我们感谢提供的pSil-GFP的结构图1的插图，博士阿尔珀乌尊，共聚焦显微镜勒杜克生物成像设备，博士希林Bonni。电解金属锰支持从巴勒斯惠康基金，NARSAD奖的年轻研究者，美国国立卫生研究院NCRR COBRE P20的RR018728-01职业医学科学奖。借券支持由美国国立卫生研究院NCRR COBRE P20的RR018728-01，并已收到小灵通NRSA 5T32MH019118-20的支持。

1。准备培养液和媒体（不是视频） <ol><li>准备完整汉克平衡盐溶液（HBSS中）含有1X HBSS中，2.5毫米HEPES液（pH7.4），30毫米D-葡萄糖，1毫米氯化钙 ，硫酸镁4 1毫米和4毫米碳酸氢钠3 1升。加入双蒸水（DDH 2 O）的。过滤消毒用0.2微米的过滤器，并储存于4°C。 </li><li>准备片用35毫升基础培养基鹰媒体的培养基，完整的HBSS 12.9毫升，20毫米D-葡萄糖（1.35毫升的1 M的解决方案），1毫米Glutamax 200毫米的解决方案（0.25毫升），0.5 ml青霉素链霉素100X股票的。 0.2微米的过滤器过滤消毒，然后加入热灭活马血清终浓度为5％。 </li><li>准备1毫克/毫升原液用无菌蒸馏水去离子水层粘连蛋白，层粘连蛋白工作的解决方案。准备在0.5毫升离心管加入100μL分装和冻结在-80°C。 </li><li>所以准备聚-L-赖氨酸的工作lution通过加入聚-L-赖氨酸5毫克，1毫克/毫升原液5毫升无菌H 2 O。准备1毫升分装和冻结，在-20°C </li><li>准备到终体积12毫升无菌水稀释1毫升聚-L-赖氨酸和100μL粘连涂层解决方案。每次作出新的这一解决方案。 </li></ol> 2。准备器官切片插入（没有视频） <ol><li>准备一个文化的插入，每孔用无菌镊子两个六孔板。加入2毫升无菌DDH 2 O的下方的文化插入。 </li><li>加入1毫升的照顾，不穿刺膜的膜之上的涂层解决方案。在饱和湿度培养箱隔夜在37°C和5％的CO 2。 </li><li>除去包衣介质，并用无菌H 2 O三次膜。让我们在使用前插入干。加入1.8毫升片培养基，在37°C间孵化器的地方。用封口膜包住未使用的板块在4°C存储长达4周。 </li></ol> 3。电穿孔的准备（没有视频） <ol><li>准备电击的RNAi构造。双链RNA发夹插入克隆到pSilencer载体。质粒含有：1）1 U6启动子驱动的双链RNA代和2）的GFP表达盒由CMV启动子8,9驱动。这种质粒以前一直由小西和他的同事和其他7,8描述。质粒纯化使用Qiagen公司的最大准备套件，并在1毫克/毫升的浓度使用。 </li><li>以可视化的DNA，而注射，准备了0.5％的快速绿色染料溶液和使用它的DNA被注入（通常为20μL的DNA 1μL快速绿色）在1:20。超过离开的DNA快速绿色染料混合物可以储存在-20°C到一个星期。 </li><li>清洁的清扫面积，dissecti吴用70％乙醇的工具和vibratome船只。冷藏完整的HBSS，以便它是冰冷的。的寒意vibratome船只围绕它包装内快速冷却的vibratome一些水冰。使用完整的HBSS，准备3％低熔点琼脂糖。微波1分钟。避免了沸点。保持在42℃水浴，直至使用。 </li><li>电参数设置如下。 1 E15胚胎使用35伏特，5个脉冲，长100毫秒，900毫秒脉冲之间的间隔。对于年龄较大的动物，以确保电，使用更高的电压高达50 V或增加脉冲数高达8个脉冲。为了防止损坏，在年轻的动物组织，使用更少的脉冲或2脉冲和低电压高达25 V。这些参数可能是多种多样的，凭经验确定取决于动物的年龄。 </li></ol> 4。解剖和电（视频） <ol><li>安乐死女性怀孕后，解剖胚胎放进冰水里完成的HBSS。柯EP在各自的胎盘囊胚胎。 </li><li>解剖胚胎，并切断后的第一颈椎头。保持在冰冷的完整的HBSS。 </li><li>注射，头放在一块培养皿上的封口膜。使用自定义Hamilton注射器（ 见表一 ）注入约6至8μL的DNA：通过第三脑室快速绿色染料混合，以填补皮质囊泡两侧脑室。另外，注射可以直接进入每一个侧脑室。 </li><li>为体外电击，使用BTX-镊子铂电极。背皮质即头顶部electroporate的皮质，你想向一侧放置正极。 </li><li>电击后，静置至少5分钟前解剖上冰元首。 </li><li>解剖大脑在使冰冷HBSSby的头部一侧的一个小切口，剥离头的两侧皮肤的折扣。下一步，细镊子轻轻剥离从脑软。从头骨完整的大脑取出，注意不要损坏皮质。 </li></ol> 5。嵌入和电穿孔皮质切片（视频） <ol><li> 3％低熔点琼脂糖转移到冰上放置了一个大型模具。模具的底部将开始固化速度更快，这将防止下沉模具底部的大脑。轻轻地，转移与细镊子取出后多余的缓冲，与Kimwipe或滤纸的大脑。使用10微升吸管尖漩涡的大脑内的模具，以确保琼脂糖和脑组织之间的最大接口。 </li><li>东方的大脑，以确保所有的大脑都在同一方向，并在同一水平在琼脂糖。让琼脂糖巩固约5分钟。使用粘接胶（疯狂胶），使嗅球站在重视琼脂糖块。一旦块被重视，立即加我每个大脑获得CE冷的HBSS和琼脂糖修剪，以确保个人片。 </li><li>切块，低速（约一半的最大值）设置的vibratome的速度和叶片的振动频率设置在最高设置。产生250微米厚的冠状切片。检索切片，用弯曲的细铲，并用细刷或镊子转移他们组织井。 </li><li>在组织文化的引擎盖，转移到片涂层刀片。加入500μL片培养基每个插入容易转移。每个刀片可放置多达5片。删除多余的介质，从切片的顶部，并在37°C在加湿的孵化器孵化。 </li></ol> 6。文化和器官切片分析（视频） <ol><li>为了保持健康的切片，新鲜媒体应增加至少隔日膜取代一半的媒体每次下方。 </li><li>为了分析切片后的d文化esired天，固定在膜切片。 1X的磷酸盐缓冲液（PBS）的37°C间三次，每次10分钟洗净。接下来，4％多聚甲醛（PFA）过夜固定在4°C或在室温下为1小时。 </li><li>片可以分析不同的细胞标记或单独用Hoechst染色，电穿孔和电穿孔非细胞可视化。通透和阻止片2小时，在室温下用10％山羊血清，0.1％，在1X PBS轻轻摇动TRITON。 </li><li>用Hoechst染色在室温下为1小时，用3次，每次10分钟1X PBS轻轻摇动。 </li><li>要挂载切片用手术刀膜，并用细镊子转移一个磨砂玻璃片膜在水室的幻灯片。可以置于载玻片每到5片。删除多余的水，并添加一个解决方案，每个大脑切片Flourmount下降。轻轻地放在大脑切片上盖玻片，并删除Ÿ气泡。利用共聚焦显微镜分析片。 </li></ol> 7。替代器官切片石蜡包埋（没有视频） <ol><li>也可以被嵌入器官切片石蜡更精细的形态分析，为此包含的器官切片的膜，可固定在煤灰作为以上所述的4％。 </li><li>固定片嵌入在1％琼脂糖（预先加热至37°C），并在冰凝固为30分钟。琼脂糖块，然后可以在4℃后固定为30分钟，在4％的煤灰。 </li><li>琼脂糖块包含的器官切片，然后将石蜡包埋免疫处理，如先前所述10。 </li></ol> 8。代表结果 如图1所示的小鼠皮层和文化的器官切片电图解。这种方法是一个RAPI有用的战略ð评估功能在神经元发育有关的基因11。根据量DNA的电穿孔和电在萌芽阶段，转染效率会有所不同。片会开始表达绿色荧光蛋白至少8个小时后，电和细胞将进行正常的神经活动，在文化（增殖，迁移和早期神经细胞分化）序列图2显示了一个电脉冲的大脑切片，表达了控制pSilencer-GFP的向量和一个可以观察到神经元祖细胞，神经元迁移和分化的神经元在片。器官切片将保持其形态，只要他们保持在一个良好的媒体空气膜接口，可以使用至少5天的文化。 <img alt="图1" src="/files/ftp_upload/3621/3621fig1.jpg" /> 图1。插图体外电击和器官切片培养法。E14.5胚胎解剖，快速的绿色染料混合的DNA与单独注射，以可视化的注射部位。 DNA可在两个侧脑室注射插图描绘，以填补侧脑室或第三脑室。注射后大脑电穿孔方波electroporator，正电极放在大脑所需的一面。大脑在3％低溶点琼脂糖和切片使用vibratome嵌入。 250微米的大脑切片放在0.4微米插入，并培养了一个星期。转染后8小时后可观察到绿色荧光蛋白。 <img alt="图2" src="/files/ftp_upload/3621/3621fig2.jpg" /> 图2。电脉冲的大脑切片分析。电穿孔的大脑切片进行染色赫司特。背的皮质神经祖细胞电穿孔在脑室区（VZ）。 CORTI中的神经元CAL板（CP）是由边缘区（MZ）分隔。白色箭头显示移植的神经元。在这种情况下，被注入大脑在E15.5与pSilencer GFP控制向量与电穿孔。部分代表电后4天的皮质外植体。比例尺100微米。 故障排除： <ol><li> 转染效率低：调整用于至少为1μg/μL的DNA浓度。总是从马克西准备使用非常干净的DNA，如果必要使用内免费Quiagen的试剂盒纯化的DNA。 </li><li> 转染细胞在不同的大脑区域，一个比一个期望：确保向一侧被电脑的正极位置正确，电极定位。 </li><li> 片失去器官形态变化的媒体每天，确保片漂浮在媒体</li><li> 琼脂糖片脱落，因为他们正在削减在vibratome：确保良好的界面嵌入在低熔点琼脂糖时的大脑。 </li></ol>

<ol>
	<li>Angevine, J. B., Sidman, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoradiographic+study+of+cell+migration+during+histogenesis+of+cerebral+cortex+in+the+mouse.">Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse.</a> Nature. 192, 766-766 (1961).</li><li>Kriegstein, A. R., Noctor, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+neuronal+migration+in+the+embryonic+cortex.">Patterns of neuronal migration in the embryonic cortex.</a> Trends Neurosci. 27, 392-392 (2004).</li><li>Barnes, A. P., Polleux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+axon-dendrite+polarity+in+developing+neurons.">Establishment of axon-dendrite polarity in developing neurons.</a> Annu. Rev. Neurosci. 32, 347-347 (2009).</li><li>Haydar, T. F., Bambrick, L. L., Krueger, B. K., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+slice+cultures+for+analysis+of+proliferation,+cell+death,+and+migration+in+the+embryonic+neocortex.">Organotypic slice cultures for analysis of proliferation, cell death, and migration in the embryonic neocortex.</a> Brain Res. Brain Res. Protoc. 4, 425-425 (1999).</li><li>Polleux, F., Ghosh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+slice+overlay+assay:+a+versatile+tool+to+study+the+influence+of+extracellular+signals+on+neuronal.">The slice overlay assay: a versatile tool to study the influence of extracellular signals on neuronal.</a> Sci. STKE. 2002, pl9-pl9 (2002).</li><li>Guerrier, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+F-BAR+domain+of+srGAP2+induces+membrane+protrusions+required+for+neuronal+migration+and+morphogenesis.">The F-BAR domain of srGAP2 induces membrane protrusions required for neuronal migration and morphogenesis.</a> Cell. 138, 990-990 (2009).</li><li>Stegmuller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-intrinsic+regulation+of+axonal+morphogenesis+by+the+Cdh1-APC+target+SnoN.">Cell-intrinsic regulation of axonal morphogenesis by the Cdh1-APC target SnoN.</a> Neuron. 50, 389-389 (2006).</li><li>Sepp, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+neural+outgrowth+genes+using+genome-wide+RNAi.">Identification of neural outgrowth genes using genome-wide RNAi.</a> PLoS Genet. 4, e1000111-e1000111 (2008).</li><li>Konishi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdh1-APC+controls+axonal+growth+and+patterning+in+the+mammalian+brain.">Cdh1-APC controls axonal growth and patterning in the mammalian brain.</a> Science. 303, 1026-1026 (2004).</li><li>Vankelecom, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+and+paraffin-embedding+of+mouse+tissues+for+GFP+visualization.">Fixation and paraffin-embedding of mouse tissues for GFP visualization.</a> Cold Spring Harb Protoc. 2009, 5298-5298 (2009).</li><li>Hand, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+Neurogenin2+specifies+the+migration+properties+and+the+dendritic+morphology+of+pyramidal+neurons+in+the+neocortex.">Phosphorylation of Neurogenin2 specifies the migration properties and the dendritic morphology of pyramidal neurons in the neocortex.</a> Neuron. 48, 45-45 (2005).</li><li>Taniguchi, Y., Young-Pearse, T., Sawa, A., Kamiya, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Utero+Electroporation+as+a+Tool+for+Genetic+Manipulation+in+Vivo+to+Study+Psychiatric+Disorders:+From+Genes+to+Circuits+and+Behaviors.">In Utero Electroporation as a Tool for Genetic Manipulation in Vivo to Study Psychiatric Disorders: From Genes to Circuits and Behaviors.</a> Neuroscientist. 18, 169-179 (2012).</li></ol>

我们什么都没有透露。

这些方法包括体外电击质粒编码双链RNA发夹和器官切片4提供了几个明显的优势文化。首先，这些方法可以快速评估为RNAi派生的表型的。 pSilencer载体包含U6启动子驱动的双链RNA发夹编码绿色荧光蛋白表达盒中列入允许细胞电穿孔与RNAi载体的快速识别和表征。除了时间效率，这些方法具有高度成本效益的几个淘汰赛行产生相比。第二，与生存电技术12，需要较长的培训和技能?...

<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>汉密尔顿注射器</td><td>哈密​​尔顿</td><td> 80008 </td><td> 31计，0.5英寸长，PT-4（点斜边的水平），体积10μL </td></tr><tr><td>白金tweezertrodes </td><td> A型肉毒毒素</td><td> 45-0489 </td><td> 5mm大小</td></tr><tr><td> ECM830 electroporator </td><td> A型肉毒毒素</td><td> 45-0002 </td><td></td></tr><tr><td> BTX的脚踏</td><td> A型肉毒毒素</td><td> 45-0208 </td><td> ECM830 electroporator </td></tr><tr><td>振动刀片切片机</td><td>徕卡</td><td> VT1000小号</td><td></td></tr><tr><td> 6 - 以及菜用插入</td><td>鹰</td><td> 353502 </td><td>包含适合插入的缺口</td></tr><tr><td>组织培养插入</td><td>鹰</td><td> 353090 </td><td> 0.4微米</TD&gt; </tr><tr><td>快绿</td><td>西格玛</td><td> F7252 </td><td></td></tr><tr><td>低融点琼脂糖</td><td>稳</td><td> BP165-25 </td><td> DNA的等级</td></tr><tr><td>层粘连蛋白</td><td> Sigma-Aldrich公司</td><td> L2020 </td><td></td></tr><tr><td>聚-L-赖氨酸</td><td> Sigma-Aldrich公司</td><td> P5899 </td><td></td></tr><tr><td>培养基鹰</td><td> Sigma Aldrich公司</td><td> B - 1522 </td><td></td></tr><tr><td>无钙，镁10倍的HBSS </td><td> GIBCO公司</td><td> 14180-046 </td><td></td></tr><tr><td>肝素钠的游离酸</td><td> Sigma-Aldrich公司</td><td> H4034 </td><td></td></tr></tbody></table>

methods for study of neuronal morphogenesis: ex vivo rnai electroporation in embryonic murine cerebral cortex

大脑皮质的指示较高的认知功能。这6个层状结构会产生一个内部第一，外部最后的方式，其中第一个出生的神经元保持密切的心室，而去年出生的神经元迁移过去的第一个出生的神经细胞向大脑表面1。除了 ​​神经细胞迁移2，皮质功能正常的一个关键过程是调节神经元的形态3。虽然神经元形态，可在原代培养体外研究，有多少要了解到，从这些过程是如何在组织环境监管。 我们描述的技术来分析神经细胞迁移和/或在大脑皮层的器官切片4,6形态。用于修改一个pSilencer载体，其中包含同时U6启动子驱动的双链发夹RNA和一个单独的编码绿色荧光蛋白表达盒驱动b雅CMV启动子7-9。我们的方法允许候选基因的具体击倒后突起生长的缺陷快速评估，并已成功用于在屏幕上一个突起生长8监管。因为只有一部分细胞会表达的RNAi构造，器官切片允许镶嵌一个潜在的表型分析。此外，因为这种分析是在附近近似体内环境，它提供了一个低成本为皮质功能未知的基因，并迅速替代代转基因或基因敲除动物。最后，在体内电穿孔技术相比， 体外电击实验的成功是不依赖于熟练的手术技巧的发展，可以用更短的训练时间和技能进行。

Watch this Scientific Journal Video about Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex at JoVE.com

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

我们描述涉及的方法进行快速评估的功能基因在大脑皮层的发展，<em&gt;体外</em&gt;电质粒共表达抑制核糖核酸（RNA干扰）和绿色荧光蛋白在小鼠胚胎皮层。该协议是适合研究神经发育的各个方面，如神经，神经细胞迁移和神经元的形态，包括树突和轴突生长。

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 ...

methods-for-study-neuronal-morphogenesis-ex-vivo-rnai-electroporation

Research

JoVE Journal

Neuroscience

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

11.7K 次观看.  Brown University. 我们描述涉及的方法进行快速评估的功能基因在大脑皮层的发展，体外电质粒共表达抑制核糖核酸（RNA干扰）和绿色荧光蛋白在小鼠胚胎皮层。该协议是适合研究神经发育的各个方面，如神经，神经细胞迁移和神经元的形态，包括树突和轴突生长。

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

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

科研

神经科学

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

在发展中国家小鼠视网膜体内电穿孔

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

检测内源NADH的荧光双光子成像的小鼠大脑皮层Microregional缺氧

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

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 ...

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 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 ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

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

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

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

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

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

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

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

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

在子宫内电途径研究神经元亚群的兴奋性和单细胞连接

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

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To do so requires generating a mosaic in which neurons with loss or gain of function of a gene are surrounded by genetically unperturbed tissue. Here, we combine the Cre-lox recombination system with in utero electroporation in order to generate mosaic brain tissue that can be used to study the cell-autonomous function of genes in neurons. DNA constructs (available through repositories), coding for a fluorescent label and Cre recombinase, are introduced into developing cortical neurons containing genes flanked with loxP sites in the brains of mouse embryos using in utero electroporation. Additionally, we describe various adaptations to the in utero electroporation method that increase survivability and reproducibility. This method also involves establishing a titer for Cre-mediated recombination in a sparse or dense population of neurons. Histological preparations of labeled brain tissue do not require (but can be adapted to) immunohistochemistry. The constructs used guarantee that fluorescently labeled neurons carry the gene for Cre recombinase. Histological preparations allow morphological analysis of neurons through confocal imaging of dendritic and axonal arbors and dendritic spines. Because loss or gain of function is achieved in sparse mosaic tissue, this method permits the study of cell-autonomous necessity and sufficiency of gene products in vivo.

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous functions of genes in the brain can be studied by inducing loss or gain of function in sparse populations of cells. Here, we describe in utero electroporation to deliver Cre recombinase into sparse populations of developing cortical neurons with floxed genes to cause loss of function in vivo.

宫内电穿孔诱导小鼠大脑皮质中氧合酶的重组

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To ...

Methods Article

神经元形态的研究方法：