这项工作是支持NINDS（NS066071）和NIAAA的补助金。 （P50AA017823）ECO。作者感谢罗伯特·奎因和爱护动物实验室动物资源部的工作人员。我们感谢贾德森·贝尔蒙特的技术支持，妮可Belletier作为暑期大学生研究员（SURF）的援助。我们也感谢戴维·卡梅伦博士的意见和编辑的早期版本的手稿。

1。 前的子宫内 Electroporations与绿色荧光蛋白表达载体的构建<ol><li>质粒DNA注射溶液的制备与CAG-eGFP的DNA 27 DDH 2 O中稀释至浓度为0.33毫克/毫升的最后工作使用Qiagen公司的Endo-免马克西Preps中纯化该质粒转化的细菌。快速绿色染料在约0.02％（w / v的最终）被添加到作为注射示踪的DNA溶液。 </li><li>为了准备手术区，喷绒与台式和解剖显微镜下阶段的70％的乙醇溶液中，并擦干。喷手术剪刀和镊子解剖前，用70％的乙醇，并擦干。 </li><li>定时怀孕的瑞士/韦伯斯特水坝被处死E13通过转移到一个腔室填充有CO 2从加压缸和监测大坝至少为1分钟，直到所有的运动停止。通过CO 2吸入牺牲后的胚胎从子宫中取出，并放置在一个10厘米的培养皿中含有冰冷的Hanks平衡盐溶液（HBSS）中。 </li><li>小心解剖每个胚胎从周围的胚外膜，并保持在冰冷的HBSS中。 </li><li>单独每个胚胎转移到第二个10厘米培养皿含有冷的HBSS。用微量注射器注入2-3微升DNA快速绿色混合物入脑室，左侧大脑半球，照顾注入皮质区在空间上分开的皮质区，供日后分析。 </li><li>若要electroporate，用钳子轻轻地按住胚胎，并轻轻放置在正的镊子电极的头部背中线并轻轻放置负胚胎下巴下面桨桨。 Electroporations实现一个BTX830电穿孔编程提供50 30 V的脉冲持续时间为50毫秒，950毫秒的间隔分开。这些设置的BTX830模型。根据制造商的指导书，其它的系统也可以使用纳秒。 </li></ol> 2。整个半球外植体准备<ol><li>电穿孔后的胚胎被放回冰冷的HBSS。从胚胎头使用两个＃5珠宝商钳去除皮肤和软骨颅骨，大脑被删除。一个镊子，然后滑动下方的大脑从头骨中取出完整的大脑。然后，将电穿孔的半球（左）是远离大脑解剖和附加的脑组织中除去。在整个解剖过程的护理，以不损伤脑膜覆盖的左侧皮质半球。 </li><li>一旦解剖每个胚胎转移用移液器有切口1毫升移液管尖，在HBSS中。半球轻轻地驱逐到胶原蛋白包被的培养插入。最多6个半球植安排内侧，每24毫米的插入。一旦设置后，除去过剩的HBSS从插入件和插入到一台35毫米的6孔板中，然后放置。以及包含EXActly 2.7毫升介质：DMEM-F12培养基含有Glutamax和补充有2％B-27，1％G5和1％青霉素 - 链霉素。可以滴上几滴以及媒体的每个外植体的顶部，但不超过原来的2.7毫升，每孔添加媒体。 </li><li>一旦胶原过滤器已被放置到外植体的6孔培养皿中的外植体被放置到一个比卢普斯罗森堡（BR）的腔室（即还包含一个加湿水盘）。密封腔阻断和断开的95％/ 5％的氧/ CO 2气体供给管之前至少1分钟的95％/ 5％的氧/ CO 2气体的混合物被注入到腔室。 BR室，然后放入37℃的组织培养箱的培养周期的其余部分，1-2 DIV。 </li></ol> 3。固定的外植体组织的组织学<ol><li>在通风橱中准备的的帕加诺固定溶液由第一解磷8克多聚甲醛在100毫升预热的（〜80和de克; C）DDH 2 O。加入1-3滴浓NaOH，以促进溶，轻轻搅拌。一旦溶解，混合8％多聚甲醛溶液以1:1的溶液是500mM的蔗糖，100毫摩尔的Hepes，pH为7.4的50mM的MgCl 2，5 mM KCl中的最终浓度为4％的多聚甲醛在250 mM蔗糖的50mM的Hepes ，25毫摩尔MgCl 2，2.5 mM KCl中，pH值7.4。 </li><li>要解决外植体的的帕加诺修复解决方案温暖至37℃。快速去除大部分的DMEM/F12媒体的每一种文化和每口井的外植体加5毫升的帕加诺修复的。确保完全覆盖每个外植体在修复。修正在室温下1小时。不要删除外植体过滤器前，固定1小时。 </li><li>固定后删除Pagano的修复溶液和Pagano的修复（250 mM蔗糖的50mM的Hepes，25毫摩尔MgCl 2，2.5 mM KCl中，pH值7.4）的解决方案，无需更换。加入几滴10％的叠氮化钠，并储存于4℃下，直到嵌入。 </li></ol> 4。嵌入和Sectioning组织学<ol><li>准备10％的小腿皮肤明胶溶液的小腿皮肤明胶10克溶解在100毫升的温开水（55-60°C）。将明胶溶液的热板上设置为60℃，旋流定期直至溶解的。 </li><li>倒入约10毫升的10％的明胶溶液中成一个10厘米的陪替氏培养皿的底部，允许30分钟固化。这将形成一个“垫”嵌入外植体。这些垫可以是预先制备的天，并在4℃下存储用尺子画一个表格，在培养皿的底部和个人外植体转移到每个方块的网格。取出残余的Pagano的溶液，用移液器，覆盖每个外植体用温水10％明胶溶液，并允许固化。恢复缓慢加入明胶溶液，直到每个外植体被完全包围由明胶，照顾，不熔化垫由添加太多温热一次明胶。允许明胶硬化〜1小时。一旦硬化的individUAL外植体，可以切割​​出小块明胶。块后固定在帕加诺解决之前切片用一个vibratome的24-48小时。 </li><li>外植体在明胶块位于嗅球和在冠状面，在100微米厚的切片。的部分被收集和存储在PBS + 0.1％叠氮化钠，在4℃直至免疫组化处理。 </li><li>通过传送部到24孔板中（每孔2-3节）进行免疫组化检测。第一数据块的非特异性结合的抗体，通过加入0.5毫升PBST + B（PBS + 0.5％的Triton X 100和2％BSA）到每个孔中，培养1小时，轻轻摇动。适当的主要抗体，然后稀释在PBST + B和0.4毫升，每孔中加入在RT下孵育过夜。主要是洗出与3X PBS洗至少10分钟。的二次抗体和2微克/毫升赫斯特33342核染色，然后在PBST + B和稀释样品在室温孵育2小时轻轻摇动。三的PBS洗涤（每次10分钟）被执行，并且然后幻灯片上使用90％甘油的50mM的Tris，pH7.4的，安装媒体被安装的部分。 </li></ol>

作者宣称，他们没有竞争的金融利益。

我们改进和评价的实验模型 - 整个半球外植体 - 皮质发育早期的研究（E13-E15）。该模型已被证明有用的兴奋性神经元构成的大脑皮质25,37层的血统，迁移和分化的分析。的原理的系统的优点是：1）器官增长2 DIV，2）简单的准备，和3）的神经元的电穿孔，药理操纵和成像实验获得在这个早期，大脑发育的关键时期。我们使用文件系统的形态，方向和枝晶生长的6层皮层神经元过程中的分裂?...

哺乳动物的大脑皮层形式，通过连续产生的神经元的一致的增殖，迁移和分化。每个神经元是出生在脑室区（VZ）和迁移的VZ到中间区（IZ），形成皮质板（CP）1。他们通过不同的皮层区域，迁移神经元显示多种模式的迁移2,3依赖于细胞外环境和发展组织内的其他细胞成分（ 如放射状胶质）。皮层神经元，然后逮捕迁移顶部的形成皮质板重合的过程神经元迁移逮捕和dendritogenesis 4。 皮质发育胚胎天11-13（E11-13）通过建立的原始丛状层或 preplate（PP），一个的先驱神经元层覆盖VZ之间开始。预期6层皮质神经元（ 即第一个出生的皮层神经元在VZ），然后定位在一个刻板的模式，其胞体合并成一个不同的层内的PP 7。这些事件的preplate分割表面的边际（未来的皮质层1）和深区，后者组成的底板细胞（瞬态皮质层7）。这个过程，是一个基础性的事件在未来的增长大脑皮层8，被称为preplate分裂。 许多基因的突变已被确定，破坏皮质发育的各个方面，9。皮质发育也受到负面影响暴露摄入的毒素，如10可卡因和酒精11。由于在开发过程中出现的皮质畸形，有可能造成神经系统疾病（ 如自闭症，精神分裂症），皮质发育的扰动所固有的实证调查LY重要的。因此，这是相当重要的建立方法来研究大脑皮质的发展，使快速检测基因或毒素的影响，但也保持分化的神经元，其他类型的细胞和细胞外基质（ECM）之间的相互作用，在这个早期阶段大脑发育12 。 切片外植体有13提供这样一个系统，并已被广泛用于测定皮层神经元的发展14-16。然而，切片化验可能遭受的缺点是不正常的神经元的迁移以及分层17可能是由于损坏的大脑发育和锚放射状胶质细胞的脑膜细胞包围。由于放射状胶质纤维是一种重要的基板皮质神经元迁移18中断的基底层，通过切片局部破坏放射状胶质架构和，导致皮质迁移改变。此外，SLI土木工程署的外植体表面的死细胞，可能会改变正常的组合物，在这些领域中的ECM提供的区域。 最近的方法都集中分析细胞在适当的健康的细胞类型和细胞外基质所包围的片位于深。然而，在某些情况下，这些新的方法可以要求原始厚培养切片冷冻切片或石蜡切片，使固定后的切片相对正常的内部提供用于分析19-21。原vibratome切片准备活的文化片固定片进行分析以及随后的cryosectioning，要求这些实验的关怀和努力工作。 皮质发育早期的研究提供了一种简单的，互补的方法，我们已经修改了现有的切片的方法13早期视觉皮层的发展促进研究。我们已经开发了一个whOLE半球外植体模式类似于现有的E14，在65转每分钟，并允许器官增长16〜18小时22,23涉及颤抖文化的整个半球模型。在我们的方法中，整个半球外植体放置在半透膜13与在高氧培养气氛21,24延长器官皮质生长48小时。这种方法还可以开发大脑皮质神经元的电一致。将胚胎从子宫中取出，并电穿孔引入质粒DNA和前脑，然后解剖。每个半球被隔离并置于内侧上的胶原蛋白涂覆的滤波器。然后，将外植体培养48小时的期间，一段时间，它包括第8 preplate分裂。在培养过程中，L6神经细胞前体分化的神经细胞，正确定位发展的皮质骨内。在此期间发展神经元周围环绕着相应的ECM和细胞类型，将对抗体内的相应单元格中 。该系统已经证明了宝贵的破译细胞事件，26层6的形成和preplate分裂7,25乙醇毒性的基础。

<table border="1">
	<tbody>
		<tr>
			<td>Name </td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM/F12 + GlutaMAX</td>
			<td>GIBCO</td>
			<td>10565</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>G5 Supplement 100X</td>
			<td>Invitrogen</td>
			<td>17503-012</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>B27 Serum-Free Suppl. 50X</td>
			<td>Invitrogen</td>
			<td>1504-044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pen / Strep Liquid 100X</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HBSS 500 ml</td>
			<td>GIBCO</td>
			<td>14025</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Culture insert collagen coated</td>
			<td>Costar</td>
			<td>3492</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bovine skin gelatin</td>
			<td>Sigma</td>
			<td>G9382</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Invitrogen</td>
			<td>H1399</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>7906</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit</td>
			<td>Qiagen</td>
			<td>12362</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BTX 830 Electroporator</td>
			<td>Harvard Apparatus</td>
			<td>450052</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tweezer electrodes 10mm</td>
			<td>Harvard Apparatus</td>
			<td>450166</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Billups Rothenberg</td>
			<td>MIC-101</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hamilton syringe (5 uL)</td>
			<td>Hamilton</td>
			<td>87930</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hamilton syringe needle</td>
			<td>Hamilton</td>
			<td>7803-04</td>
			<td>Specify 1" and style 4</td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>FST</td>
			<td>11251-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fine Scissors Tough Cut 9 cm</td>
			<td>FST</td>
			<td>14058-09</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

ex utero electroporation and whole hemisphere explants: a simple experimental method for studies of early cortical development

皮层的发展涉及神经元和非神经元的元素，包括前体细胞，血管，脑膜和相关的细胞外基质之间的复杂的相互作用。因为它们提供了一个合适的器官环境，皮质切片外植体经常被用来调查这些控制神经细胞的分化和发育的相互作用，。虽然有益，片植模型可能遭受的缺点，包括的异常细胞层压和迁移。在这里，我们报告整个大脑半球外植体系统研究的早期视觉皮层的发展是比较容易准备比皮质片，并显示一致的器官迁移以及分层。在这个模型系统，早期的分层和迁移模式正常进行为期两天的体外 ，包括分裂的preplate期间，在未来的皮质层的六种形式。然后，我们开发前子宫穿孔 （EUEP）达到〜80％的成功率，针对发展中的背内侧皮质神经元GFP表达的方法。 的整个半球外植体模型皮质发育早期，电，药物干预和实时成像方法访问。这种方法避免了生存手术中子宫穿孔 （IUEP）的方法，同时提高转染和面目标的一致性。这种方法将有利于神经细胞增殖，迁移和分化的实验研究。

在整个发育期胚胎的啮齿动物皮层表现出的横向神经源性梯度，这样，横向的新大脑皮层是比较成熟的新皮层比背内侧28约1天。层6神经元的大部分被由此而产生（ 即表现出他们的最终S相）的E12在外侧皮质（也称为场40 5）和E13在背内侧皮质（也称为字段1 5）中。 Preplate分裂开始后约1天，第6层神经元的产生，从而开始在外侧皮质皮层E14 E13和背。为了研究preplate分裂?...

Watch this Scientific Journal Video about Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development at JoVE.com

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

该协议介绍了一种改进的外植体的过程，涉及到<em&gt;子宫外</em电，从小鼠胚胎的整个大脑半球的解剖和文化。编制有利于早期皮质发育过程中的基因功能的药理研究和分析。

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 ...

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JoVE Journal

Neuroscience

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

12.8K 次观看.  SUNY Upstate Medical University. 该协议介绍了一种改进的外植体的过程，涉及到子宫外

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

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

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

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

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

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

在子宫内电穿孔：鼠标控制时空基因Transefection

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

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

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

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

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

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

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

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

外用转基因大鼠通过代<em&gt;在子宫内</em&gt;电穿孔和<em&gt;在体内</em&gt;生物发光筛选

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF differentiates from the amniotic fluid upon closure of the anterior neural tube. CSF volume then increases over subsequent days as the neuroepithelial progenitor cells lining the ventricles and the choroid plexus generate CSF. The embryonic CSF contacts the apical, ventricular surface of the neural stem cells of the developing brain and spinal cord. CSF provides crucial fluid pressure for the expansion of the developing brain and distributes important growth promoting factors to neural progenitor cells in a temporally-specific manner. To investigate the function of the CSF, it is important to isolate pure samples of embryonic CSF without contamination from blood or the developing telencephalic tissue. Here, we describe a technique to isolate relatively pure samples of ventricular embryonic CSF that can be used for a wide range of experimental assays including mass spectrometry, protein electrophoresis, and cell and primary explant culture. We demonstrate how to dissect and culture cortical explants on porous polycarbonate membranes in order to grow developing cortical tissue with reduced volumes of media or CSF. With this method, experiments can be performed using CSF from varying ages or conditions to investigate the biological activity of the CSF proteome on target cells.

The ventricular cerebrospinal fluid (CSF) bathes the neuroepithelial and cerebral cortical progenitor cells during early brain development in the embryo. Here we describe the method developed to isolate ventricular CSF from rodent embryos of different ages in order to investigate its biological function. In addition, we demonstrate our cerebral cortical explant dissection and culture technique that allows for explant growth with minimal volumes of culture medium or CSF.

脑脊液分离解剖大脑皮质外植体使用的啮齿动物胚胎

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF ...

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 ...

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of intellectual disability and account for 20-40% of drug-resistant epilepsy in childhood. High-resolution brain imaging has facilitated in vivo identification of a large group of MCD phenotypes. Despite the advances in brain imaging, genomic analysis and generation of animal models, a straightforward workflow to systematically prioritize candidate genes and to test functional effects of putative mutations is missing. To overcome this problem, an experimental strategy enabling the identification of novel causative genes for MCD was developed and validated. This strategy is based on identifying candidate genomic regions or genes via array-CGH or whole-exome sequencing and characterizing the effects of their inactivation or of overexpression of specific mutations in developing rodent brains via in utero electroporation. This approach led to the identification of the C6orf70 gene, encoding for a putative vesicular protein, to the pathogenesis of periventricular nodular heterotopia, a MCD caused by defective neuronal migration.

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Periventricular nodular heterotopia (PNH) is the most common form of malformation of cortical development (MCD) in adulthood but its genetic basis remains unknown in most sporadic cases. We have recently developed a strategy to identify novel candidate genes for MCDs and to directly confirm their causative role in vivo.

一种新的结合阵列全息、全外测序和宫内电穿孔的方法来识别脑畸形的致病基因

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of ...

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, ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

全细胞电压钳记录的电生理方法<em&gt;果蝇</em&gt;光感受器

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. Transcytosis across the BBB regulates the delivery of hormones, metabolites, and therapeutic antibodies to the brain parenchyma. Here, we present a protocol that combines immunofluorescence of free-floating sections with laser scanning confocal microscopy and image analysis to visualize subcellular organelles within endothelial cells at the BBB. Combining this data-set with 3D image analysis software allows for the semi-automated segmentation and quantification of capillary volume and surface area, as well as the number and intensity of intracellular organelles at the BBB. The detection of mouse endogenous immunoglobulin (IgG) within intracellular vesicles and their quantification at the BBB is used to illustrate the method. This protocol can potentially be applied to the investigation of the mechanisms controlling BBB transcytosis of different molecules in vivo.

Here we present a microscopy-based protocol for high-resolution imaging and a three-dimensional reconstruction of the mouse neurovascular unit and blood-brain barrier using brain free-floating sections. This method allows for the visualization, analysis, and quantification of intracellular organelles at the BBB.

血脑屏障的高分辨率共聚焦成像: 成像、3D 重建和 Transcytosis 的量化

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...