Name: in utero electroporation approaches to study the excitability of neuronal subpopulations and single-cell connectivity
Uploaded: 2017-02-15T13:00:00.000+00:00
Duration: 10 min 49 s
Description: Watch this Scientific Journal Video about In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity at JoVE.com

我们感谢R.古铁雷斯和A.莫拉莱斯的出色技术援助和洛杉矶魏斯进行编辑。 CGB是由西班牙部:西恩西亚ËINNOVACIÓN（MICINN），FPI-BES-2012-056011资助。这项工作是由来自BBVA基金会和SAF2014-58598-JIN（MINECO）到M.纳瓦雷特的资助，并从拉蒙阿雷塞斯基金会和赠款SAF2014-52119-R和BFU2014-55738-REDT（从MINECO）向基金资助M.涅托。

所有动物的程序是由马德里动物护理和使用委员会，共同体符合国家和欧洲法规认可（PROEX一十四分之一百十八; PROEX15分之331）。保持在手术过程中在无菌条件。 1. 在子宫内的电注意:此协议为Iue时从先前已公布的5，6，7更为适合。该原稿描述了一种协议，用于E15.5的IUE的胚胎，在记者策略，其允许使用标准的GFP报告质粒单个神经元8和在一个单独的实验中其电生理学特性的形态的研究修改。 <ol><li>准备DNA <ol><li>准备使用不含内毒素的分离试剂盒的DNA质粒按照制造商的说明和稀他们在1×phosphate缓冲盐水（PBS）中。 <ol><li>对于单细胞标记，准备每个手术DNA混合液10μL使用下面的构造和最终浓度（每胎1μL）:质粒编码荧光蛋白报告（ 例如，CAG-的DsRed2 9），1微克/μL作为控制对于电效率;实验的质粒进行测试，通常在1微克/微升; LoxP位一站式loxP的荧光蛋白的质粒（CALNL-GFP 10），1微克/μL;构建编码Cre重组酶10，1 - 4纳克/μL。添加0.1％的快速绿色1μL水（重量/体积），以可视化的注射DNA。 注:将GFP构建体的Cre重组只在少数神经元，其允许可视化和个人轴突突起（ 图1A）的重建发生。 </li><li>对于标准的膜片钳研究，准备手术每DNA混合物10μL（每1 EMBRμL哟）编码荧光蛋白（如 CAG-GFP 11），1微克/μL作为记者对照以下质粒;实验的质粒，通常为1微克/微升;和0.1％坚牢绿的1μL的水（重量/体积）。 注意:这些是分析含有所需薄层内的大量标记的兴奋性神经元的急性切片的标准电穿孔条件。在单细胞进行膜片钳研究，如在步骤1.1.1.1中所述制备的DNA。 </li></ol></li></ol></li></ol> 2.准备手术<ol><li>通过使用无菌程序执行的生存术。确保无菌条件下，包括面罩，手套，仪器和外科领域。消毒手术器械（手术刀，镊子斜角肌挤压，硬化精剪刀，剪子弯，杜蒙钳和持针器）。 </li><li> 1 / 0.58毫米外/内径选择硼硅玻璃毛细管。拉帽illaries 3。指定用于拉动后1厘米最佳尖端长度。切针尖在使用罚款钳（ 图1B）约30°角。 </li><li>制备500mL的无菌等渗溶液（1×PBS或Hank&#39;s平衡盐溶液（HBSS））。加青霉素 - 链霉素1:100和温热此溶液至37℃。它可以在手术后保存在4℃。 </li><li>皮下注入镇痛药的术前剂量（ 例如，卡洛芬，5毫克/公斤体重）。 </li><li>通过将它们放置在一个加热垫保持温暖手术的动物。温暖干净的笼子至37℃，术后恢复。 </li></ol> 3.手术<ol><li>一个麻醉C57BL / 6 E15.5孕鼠异氟醚。首先，在0.8升/分钟氧气注入用3％异氟烷的密闭室和离开鼠标内部，直到它是睡着了。鼠标转移到一个加热垫，并放置在鼻子和嘴的掩模内用于递送异氟醚年。逐渐减小麻醉过经由掩模手术至1.5％的异氟醚的过程。通过观察踏板反射（趾捏）损失确认合适的麻醉。最佳过程需要大约20分钟，不超过45分钟。 </li><li>适用眼膏，以防止眼睛从过程中干燥。 </li><li>从腹部（使用电动剃须刀或脱毛霜）的〜3厘米的区域除去毛。洗用70％的乙醇注入棉签，后跟一个碘注入棉签手术区。重复三次。 </li><li>覆盖消毒纱布，防止感染手术区。 </li><li>使用手术刀，使透过皮肤长2cm至中线垂直开口和平行。分开使用钝剪子弯腹部的皮肤和肌肉。保持与镊子肌肉和削减它通过白线，以暴露腹腔。 </li><li>找到的帮助澳胚胎F中的镊子。滋润2湿棉签与在步骤2.3中制备的无菌生理盐水溶液，并用它们来操作可访问的胚胎出开口。周围放置胚胎的棉签轻轻提取从腹腔两个子宫角。保持用温生理盐水溶液水合的胚胎和打开腹腔整个过程中，以防止它们干燥。 </li></ol> 4.注射DNA和电的<ol><li>用手指来定位端脑轻轻操纵胚胎（可以由眼睛作为脑的两个前囊泡被清晰显示）。发生在口腔吸管准备硼硅毛细管。通过针尖通过子宫，避免血管，直到它到达一个横向心室。直到大蓝点观察缓缓注入大约1快速绿色的DNA溶液微升。 </li><li>放置7毫米铂电极横向地在th胚胎（ 图1C）电子负责人。 注:DNA被带负电荷;因此，当铂电极之间施加电压移向正极。通过改变电极的位置，大脑的不同区域可以有针对性。 </li><li> （:这些条件取决于发育阶段5个脉冲，38伏，50毫秒时间间隔周期的长度，950毫秒的时间间隔的暂停为E15.5胚胎。）12经由铂电极施加电压。 注:请参阅表1的电压条件和电极，用于不同的胚胎阶段。 </li><li>每个胚胎4.3 - 重复步骤4.1。 </li></ol> 5.结束了手术，术后<ol><li>用棉签操纵子宫回母亲。填用温热生理盐水溶液腹腔（加入约2毫升）中。 </li><li>缝合单纯间断缝合或连续缝合肌肉。使用＃6-0缝线秒。 </li><li>使用订书钉关闭外部伤口。小心装订之前从肌皮分离。取下鼻罩。 </li><li>让鼠标放置在动物设施室之前在加热的，洁净笼30分钟恢复。无人看管，直到它已经恢复了足够的意识，以保持胸骨斜卧不要让动物。不要将已动过手术公司中的其他动物，直到完全康复的动物。 </li><li>监督在手术后的几天动物。皮下应用镇痛药（卡洛芬，5毫克/千克体重）为2天或根据动物立法每12小时。没有额外的术后护理是必需的幼崽。 </li></ol> 6.样品的制备和分析<ol><li>可选:在P2，检查荧光用荧光显微镜电穿孔幼崽的表达;当IUE是成功的，荧光可以清楚地看出我n中的头13。标记或分开正在积极对在下面的步骤利用电穿孔的小鼠。请标准动物设施条件水坝和幼仔。 </li><li>在研究的期望阶段，灌注小鼠transcardially。通常情况下，树突和轴突生长的最活跃的相位对应于前三产后周2，14，15。 <ol><li>在1X PBS准备30毫升4％多聚甲醛（PFA）的每鼠标和寒意在冰上。 注意:PFA是一种已知的过敏原和致癌物质。它是有毒的。穿戴合适的个人防护装备。 注意:需要的PFA的量取决于鼠标（ 例如 ，P16，20毫升用于灌注和10ml的postfixation需要）的年龄。 </li><li>制备氯胺酮 - 赛拉嗪混合物用1至麻醉的动物:1:100微克/毫升氯胺酮8体积比:100微克/毫升×ylazine:PBS（准备每只小鼠约0.2毫升）。 </li><li>腹腔注射0.2毫升步骤6.2.2准备的混合麻醉鼠标。 </li><li>将在它的后面麻醉鼠标。使用手术刀胸部之上的水平切口。切开肌肉和隔膜用细剪刀直到心脏可以观察到。 <ol><li>制作与精细剪刀右心房切口。缓缓注入20毫升的PBS在用25号针头在左心室以除去血液。注射20毫升PFA（步骤6.2.1），直到鼠标是刚性的器官变成白色。 </li><li>立刻移开磁头，使正中切口，从颈部到颅骨皮肤。用弯钳轻轻剥离颅骨，并提取大脑。请特别小心不要刺破或切除颅骨时损坏大脑。把脑在10mL的4％的PFA的在4℃下过夜与postfix它。 </li></ol></li></ol></li><li> Cryoprotec吨于4℃固定脑在10毫升PBS中30％蔗糖的1 - 2天，直到它们下沉。制备1厘米3铝箔立方体。填充立方体与华侨城的方式2/3。把大脑内，通过将立方体上的干冰冷冻。储存在-80℃的冷冻大脑。 </li><li>样品盘的表面上的OCT的地方下降，剥离从组织学立方体的铝箔，并在所需方向上的液体OCT的顶部试样磁盘立方体定位。直到它被固定应用公司的压力。将样品光盘放入低温恒温器的样品头。定向标本（它移动到相对刀/刀片有利的位置）。 <ol><li>部分在低温恒温器的大脑。选择50 - 100微米厚的冰冻切片上浮16用细刷，并放置在PBS。 </li></ol></li><li>如果需要的污点。 注:对于轴突形态的研究，染色用绿色荧光蛋白抗体强烈推荐<s补课="外部参照"&gt; 2。 <ol><li>方框1小时在室温下用5％胎牛血清的PBS中含有0.5％的Triton-X 100（封闭溶液）的部分。在4℃下用1孵育过夜:500的第一抗体（ 例如，兔抗GFP）的封闭溶液稀释。 </li><li>洗部分三次于PBS中。添加1:500的二级抗体（ 例如，山羊抗兔的Alexa Fluor 488）在阻断溶液并孵育在室温下1小时稀释。洗部分三次于PBS中。 </li><li>在含有0.5％的Triton-X 100 10分钟的PBS:用DAPI（10,000 1）染剂。用PBS冲洗切片。安装在水性安装介质16的部分。 </li></ol></li></ol> 7.成像和分析<ol><li>荧光或共聚焦显微镜<ol><li>为了重建完整的神经元，以高倍率（至少40X）和高分辨率（最小1024x1024个）获取图像。选择"瓷砖扫描"，或在采集软件等效选项以覆盖感兴趣的区域，跨越所有树突和轴突的过程。获取足够数量的Z轴堆叠的，以避免信息丢失。 注意:通常情况下，在显微镜的软件有一个"优化栈"选项，但如果不是这种情况下，测试手动确定多少步骤是必要的图像，以便正确地确定各个凸起重叠。两个重要参数是针孔和物镜;考虑到"更高的放大倍率，更高的分辨率，更需要的z的步骤。" </li></ol></li><li>分析 注意:尽管几个参数可以被分析，步骤7.2.1集中于两个:树突形态和轴突分枝。下载斐济（ <a href="http://fiji.sc/" target="_blank">http://fiji.sc/</a> ）。 <ol><li>打开图像，从菜单中选择了"分段线"选项。画一条线（沿次上下移动Ëz轴通过滚动鼠标）的神经元的结构如下。进入"分析，工具，投资回报率经理，添加"（或者按"T"）来保存就行了。重复这个过程中所分析的神经元2的轴突每树突或。 </li><li>在ROI管理器菜单按"办法"按钮，得到的长度（或其他参数）。导出测量到一个文本文件或电子表格来分析它们。 </li></ol></li></ol> 8.电注:此协议的目标是从层获得全细胞电流钳记录中的GFP电穿孔小鼠大脑（或先前电穿孔的任何其它荧光蛋白）通过GFP表达视觉上识别的II / III锥体细胞的神经元。这是先前公布的方法17，18改编。使用此协议是可能的研究遗传改性的效果ication由IUE对神经元的电特性引入的。具体的发射模式的收购是分化的一个渐进的过程，涉及的离子通道的广泛的曲目的动态表达，导致瞬态射击模式的表达已故前产后阶段。例如，成熟的电反应不是在层的躯体感觉皮质鼠标的II / III P16 2，19之前观察到的。 <ol><li>先决条件急性片<ol><li>准备无菌手术工具从小鼠取出大脑:断头台，去除头部;小剪刀，通过头骨削减;镊子，分离从组织头骨;锅铲，以微妙从包装中取出脑组织;金属切片机，解剖皮质成相等的两半;和巴斯德吸管，从vibratome（将它们放置到含有人工脑脊液中的溶液移至片（交流SF）进行检查，然后从ACSF转移切片孵育保持区域）。 </li><li>制备1升使用含有119 mM氯化钠，26毫的NaHCO 3的高纯度的（双蒸水）水ACSF的，11毫米的葡萄糖，2.5mM的氯化钾，1.2mM的MgCl 2的，2.5mM的氯化钙和1mM的NaH 2 PO 4。滴定pH值至7.3 - 7.4用HCl或NaOH。调节渗透压至290毫渗量。 </li><li>气泡ACSF与卡波金（95％O 2/5％CO 2）15 - 7.4 -用特氟隆管（〜1毫米），以稳定pH至7.3 20分钟。 </li><li>冻结在-80℃，10与卡波金饱和ACSF溶液200毫升 - 15分钟，以产生泥泞解剖溶液。广场上冰100-×20毫米组织培养皿切断用大脑。 </li><li>只是在实验开始前制备100毫升的1％的低熔点琼脂糖溶液。冷却该溶液，切出一块方形琼脂糖（尺寸:1×1×0.5mm）的，并且它强力胶的回来的平台，后面那里的大脑切片（琼脂糖凝胶切片过程中提供支持，为大脑）。使前尽可能平坦。 </li></ol></li><li>获取急性片<ol><li>固定鼠标和用异氟烷2％麻醉它。将头部到断头台开放，迅速杀头的。与使用骨护林员或细镊子的尽可能快地取出它的头骨。把大脑进入冷冻学联。 注意:要快速执行此步骤是非常重要的。 </li><li>放置在大脑冷冻培养皿。切断与小剪刀小脑。 </li><li>拿起大脑用抹刀和吸干水分在纸巾上。 </li><li>胶上的vibratome支架大脑的ventrocaudal平面。放置保持器上装有冰冷ACSF一个vibratome。 </li><li>通过削减300微米冠状切片（确保持续carbogenation整个过程中， 例如，将一个起泡器（1毫米获取急性片使用以下vibratome设置聚四氟乙烯管）在vibratome室）:0.06毫米的振幅和0.08 - 0.10毫米/秒的速度。将推进到最慢的速度（每个通第22号）。修改这些最佳设置，并获得对每一台机器经验，如果有必要的最佳条件。 </li><li>孵育急性切片ACSF中至少60分钟补充有3毫肌醇 ，0.4毫抗坏血酸，和2mM丙酮酸钠，同时用95％ 的 O 2/5％CO 2的气体鼓泡。保持在25℃的温度。 </li><li>在不到15分钟进行切片过程。被转移到记录室，用于在使用前7小时 - 如果需要，该切片可被存储为1。 </li></ol></li><li>先决条件全细胞记录<ol><li>确保该电站配备的记录室中，灌注系统，显微镜，电极（记录，刺激，和地面），宏观和MICRomanipulators，刚性抗振台式和法拉第笼，刺激器，放大器和模拟 - 数字（A / D）转换器，以采集软件的计算机，以及一GFP（或任何其他荧光染料）过滤器，用于分析遗传修饰的神经元18（ 图2A）。 </li><li>制备细胞内溶液，含有115毫葡糖酸钾，2mM的MgCl 2的，10毫米的HEPES，20mM的氯化钾，4毫的 Na 2 ATP和0.3mM的钠3 GTP，由KOH和至290毫渗透摩尔浓度氯化钾调节至pH 7.2。 </li><li>通过拉硼硅玻璃毛细管使补丁吸管。准备使用微量拉马块电极。使用硼硅酸盐毛细管（1.5毫米外径，0.86毫米内径，10厘米长）。使电极贴片显示在充满细胞内液3-10MΩ的电阻。 </li><li>以2毫升/分钟的速率灌注与ACSF记录室中。保持腔室的温度保持在approximately 33℃。 </li></ol></li><li>全细胞记录<ol><li>转移片放入使用巴氏吸管（切断长尖）或小刷子录音室。按住与竖琴的切片。灌注不断地与ACSF切片以2毫升/分钟的速率。 </li><li>修补GFP阳性神经元。 <ol><li>把片到记录室，并通过在低放大倍数（10×）的显微镜找到感兴趣的区域。然后，找到一个GFP阳性细胞使用60倍的目标修补。 </li><li>填与细胞内溶液的记录电极。使用链接到所述过滤器（4毫米过滤器）的注射器和微装载机尖填补与细胞内溶液的记录电极。 </li><li>放置在移液管固定器的玻璃吸管。将枪头在洗澡和重点提示。一旦吸管是在浴，通过回压控制系统施加正压。 </li><li>修补神经元是荧光（ 图URE 2B）。 <ol><li>接近所关注的细胞的视觉指导下，同时保持背在吸管压力。在细胞表面上的小凹坑的外观，释放压力。在这一点上，可以形成一个紧密的密封（电阻大于1GΩ大）。否则，应用光负压（吸气），以促进它。 </li><li>而正在形成的密封，使保持电压钳位至-60 mV的。一旦GΩ密封形成，施加吸力的脉冲破裂细胞膜吸管下方并进入全细胞模式。见参考文献20的更多细节。 </li></ol></li><li>记录使用电流钳的条件21的活性。一旦在全细胞模式，从电压钳切换到电流钳模式，并开始记录。例如，为了记录细胞的兴奋性，适用于500毫秒之久的去极化电流注入（100 - 400帕）。 <ol><li>计算燃烧率bŸ绘制动作电位数一起列车增加输入电流。 注:静息膜电位，输入电阻和膜电容，也可以从记录21计算。 </li></ol></li></ol></li></ol></li></ol>

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作者宣称没有利益冲突。

这个协议在详细描述了如何标记C75BL / 6小鼠的躯体感觉皮层的神经元，以分析它们的连接和它们的兴奋性。相对于现有的方法，它可视连通的识别方面，如每神经元轴突的分支数，它们的精确地形，和它们的解剖位置。通过改变电极的位置，有可能指定其他神经元群体，如扣带皮层（保持电极和大脑之间的角度相同，但改变磁极的取向）或海马5，并且执行类似实验标记单个神经...

树突和轴突结构的发展是电路调节在神经系统中的一个重要方面，包括在大脑皮层。它起着多样化神经元亚群的选择性布线过程中起关键作用。若干最近的报告表明，除了连接性，神经元的分子多样性由获取烧成的高度特异性的方式体现出来。然而，机制确定发育过程中不同的神经元亚型的兴奋性和连接性，以及它们的协调程度，仍然知之甚少1,2。 体内损失-和增益的功能分析允许对特定基因的表达水平和其在电路的发展的影响之间的关系的研究。 在子宫内电（IUE）是广泛用于研究的技术在特定神经元群体的兴趣的基因的功能，并研究它们的连接的总体图案。然而，为了确定在活的小鼠轴突和树突皮质层的形态特征，是必不可少的疏标记神经元。一个的Cre重组系统用IUE组合可用于在足够低的密度来标记的神经元的稀疏人口来解析所识别的皮质椎板的各个细胞发出的突起。此方法标记足够数目的每皮质神经元的电脑（ 图1）的合理数量的分析之后，得到定量数据。这份手稿提出了连通性等精细分析的方法。它也提供了一个类似的策略来分析，在单独的实验中，通过在绿色荧光蛋白（GFP）进行电流钳记录神经元的电性能-electroporated从急性皮层切片的细胞。这些亲母育酚是通用的，可以适用于WT和转基因动物的神经元的兴奋性和连通性的研究中，以及在其损失和功能增益由附加质粒IUE期间引入神经元。 虽然该协议描述了在胚胎天（E）15.5小鼠的电穿孔，该技术可以在任何年龄E9.5 3和日龄（P）的2 4之间执行。虽然电在早期阶段目标的神经元和丘脑的前体和皮质的深层，后期电痕更多的表层（如 E15.5 IUE目标层II-III神经元）。总之，IUE的具有单细胞形态学分析和电的组合是阐明在神经系统的基本神经元的巨大结构和功能的多样性的分子机制的有用工具。

<table><tbody><tr><td>pCAG-Cre</td><td>Addgene</td><td>13775</td><td></td></tr><tr><td>pCALNL-GFP</td><td>Addgene</td><td>13770</td><td></td></tr><tr><td>pCAG-DsRed2</td><td>Addgene</td><td>15777</td><td></td></tr><tr><td>pCAG-GFP</td><td>Addgene</td><td>11150</td><td></td></tr><tr><td>Fast Green</td><td>Carl Roth</td><td>301.1</td><td></td></tr><tr><td>EndoFree Plasmid Maxi Kit</td><td>QIAGEN</td><td>12362</td><td></td></tr><tr><td>Carprofen (Rimadyl)</td><td>Pfizer GmbH</td><td>1615 ESP</td><td></td></tr><tr><td>Isoflurane (IsoFlo)</td><td>Abbott (Esteve)</td><td>1385 ESP</td><td></td></tr><tr><td>Ketamine (Imalgene)</td><td>Merial</td><td>2528-ESP</td><td></td></tr><tr><td>Xylazine (Xilagesic)</td><td>Calier</td><td>0682-ESP</td><td></td></tr><tr><td>Povidone Iodine</td><td>Meda</td><td>694109.6</td><td></td></tr><tr><td>Eye Ointment (Lipolac)</td><td>Angelini</td><td>65.277</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt Solution (HBSS)</td><td>Gibco by Life Technologies</td><td>24020-091</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma -Aldrich</td><td>P4333</td><td></td></tr><tr><td>Scalpel Handle # 3 - 12 cm</td><td>Fine Science Tools</td><td>10003-12</td><td></td></tr><tr><td>Scalpel Blades # 10</td><td>Fine Science Tools</td><td>10010-00</td><td></td></tr><tr><td>Adson Forceps-Serrated - Straight 12 cm</td><td>Fine Science Tools</td><td>1106-12</td><td></td></tr><tr><td>Hardened Fine Scissors - Straight 11 cm</td><td>Fine Science Tools</td><td>14090-11</td><td></td></tr><tr><td>Scissors Mezenbaum-Nelson Curved L=14.5 cm</td><td>Teleflex</td><td>PO143281</td><td></td></tr><tr><td>Thin curved tips - Style 7 Dumoxel</td><td>Dumont</td><td>0303-7-PO</td><td></td></tr><tr><td>Dumont #5 Forceps-Inox</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>Mathieu Needle Holder - Serrated</td><td>Fine Science Tools</td><td>12010-14</td><td></td></tr><tr><td>AutoClip Applier</td><td>Braintree scientific, Inc</td><td>ACS&amp;nbsp;APL</td><td></td></tr><tr><td>9 mm AutoClips</td><td>MikRon Precision, Inc.</td><td>205016</td><td></td></tr><tr><td>Sutures - Polysorb 6-0</td><td>Covidien</td><td>UL-101</td><td></td></tr><tr><td>Electric Razor&amp;nbsp;</td><td>Panasonic</td><td>ER 240</td><td></td></tr><tr><td>Borosilicate glass capillaries (100 mm, 1.0/0.58 Outer/Inner diameter)</td><td>World Precision Instrument Inc.</td><td>1B100F-4</td><td></td></tr><tr><td>Aspirator tube assemblies for calibrated microcapillary pipettes</td><td>Sigma -Aldrich</td><td>A5177-5EA</td><td></td></tr><tr><td>Gauze (Aposan)</td><td>Laboratorios Indas, S.A.U.</td><td>C.N. 482232.8</td><td></td></tr><tr><td>Cotton Swabs (Star Cott)</td><td>Albasa</td><td>-</td><td></td></tr><tr><td>Needle 25 G (BD Microlance 3)</td><td>Becton, Dickinson and Company</td><td>300600</td><td></td></tr><tr><td>Sucrose</td><td>Sigma -Aldrich</td><td>S0389</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma -Aldrich</td><td>158127</td><td></td></tr><tr><td>OCT Compound</td><td>Sakura</td><td>4583</td><td></td></tr><tr><td>Tissue Culture Dish 100 x 20 mm</td><td>Falcon</td><td>353003</td><td></td></tr><tr><td>GFP Tag Polyclonal Antibody</td><td>Thermo Fisher Scientific</td><td>A-11122</td><td></td></tr><tr><td>Secondary Antibody, Alexa Fluor 488 conjugate</td><td>Thermo Fisher Scientific</td><td>A-11008</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542&amp;nbsp;</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Fisher Scientific</td><td>10270106&amp;nbsp;</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100-500ML</td><td></td></tr><tr><td>Electroporator ECM 830&amp;nbsp;</td><td>BTX Harvard Apparatus</td><td>45-0002</td><td></td></tr><tr><td>Platinum electrodes 650P 7 mm</td><td>Nepagene</td><td>CUY650P7</td><td></td></tr><tr><td>Microscope&amp;nbsp;for Fluorescent Imaging - MZ10F</td><td>Leica</td><td>-</td><td></td></tr><tr><td>VIP 3000 Isofluorane Vaporizer</td><td>Matrx</td><td>-</td><td></td></tr><tr><td>TCS-SP5 Laser Scanning System</td><td>Leica</td><td>-</td><td></td></tr><tr><td>Axiovert 200 Microscope</td><td>Zeiss</td><td>-</td><td></td></tr><tr><td>Cryostat - CM 1950</td><td>Leica</td><td>-</td><td></td></tr><tr><td>P-97 Micropette Puller</td><td>Sutter Instrument Company</td><td>P-97</td><td></td></tr><tr><td>Patch clamp analysis softwarw (p-Clamp Clampfit 10.3)</td><td>Molecular Devices</td><td>-</td><td></td></tr><tr><td>Acquisition software (MultiClamp 700B Amplifier)</td><td>Molecular Devices</td><td>DD1440A</td><td></td></tr><tr><td>Motorized Micromanipulator + Rotating Base&amp;nbsp;</td><td>Sutter Instrument</td><td>MP-225</td><td></td></tr><tr><td>Air Table</td><td>Newport</td><td>-</td><td></td></tr><tr><td>Miniature Peristaltic Pumps</td><td>WPI</td><td>-</td><td></td></tr></tbody></table>

in utero electroporation approaches to study the excitability of neuronal subpopulations and single-cell connectivity

神经系统是由不同的神经元类型的一个巨大的范围。这些神经元亚群的特征在于，其它的特征，其独特的树突形态，其轴突连通的特定的模式，和它们的选择性点火的反应中。负责开发期间分化的这些方面的分子和细胞机制仍然知之甚少。 在这里，我们描述了联合协议标签和表征皮层神经元的结构连接性和兴奋性。在子宫内电（IUE）协议的修改使得神经元人口稀少的标签。这，反过来，使树突和单个神经元，轴突突起的层位置的精确表征，和形态分析的轴突的识别和跟踪。 IUE也可以用来研究在的兴奋性变化野生型（WT），或通过将其与全细胞记录从电脑的急性切片结合遗传修饰的神经元。这两种技术有助于更好地理解的结构和功能连接的耦合和分子机制控制发育过程中的神经元的多样性。这些发育过程对轴突布线，神经元的功能的多样性，以及认知障碍的生物学重要的影响。

为了表征在细节和整个开发神经元的形态变化，有必要稀疏标记神经元。一个Cre重组稀释系统允许感兴趣的基因在神经元的人口稀少的表达，因此，只有那些掺入这种酶的神经元表达GFP（ 图1A）。使用这种策略，层II-III是针对在E15.5通过IUE标记。 CAG-的DsRed2在1微克/μL，是共电为控制和动物活体识别正面电大脑。重要的是，抗GFP抗体染色后，信号足够强，让他?...

Watch this Scientific Journal Video about In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity at JoVE.com

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

这份手稿规定， 在子宫内电（IUE）用来描述在单细胞水平与荧光标记的神经元兴奋性神经元的结构连接协议。组织学是用于表征树突和轴突突起。在急性切片全细胞记录被用来研究兴奋性。

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, ...

in-utero-electroporation-approaches-to-study-excitability-neuronal

Research

JoVE Journal

Neuroscience

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

9.6K 次观看.  Centro Nacional de Biotecnología (CNB-CSIC). 这份手稿规定， 在子宫内电（IUE）用来描述在单细胞水平与荧光标记的神经元兴奋性神经元的结构连接协议。组织学是用于表征树突和轴突突起。在急性切片全细胞记录被用来研究兴奋性。 

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

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 ...

科研

神经科学

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 ...

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 ...

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and associated extracellular matrix. Because they provide a suitable organotypic environment, cortical slice explants are often used to investigate those interactions that control neuronal differentiation and development. Although beneficial, the slice explant model can suffer from drawbacks including aberrant cellular lamination and migration. Here we report a whole cerebral hemisphere explant system for studies of early cortical development that is easier to prepare than cortical slices and shows consistent organotypic migration and lamination. In this model system, early lamination and migration patterns proceed normally for a period of two days in vitro, including the period of preplate splitting, during which prospective cortical layer six forms. We then developed an ex utero electroporation (EUEP) approach that achieves ~80% success in targeting GFP expression to neurons developing in the dorsal medial cortex.
The whole hemisphere explant model makes early cortical development accessible for electroporation, pharmacological intervention and live imaging approaches. This method avoids the survival surgery required of in utero electroporation (IUEP) approaches while improving both transfection and areal targeting consistency. This method will facilitate experimental studies of neuronal proliferation, migration and differentiation.

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.

前子宫穿孔和整个半球的外植体早期皮质发育的研究：一个简单的实验方法

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and ...

Neonatal Pial Surface Electroporation

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and gliogenesis, including after injury. However, methods for genetically interrogating these progenitor populations and tracking their lineages had been limited owing to a lack of specificity or time consuming production of viruses. Thus, progress in this region has been relatively slow with only a handful of investigations of this location. Electroporation has been used for over a decade to study neural stem cell properties in the embryo, and more recently in the postnatal brain. Here we describe an efficient, rapid, and simple technique for the genetic manipulation of pial surface progenitors based on an adapted electroporation approach. Pial surface electroporation allows for facile genetic labeling and manipulation of these progenitors, thus representing a time-saving and economical approach for studying these cells.

The pial surface is a unique progenitor zone in the CNS that is receiving increasing attention. Herein, we detail a method for rapid genetic manipulation of this progenitor zone using a modified electroporation method. This procedure can be used for cellular and molecular investigations of cell lineages and signaling pathways involved in cell differentiation and to elucidate the fate and properties of daughter cells.

新生儿软脑膜表面电穿孔

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

皮质的interneurons的亚型选择性电穿孔

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

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 ...

Double In Utero Electroporation to Target Temporally and Spatially Separated Cell Populations

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian corticogenesis. This procedure takes advantage of the brain ventricles to allow the introduction of DNA of interest and uses a pair of electrodes to direct the entrance of the genetic material into the cells lining the ventricle, the neural stem cells. This method allows researchers to label the desired cells and/or manipulate the expression of genes of interest in those cells. It has multiple applications, including assays targeting neuronal migration, lineage tracing, and axonal pathfinding. An important feature of this method is its temporal and regional control, allowing circumvention of potential problems related with embryonic lethality or the lack of specific CRE driver mice. Another relevant aspect of this technique is that it helps to considerably reduce the economic and temporal limitations that involve the generation of new mouse lines, which become particularly important in the study of interactions between cell types that originate in distant areas of the brain at different developmental ages. Here we describe a double electroporation strategy that enables targeting of cell populations that are spatially and temporally separated. With this approach we can label different subtypes of cells in different locations with selected fluorescent proteins to visualize them, and/or we can manipulate genes of interest expressed by these different cells at the appropriate times. This strategy enhances the potential of in utero electroporation and provides a powerful tool to study the behavior of temporally and spatially separated cell populations that migrate to establish close contacts, as well as long-range interactions through axonal projections, reducing temporal and economic costs.

Double in utero electroporation allows targeting cell populations that are spatially and temporally separated. This technique is useful to visualize interactions between those cell populations using fluorescent proteins in normal conditions but also after functional experiments to perturb genes of interest.

双电穿孔，以定时和空间分离的细胞群

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian ...

In Vivo Targeting of Neural Progenitor Cells in Ferret Neocortex by In Utero Electroporation

Manipulation of gene expression in vivo during embryonic development is the method of choice when analyzing the role of individual genes during mammalian development. In utero electroporation is a key technique for the manipulation of gene expression in the embryonic mammalian brain in vivo. A protocol for in utero electroporation of the embryonic neocortex of ferrets, a small carnivore, is presented here. The ferret is increasingly being used as a model for neocortex development, because its neocortex exhibits a series of anatomical, histological, cellular, and molecular features that are also present in human and nonhuman primates, but absent in rodent models, such as mouse or rat. In utero electroporation was performed at embryonic day (E) 33, a midneurogenesis stage in ferret. In utero electroporation targets neural progenitor cells lining the lateral ventricles of the brain. During neurogenesis, these progenitor cells give rise to all other neural cell types. This work shows representative results and analyses at E37, postnatal day (P) 1, and P16, corresponding to 4, 9, and 24 days after in utero electroporation, respectively. At earlier stages, the progeny of targeted cells consists mainly of various neural progenitor subtypes, whereas at later stages most labeled cells are postmitotic neurons. Thus, in utero electroporation enables the study of the effect of genetic manipulation on the cellular and molecular features of various types of neural cells. Through its effect on various cell populations, in utero electroporation can also be used for the manipulation of histological and anatomical features of the ferret neocortex. Importantly, all these effects are acute and are performed with a spatiotemporal specificity determined by the user.

Presented here is a protocol to perform genetic manipulation in the embryonic ferret brain using in utero electroporation. This method allows for targeting of neural progenitor cells in the neocortex in vivo.

通过子宫电位对脑神经祖细胞进行神经祖细胞的振动定位

Manipulation of gene expression in vivo during embryonic development is the method of choice when analyzing the role of individual genes during mammalian ...

Murine In Utero Electroporation

In utero electroporation is an important technique for studying the molecular mechanisms that guide the proliferation, differentiation, migration, and maturation of cells during neural development. Electroporation enables the rapid and targeted delivery of material into cells by utilizing electrical pulses to create transient pores in cell membranes. Although electroporation has traditionally been used in in vitro studies, scientific advancements have now broadened its utilization to intact organs, such as those found in mouse embryos developing in utero. This video will introduce the key principles behind in utero electroporation in addition to reviewing the basic surgical techniques required to access developing embryos within a pregnant rodent. Details of the injection and electroporation steps are provided along with important considerations for directing gene delivery to specific brain regions. Finally, neurobiological applications of in utero electroporation are presented, such as investigating how specific genes contribute to neural development and how connections form between developing neurons.

Murine In Utero Electroporation

小鼠子宫内电穿孔

In utero electroporation is an important technique for studying the molecular mechanisms that guide the proliferation, differentiation, migration, and ...