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
Summary
该协议介绍了一种改进的外植体的过程，涉及到<em>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。
Abstract
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。
Introduction
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。
Protocol
1。 前的子宫内 Electroporations与绿色荧光蛋白表达载体的构建质粒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。 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。?…
Representative Results
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?…
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Name | Company | Catalog Number | Comments (optional) |
| Reagents | |||
| DMEM/F12 + GlutaMAX | GIBCO | 10565 | |
| G5 Supplement 100X | Invitrogen | 17503-012 | |
| B27 Serum-Free Suppl. 50X | Invitrogen | 1504-044 | |
| Pen / Strep Liquid 100X | Invitrogen | 15140-122 | |
| HBSS 500 ml | GIBCO | 14025 | |
| Culture insert collagen coated | Costar | 3492 | |
| Bovine skin gelatin | Sigma | G9382 | |
| Hoechst 33342 | Invitrogen | H1399 | |
| Bovine Serum Albumin | Sigma | 7906 | |
| EndoFree Plasmid Maxi Kit | Qiagen | 12362 | |
| Equipment | |||
| BTX 830 Electroporator | Harvard Apparatus | 450052 | |
| Tweezer electrodes 10mm | Harvard Apparatus | 450166 | |
| Incubator | Billups Rothenberg | MIC-101 | |
| Hamilton syringe (5 uL) | Hamilton | 87930 | |
| Hamilton syringe needle | Hamilton | 7803-04 | Specify 1″ and style 4 |
| Dumont #5 Forceps | FST | 11251-10 | |
| Fine Scissors Tough Cut 9 cm | FST | 14058-09 |
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Cite This Article
Nichols, A. J., O’Dell, R. S., Powrozek, T. A., Olson, E. C. Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development. J. Vis. Exp. (74), e50271, doi:10.3791/50271 (2013).