style="width:111px;">M2643</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">sodium bicarbonate</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:111px;">S6297</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">square wave electroporator</td>
			<td style="width:171px;">Sonidel</td>
			<td style="width:111px;">CUY21EDIT</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">tweezers with 5 mm platinum disk electrodes</td>
			<td style="width:171px;">Sonidel</td>
			<td style="width:111px;">CUY650P5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">micropipette puller</td>
			<td style="width:171px;">Sutter Instrument</td>
			<td style="width:111px;">P-97</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">box filament</td>
			<td style="width:171px;">Sutter Instrument</td>
			<td style="width:111px;">FB255B</td>
			<td>2.5 mm x 2.5 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">micro-spoon spatula</td>
			<td style="width:171px;">VWR</td>
			<td style="width:111px;">231-0191</td>
			<td>185 mm x 5 mm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">glass bottom dish, 50 mm</td>
			<td style="width:171px;">World Precision Instruments</td>
			<td style="width:111px;">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突变体神经元沿着IZ和VZ / SVZ的迁移路线停滞。此外，我们发现以Bcl11a缺失为特征的IZ中双极细胞损失的多极细胞数量的增加表明极化10的缺陷。 直接和动态地分析migraBcl11a突变体神经元的tory行为，我们使用电穿孔大脑制备的器官切片培养中迁移神经元的延时共聚焦显像。概述使用数值孔径为0.4的10X物镜的延时系列证实了我们的结果， Bcl11a突变体投影神经元在径向迁移中具有缺陷。在36h内，许多控制神经元已迁移到CP，而只有少数Bcl11a突变体神经元离开IZ并迁移到CP。有趣的是，许多Bcl11a突变体神经元似乎没有直接迁移到大脑的表面，而是减慢了迁移并且转向切向或甚至向心室转移（Movie 1）。为了进一步分析这些发现，我们使用数值孔径为0.6的40x物镜，以更高的放大倍率产生延时序列。我们的数据显示， Bcl11a突变体神经元无法有效地极化并继续径向定向迁移离子入CP。在12小时内，许多Bcl11a突变体神经元不能从多极转变为双极形态，而是将许多过程投射和收回到周围环境中（ 图1A中的电影2）。 此外，我们追踪了不同延时序列中各个神经元的迁移路径，并用它们来计算迁移神经元的特定参数。我们发现Bcl11a突变体神经元重复地经历几个小时持续时间的阶段，具有降低的迁移速度和随机取向变化（Movie 3; 图1B ，红色箭头）。特别地，与对照神经元相比， Bcl11a突变体神经元的速度曲线从较高速度（25μm/ h）转向较低速度（5μm/ h）（ 图1C ）。与此同时， Bcl11的整体迁移速度突变神经元从对照组的10.34±0.34μm/ h显着降低到6±0.54μm/ h（p = 2.4889E-05）（ 图1D ）。最后， Bcl11a突变体神经元的偏向指向迁移到脑部表面，从17.15±2.13°（40.16±4.42°）明显增加（ 图1E ）。这些结果一起表明，在器官切片培养中GFP电穿孔神经元的延时共聚焦成像是研究控制神经元迁移过程的分子机制的有价值的方法。 <img alt="电影1" src="/files/ftp_upload/55886/55886mov1.jpg" /> 电影1:GFP标记控制与皮质切片中Bcl11a突变体神经元的比较迁移。 Bcl11a flox / flox大脑电穿孔E14.5用含有IRES-GFP （A）或Cre-IRES-GFP （B）的质粒载体，切片培养物在E16.5中制备。 VZ / SVZ;心室/室下区; IZ，中间区; CP，皮质板。刻度棒=100μm。电影由5帧/秒的速率组成。从Wiegreffe 等人转载10经Elsevier许可。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55886/Movie_1.avi" target="_blank">请点击此处查看此视频。 （右键单击下载。）</a> <img alt="电影2" src="/files/ftp_upload/55886/55886mov2.jpg" /> 电影2:与皮质切片中的Bcl11a突变型神经元相比，GFP标记对照的极化。 Bcl11a flox / flox大脑在E14.5上用含有IRES-GFP （A）或Cre-IRES-GFP （B）和切片培养物的质粒载体电穿孔<html>在E16.5准备。刻度棒=10μm。电影由5帧/秒的速率组成。从Wiegreffe 等人转载10经Elsevier许可。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55886/Movie_2.avi" target="_blank">请点击此处查看此视频。 （右键单击下载。）</a> <img alt="电影3" src="/files/ftp_upload/55886/55886mov3.jpg" /> 电影3:具有代表性控制和Bcl11a突变体神经元的1小时间隔分辨率的动画跟踪。迁移神经元的踪迹是从含有IRES-GFP （ A ）或Cre-IRES-GFP （ B ）的质粒载体在E14.5上电穿孔的Bcl11a flox / flox脑的E16.5切片培养物的延时系列获得的， 。刻度棒=20μm。从Wiegreffe 等人转载&gt; 10，得到Elsevier许可。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55886/Movie_3.avi" target="_blank">请点击此处查看此视频。 （右键单击下载。）</a> <img alt="图1" class="xfigimg" src="/files/ftp_upload/55886/55886fig1.jpg" /> 图1: Bcl11a突变体上层皮质神经元的迁移行为。 （ A ）在12小时的总成像期间，用Cre-IRES-GFP或对照载体在E14.5电穿孔的Bcl11a flox / flox脑的E16.5切片培养物中迁移GFP阳性神经元的代表性图像。 （ B ）具有1小时间隔分辨率的代表性痕迹，在E16.5切片培养物中从携带 Cre-IRES-GFP的 E14.5电泳的Bcl11a flox / flox脑中迁移GFP阳性神经元或在整个成像周期长达22小时。 Bcl11a突变体神经元通常经历重复阶段的迁移速度降低和随机变化的方向（用红色箭头标记）。 （ C ）从迁移神经元的痕迹计算的速度曲线，如B.（ DE ）从Bcl11a flox / flox的 E16.5切片培养物中迁移的GFP阳性神经元的速度（ D ）和径向取向（ E ）的定量用Cre-IRES-GFP或对照载体（n = 15）以E14.5电穿孔的脑。平均值±sem;学生考试*** p &lt;0.001。刻度棒=10μm。从Wiegreffe 等人转载10经Elsevier许可。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55886/55886fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

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

10.1K 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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

在子宫内的电穿孔其次原发性神经文化为研究基因的功能在皮层神经元的一个子集

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

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 ...

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

在发展中国家斑马鱼的前脑的神经祖细胞克隆相关的定时实时成像

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

高效的基因传递到多个中枢神经系统使用新界在子宫内电穿孔

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

在子宫内电穿孔:鼠标控制时空基因Transefection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

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 ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

老鼠耳蜗外植体的长期时间推移成像

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced tools currently used to study neurons, have only partly been taken on by oligodendrocyte researchers. Cell type-specific staining by viral transduction is a useful approach to study live organelle dynamics. This paper describes a protocol for visualizing oligodendrocyte mitochondria in organotypic brain slices by transduction with adeno-associated virus (AAV) carrying genes for mitochondrial targeted fluorescent proteins under the transcriptional control of the myelin basic protein promoter. It includes the protocol for making organotypic coronal mouse brain slices. A procedure for time-lapse imaging of mitochondria then follows. These methods can be transferred to other organelles and may be particularly useful for studying organelles in the myelin sheath. Finally, we describe a readily available technique for visualization of unstained myelin in living slices by Confocal Reflectance microscopy (CoRe). CoRe requires no extra equipment and can be useful to identify the myelin sheath during live imaging.

Myelinating oligodendrocytes promote rapid action potential propagation and neuronal survival. Described here is a protocol for oligodendrocyte-specific expression of fluorescent proteins in organotypic brain slices with subsequent time-lapse imaging. Further, a simple procedure for visualizing unstained myelin is presented.

腺相关病毒和共聚焦显微镜对器官脑切片胶质细胞器的可视化和实时成像

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced ...