笔者想感谢他们的帮助凯瑟琳·马瑟斯博士，博士让-菲利普·Mocho和约兰达·萨维德拉托雷斯博士在宫内电执行无菌程序下，海莉木对她的帮助下准备的图纸。 支持的EP是由长期的欧洲生化学会联合会（FEBS）奖学金和医学研究理事会（MRC）的职业发展奖学金，MAH的威康信托基金赠款到伊丽莎白费舍尔和维克多Tybulewicz（080174/B/06/Z） ，硬件由欧洲分子生物学长短期奖学金和RA MRC的助学金。这项工作得到了从威康信托基金会（086947/Z/08/Z）项目赠款，由格兰特在急救医学研究理事会（U117570528）福源

在英国，老鼠被安置，繁殖，根据民政厅批准的指引下，动物（科学规程）行动1986年和治疗。 1。制备方法:DNA溶液和针头<ol><li>马克西准备套件内毒素纯化质粒DNA。准备质粒DNA溶液注射到所需的浓度在水中，加快绿（终浓度为0.05％），可视化注射。电效率是高度依赖的DNA浓度。一般使用的浓度为1μg/μL。这个浓度是足够的可视化，而不会影响其发展的电穿孔神经元。然而，根据质粒载体使用的启动以及大小和（低表达水平与巨细胞病毒启动子/增强，较强的表达水平与巨细胞病毒立即早期增强子和鸡β-肌动蛋白启动子融合（CAG）启动）<html>表达蛋白的稳定性，这个浓度可以调整（0.25微克/微升至5微克/微升）。 </li><li>拉使用微量拔的玻璃针。 </li></ol> 2。手术的制备<ol><li>高压灭菌的手术器械和磷酸盐缓冲液（PBS）。 </li><li>准备在PBS（丁丙诺啡，Vetergesic的，终浓度为30微克/毫升）的镇痛解决方案。称重孕鼠注射手术前至少30分钟皮下注射0.1毫克/千克Vetergesic。在这段时间内，准备手术区。打开电热垫和恢复室。温水澡和无菌单上所有无菌文书和材料放置在无菌PBS。一个PBS填充烧杯放入铂电极和连接的electroporator。使用microloader尖端的DNA解决方案填补针，针连接毛细管人，和一个镊子掐针尖。 </li></ol>e_title"&gt; 3。手术前DNA注射和电<ol><li>怀孕鼠标（E14.5-E15.5）麻醉与异氟醚氧载体中使用的麻醉诱导室（氧气2升/分钟）。等到失去翻正反射的动物。 </li><li>动物转移到了"手术前"面具。每只眼睛上，以防止眼睛的角膜溃疡，而她的母亲是在全身麻醉下放置一个眼胶下降。使用电动剃须刀剃腹部的头发。清洁光头面积一次与clorhexidine收集飞行的头发。 </li><li>转移动物在手术区的第二个面具。鼠标将其加热垫的背面。开始时，踏板的反射已经失去手术。 </li><li>戴上口罩和无菌手套。覆盖无菌悬垂（用在腹部的小孔），以防止被污染的皮肤没有被剃光和消毒领域的组织和文书的动物。清洁光头区ţ至少与clorhexidine 3倍。每次使用不同的无菌棉签。使用手术刀，通过皮肤垂直切口，沿中线（1英寸长）。用剪刀沿白线（白线主要组成胶原蛋白的结缔组织），使类似的腹部肌肉的切口。 </li><li>选择最方便的胚胎，并放置两个胚胎之间的环钳小心地拉出腹腔胚胎链。从这点上，用无菌预温PBS水合保持胚胎。 </li></ol>没有显微镜的可视化要求。 4。注射DNA和电<ol><li>最外侧​​的胚胎开始，使其更容易追踪，其中胚胎进行电穿孔。不要拉太多的胚胎，因为这会增加出血的危险。内的羊膜囊使用环钳和操纵胚胎的位置稳定环之间胚胎头。轻轻挤推胚胎在子宫壁。 </li><li>另一方面，采取毛细管持有人，并针对侧脑室半球的中间插入针仔细。按踏板注入约1μLDNA溶液混合，固绿（小于1μL研究树突）。你应该遵守的绿色染料，填补了侧脑室。关键步骤: - 锐利的针头刺破的关键正确的子宫壁。重要的是要尽量减少在子宫壁表面，入脑室后插入针的运动，因为孔的扩大将导致泄漏羊水和胚胎死亡。避免在子宫壁刺入血管，因为这会导致出血和胚胎死亡。 - 重要的是不要过大音量的DNA注入侧脑室，因为它会诱发脑积水（不超过2μL在E14.5）。根据实验的目的，调整的DNA量。如果1μL一般用于大多数实验使用，体积小的DNA（约0.5μL）枝和脊椎分析的需要。事实上，一些孤立的细胞都需要进行有针对性的，以可视化和测量电脉冲神经元的树突状乔木。 </li><li>将胚胎头部两侧电极的正极（+）注射皮质电脑室或海马电注入脑室（ 图2）对面的同一侧的桨。然后申请在1秒的时间间隔电脉冲30V（持续时间为50毫秒）。关键的一步:避免应用电流通过胎盘，因为这将导致胚胎死亡。 在孕鼠的胚胎可以被电通常具有相同的DNA，构建以避免任何混淆。然而，长期以来手术减少<html> S的胚胎成活率。腹腔不应超过30分钟打开不再。 </li></ol> 5。手术后的电<ol><li>电穿孔的胚胎后，加入腹腔PBS及使用环钳，子宫角，以取代原来的位置。 Vicryl可吸收缝合线缝合腹壁及皮肤。 </li><li>将在恢复室中的动物，直到它唤醒（通常5-10分钟），然后转移在笼子放置在加热垫。 </li></ol> 6。手术后检查小鼠的行为，以评估疼痛，​​痛苦或困扰，体重在手术后24小时和48小时的动物。如果需要，止痛药，可管理，以减少疼痛和不适。 7。组织处理实验所需的胚胎或产后阶段收集的电穿孔胚胎或幼崽。 ove_content" - 在萌芽阶段（例如，研究细胞增殖或迁移）分析: 安乐死的母亲通过颈椎脱位，收集胚胎。斩首后，选择已妥善电穿孔的大脑，通过荧光信号的数量和位置，可视化整个使用荧光双眼的头骨。解剖的头骨和大脑修复一夜之间在4％的煤灰，然后在20％蔗糖/ PBS过夜的地方。在-80°C和部分使用恒温器嵌入华侨城化合物，冻结。 - 在产后阶段（例如，研究树突和刺）分析: 麻醉幼仔或成年小鼠腹腔注射戊巴比妥（40-60毫克/千克）和执行transcardial灌注PBS，4％，在PBS PFA。大脑解剖的头骨和修复后，在4％的煤灰隔夜。洗涤在PBS节的大脑后，使用vibratome（100微米的部分为Dendrite分析）。安装在Aqua聚/安装的部分，用0.16-0.19毫米厚的盖玻片形象树突和刺。 8。代表结果 图3显示了电穿孔细胞在CA1区（ 图3C，D）的齿状回海马（ 图3E，F）的，在大脑皮质（ 图3A，B）的例子。野生型小鼠在E14.5与GFP的构造（PCA-B-EGFPm5消音器3），并在产后天（P）14收获的大脑电穿孔。注射1 DNA溶液的小体积（0.5μL或解决方案少1微克/微升），少数细胞被标记，允许在孤立的绿色荧光蛋白+细胞（ 图3）以及他们的棘树突状树枝状的可视化在更高的放大倍率（ 图4）。 <img alt="图1" src="/files/ftp_upload/4163/4163fig1.jpg" /> &lt;强&gt;图1。电协议，可以用来研究树突和棘示意图。 （一）建设GFP的电穿孔，比较野生型和突变小鼠的树突棘。 （二）绿色荧光蛋白基因的shRNA（GFP是从相同的结构表示）电穿孔比较电穿孔用的shRNA的小鼠树突和棘建设的具体利益的基因（X）或控制的shRNA。 （三）IUE位置可以用Cre重组酶诱导系统一起，以限制的shRNA表达可取一段时间。在这个实验中，表达载体形式的Cre重组酶可以激活4 -羟基（CAG-ER T2 CreER T2的 ; 1微克/微升）10，被用在Cre重组酶依赖特异性shRNA表达载体电穿孔方式（1微克/微升，与重组指标（CALNL-GFP结构，绿色荧光蛋白表达诱导的Cre; 1微克/微升）10效率CY的击倒shRNA的浓度增加，以及增加造影-ER T2 CreER T2的浓度可以提高。 <img alt="图2" src="/files/ftp_upload/4163/4163fig2.jpg" /> 图2。电空间的控制。这个数字显示定位桨根据DNA注射部位的电极，以针对大脑皮层或海马。 <img alt="图3" src="/files/ftp_upload/4163/4163fig3.jpg" /> 图3。 在子宫内的树突状乔木可视电穿孔细胞在大脑皮层和海马。 （甲，乙）显示绿色荧光蛋白+在大脑皮质锥体细胞，海马CA1锥体细胞（光盘版）和（，E，F），在齿状回颗粒细胞在P14的日冕部分。 GFP的构造（PCA-B-EGFPm5消音器3）在E14.5电穿孔。高放大倍率的照片（B，D F）的显示IUE位置，是一种有效的方法来可视化的树突。比例尺（B，D和F）的代表50微米，150微米（A，C）。 <img alt="图4" src="/files/ftp_upload/4163/4163fig4.jpg" /> 图4。可视化表达构建绿色荧光蛋白在E14.5 在子宫内 ，进行电穿孔的P14的神经元的树突棘。 （甲，乙）从基础的海马锥体神经元树突棘的高倍率图像。标尺代表5微米（A）和2微米（乙）。

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没有利益冲突的声明。

IUE位置是一个功能强大的工具来操纵基因的表达，不仅在空间上，而且在时间。在这里，我们表明，这种技术可以用于可视化和基因操纵树突棘在小鼠大脑皮层和海马。除了前面提到的优点，​​值得一提的是，IUE位置，在高尔基方法的对比，可以用免疫组化或原位杂交，例如电穿孔细胞型允许结合。同样重要的是提到，此过程不会引起明显脑畸形，尽管它相对的侵袭。此外，在细胞水平上，IUE位置不修改13神经元的电穿孔的电生理特性。虽然我们的枝和脊椎形态的可视化示范重点，在E14.5皮质或海马神经元IUE位置也用于研究其他发展活动，如轴突的形成和指导。在添加ition，同一种协议，可以实现在胚胎发育的其他阶段，针对不同的人群。例如，在E18.5发育很晚皮质电范式可以驾驶1星形细胞祖细胞中的表达。同样，而在E14.5海马电允许同时针对海马CA1-CA3区锥体神经元祖细胞和齿状回颗粒细胞祖，后期海马电（E18.5或产后早期）将允许针对不同的齿状回祖14。在这种情况下，DNA的注射量可以增加以及电流强度。 IUE位置由引进的转基因似乎仍然附加体式，并因此失去了连续的细胞分裂后的细胞。然而，在分裂后的细胞，如神经元，附加体式转基因保持活跃后电击使长期研究13个月15。在我们的研究中，我们已经观察到明亮的绿色荧光蛋白+细胞7周后出生（我们分析了最新的时间点），表明胚胎针对使用IUE位置从早期发育时间点的结果，在转基因持续表达的皮质或海马神经元前体到成年。 目前的技术限制，这是很难施加罚款的电穿孔细胞总数控制。然而，我们的DNA溶液注入量减少，表明它是可能标注少数细胞和树突状树枝状可视化的孤立的GFP +细胞以及他们的刺。转染面积的尺寸，也可以通过修改如电流和脉冲数或电桨直径强度的电参数调整。 共有IUE位置是很容易实现，快速的方法，高效和体内研究树突棘。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td></td><td></td><td></td><td colspan="4"> 注射用的针头和DNA溶液的制备 </td></tr><tr><td> endofree质粒马克西套件</td><td> Qiagen公司</td><td> 12362 </td><td></td></tr><tr><td>快绿</td><td>西格玛</td><td>架F-7258 </td><td></td></tr><tr><td>硼硅玻璃毛细管 1.0毫米外径×0.58 mm内径</td><td>哈佛仪器</td><td> 30-0016 </td><td></td></tr><tr><td> microloader秘诀</td><td> Eppendorf公司</td><td> 5242956003 </td><td>对于离心吸液管0.5μL，10μL/ 2-20微升</td></tr><tr><td></td><td></td><td> ＆NBSP; </td><td> 手术材料 </td></tr><tr><td>超薄虹膜剪刀</td><td>罚款科学工具</td><td> 14088-10 </td><td></td></tr><tr><td>弯钳</td><td>罚款科学工具</td><td> 91197-00 </td><td></td></tr><tr><td>环钳</td><td>罚款科学工具</td><td> 11103-09 </td><td></td></tr><tr><td>持针器</td><td>罚款科学工具</td><td> 12002-12 </td><td></td></tr><tr><td>格雷夫钳</td><td>罚款科学工具</td><td> 11050-10 </td><td></td></tr><tr><td> vicryl吸收缝合</td><td>爱惜康公司（强生） </td><td> W9074 </td><td></td></tr><tr><td> 30厘米x45厘米无菌单</td><td>巴斯特</td><td> 141765 </td><td></td></tr><tr><td>无菌棉签</td><td> shermond </td><td> HUBY-340 </TD&gt; <td></td></tr><tr><td>棉花棒</td><td>清洁跨有限公司</td><td> 1860 </td><td></td></tr><tr><td>丁丙诺啡（Vetergesic） </td><td> alstoe动物健康</td><td></td><td></td></tr><tr><td> clorhexidine </td><td> vetasept </td><td> XHG007 </td><td></td></tr><tr><td>眼凝胶，涉及的</td><td>诺华公司</td><td></td><td></td></tr><tr><td>异氟醚</td><td>雅培公司</td><td> B506 </td><td></td></tr><tr><td> pentoject，戊巴比妥钠20％ </td><td> animalcare </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td colspan="4"> 电 </td></tr><tr><td> electroporator </td><td> A型肉毒毒素</td><td> ECM830 </td><td></td></tr><tr><td>白金TweezertrodË5毫米</td><td> A型肉毒毒素，哈佛仪器</td><td> 45-0489 </td><td></td></tr><tr><td> femtojet微量注射器</td><td> Eppendorf公司</td><td> 5247000030 </td><td></td></tr><tr><td>脚控制Femtojet微量注射器</td><td> Eppendorf公司</td><td> 5247623002 </td><td></td></tr><tr><td>毛细管持有人</td><td> Eppendorf公司</td><td> 5176190002 </td><td></td></tr><tr><td></td><td></td><td></td><td> 组织处理 </td></tr><tr><td>多聚甲醛</td><td>西格玛</td><td> P6148 </td><td></td></tr><tr><td>蔗糖</td><td> VWR（Prolabo） </td><td> 27480.294 </td><td></td></tr><tr><td>显微镜载玻片</td><td> ThermoScientific（门泽尔，格拉泽） </td><td> J1800AMN​​Z </td><td></td></tr><tr><td>盖玻片</td><td>门泽尔，格拉泽</td><td></td><td> 22×50毫米＃1,5 </td></tr><tr><td>水族聚/安装</td><td> polysciences，公司</td><td> 18606 </td><td></td></tr></tbody></table>

visualization and genetic manipulation of dendrites and spines in the mouse cerebral cortex and hippocampus using in utero electroporation

（IUE）为何在子宫内电已成为一个强大的技术来研究胚胎神经系统1-5不同地区的发展。迄今为止，这个工具已经被广泛用于研究调控细胞增殖，分化和神经细胞迁移，尤其是在发展中国家大脑皮层6-8。这里我们详细协议electroporate在子宫内的大脑皮质和海马和提供的证据表明，这种方法可以用来研究这两个脑区的树突和棘。 在原代培养神经元的可视化和操纵，有助于更好地理解到参与在树突棘和突触发展的进程。然而神经细胞在体外生长，没有接触到所有的生理线索，期间可能会影响正常发育枝和/或脊柱的形成和维护。我们的知识枝和脊柱结构<e米在野生型或突变小鼠体内&gt;，大多是从观测使用的高尔基考克斯方法9。然而，高尔基染色被认为是不可预测的。事实上，神经细胞和神经纤维束组随机标记，与特定领域经常出现的完全染色，而相邻的地区是没有染色。最近的研究表明，荧光结构IUE位置代表一个有吸引力的替代方法研究10-11（ 图1A）突变型/野生型小鼠树突棘，以及作为突触。此外，相比一代鼠标击倒，IUE位置代表一个快速的方法进行细胞的特定人群在特定时间窗口增益和丧失功能的研究。此外，IUE位置已被成功地用于诱导基因表达或诱导的RNAi方法提炼的时间控制在表达一个基因或shRNA 12。 ，IUE这些优势，从而opened个新的尺寸，研究基因表达/抑制不仅在特定的脑结构（ 图1B），但还处于发展的特定时间点（ 图1C）对树突棘的影响。 最后，IUE位置提供了一个有用的工具，以确定枝，脊椎和/或突触发育有关的基因之间的功能互动。事实上，在其他如病毒基因转移方法相比，它是直接结合多个RNA干扰或转基因细胞的相同人口。 总之，IUE位置是一个功能强大的方法，已经促成表征的脑功能和疾病背后的分子机制，也应该在树突棘的研究有用。

Watch this Scientific Journal Video about Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation at JoVE.com

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

本文详细介绍了协议，在宫内electroporate大脑皮层和海马小鼠E14.5。我们还表明，这是一个有价值的方法来研究在这两个脑区的树突和刺。

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 ...

visualization-genetic-manipulation-dendrites-spines-mouse-cerebral

Research

JoVE Journal

Neuroscience

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

17.9K 次观看.  MRC National Institute for Medical Research. 本文详细介绍了协议，在宫内electroporate大脑皮层和海马小鼠E14.5。我们还表明，这是一个有价值的方法来研究在这两个脑区的树突和刺。

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

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

嗅觉感官神经元的慢病毒介导的遗传操作和可视化在体内</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

科研

神经科学

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 ...

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 ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

可视化本体感受器<em&gt;果蝇</em&gt;幼虫和蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

电压敏感染料记录单个神经元在大脑切片的轴突，树突和树突棘

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

同时脑电图，实时测量的啮齿动物的大脑皮质中的神经元活动的乳酸浓度与Optogenetic的操作

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

遗传操纵鼠标的发展下丘脑通过<em&gt;在子宫内</em&gt;电穿孔

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

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

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

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

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

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

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

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

双光子<em&gt;在体内</em&gt;树突棘中的鼠标皮质用稀疏的头颅成像准备

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘

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此视频中的章节

嗅觉感官神经元的慢病毒介导的遗传操作和可视化在体内

在子宫内电穿孔:鼠标控制时空基因Transefection

神经元形态的研究方法:

可视化本体感受器

电压敏感染料记录单个神经元在大脑切片的轴突，树突和树突棘

同时脑电图，实时测量的啮齿动物的大脑皮质中的神经元活动的乳酸浓度与Optogenetic的操作

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

遗传操纵鼠标的发展下丘脑通过

小脑颗粒神经元的遗传操作

双光子