Name: live imaging of mitosis in the developing mouse embryonic cortex
Uploaded: 2014-06-04T13:45:00
Description: Watch this Scientific Journal Video about Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex at JoVE.com

作者感谢NINDS /美国国立卫生研究院，R00-NS064197和NINDS /美国国立卫生研究院，R01NS083897（既DLS）的资金。

1，制备媒体（图1，步骤1） <ol><li>片培养基<ol><li> 25毫升切片培养基足以制备5玻璃底培养皿中，每孔2片。 </li><li>在50ml锥形管中，加入250微升的100×N2溶液及500微升的50倍溶液B27无维生素A的DMEM/F12添加到22.5毫升的体积。 </li><li>过滤灭菌的溶液，随后加入1.25热灭活的马血清中的溶液和1.25ml胎牛血清。 </li><li>孵育在水浴溶液在37℃下进行切片的制备的持续时间。 </li><li>添加生长因子（FGF和EGF，10终?...

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我们已经描述了协议的主要优点是，它提供了神经祖细胞的有丝分裂的动态时间分辨率。通常情况下，检测到有丝分裂可视化在脑发育所使用的固定的组织切片进行免疫。但这种方法只提供了有丝分裂的快照在一个时间点。 有几个步骤是在大脑切片成像有丝分裂最关键的：1）脑片应保持附着在琼脂糖，转让和安装过程中保留的脑切片的完整性。 2）产生和切片成像，rostro-尾?...

本协议的总体目标是描述如何在胚胎大脑切片进行神经祖细胞有丝分裂的实时成像。用文化的脑片实时成像，该协议提供了一种简单的方法来测定在一个环境非常相似， 在体内环境的神经祖细胞有丝分裂的多个方面。它可以应用到已经被操纵以在子宫内电穿孔）1-5突变的动物和/或大脑的大脑。该技术也适合用于测试药剂对神经前体“有丝分裂的作用，通过简单地增加一个代理到培养基中。总而言之，本文将做一个技术上的挑战协议访问到那些学习神经。 在神经发生，不同的神经祖细胞群进行精确分割，产生神经元，最终有助于成年人的六个皮质层大脑皮层6-8。早在皮质发育，神经前体池扩大为神经上皮（NE）细胞分裂对称地自我更新。东北细胞，然后转换成放射状胶质细胞（RGC的）。最初，视网膜神经节细胞对称分裂产生两个新的视网膜神经节细胞，但大部分神经发生过程中，视网膜神经节细胞的主要分工模式是不对称的。在不对称分裂，1 RGC产生了一个新的RGC以及一个有丝分裂后的神经元，或更专门的祖细胞（无论是短期的神经前体（SNP），外放射状胶质（ORG），或一个中间祖（INP） 2,3,7,9。综合邻舍计划，单核苷酸多态性，以及ORGS然后可以在该子心室，左心室产生的神经元，和皮层的基底区域，分别，因此，祖细胞的细胞分裂是产生的神经元的基本处理新皮层。 许多研究指出，视网膜神经节细胞的有丝分裂的具体特征和子细胞的命运之间的相关性。海达尔等和Takahashi 等。已经表明，研资局有丝分裂期和细胞周期的长度增加为神经收益，这一发现回荡在后续研究10-13。大量的研究表明，有丝分裂纺锤体相对于心室取向影响神经发生和corticogenesis的各方面，包括在大脑中，生成的类型的神经元和后代的位置分别3,10,14-16。无论是解理面取向直接影响着细胞的命运是有争议的，但结论仍然是，这种有丝分裂参数影响神经发生。进一步强调了有丝分裂的重要性是观察，涉及有丝分裂的机制的许多基因是至关重要的神经发生和进行适当的大脑发育17-20。 有丝分裂是一个动态的过程，但迄今最为详细的研究神经祖有丝分裂利用的固定的组织切片或神经祖细胞成像通过体外细胞培养分析。因此，主流的方法来评价有丝分裂只提供这一过程的快照，并不能揭示细胞如何表现在组织。神经祖细胞有丝分裂实时成像日益成为了解神经祖细胞功能的重要工具。有关示例，请参阅这些引用4,8,10,21-25。几个突出的协议已经出版的准备和脑切片26,27的成像。然而迄今为止，全面协议用于成像和有丝分裂的分析还没有被描述，也不在视频演示。 这种技术提供了一些显著优势的脑切片固定的分析。脑切片的时间推移分析使之能够以一种灵活的方式来分析显著更多的数据点产生。首先，数据被聚集在单个时间点在数分钟或数小时的过程。我们可以分析各个时间点（创建一个静态蒙太奇）或可以结合不同的时间点成电影。其次，切片共焦成像能够产生数据在脑片不同的Z部分。其结果是，各个部分可以被分析。或者，堆叠各个部分可以被组合成一个最大强度投影。第三，分析是在一个组织的背景下完成的，揭示细胞如何分裂相对于邻近的细胞和结构。第四，它是非常适合的，显示有丝分裂缺陷的一些证据突变体的分析。总之这个协议将有助于澄清，以帮助调查谁愿意开展神经祖有丝分裂实时成像在自己的实验室关键步骤。

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	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:143px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:261px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100X N2</td>
			<td>Life technologies</td>
			<td>17502048</td>
			<td style="width:261px;">for culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50X B27 without vitamin A</td>
			<td>Life technologies</td>
			<td>12587010</td>
			<td style="width:261px;">for culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F12</td>
			<td>Life technologies</td>
			<td>11320033</td>
			<td style="width:261px;">for culture medium</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Heat-inactivated horse serum</td>
			<td>Sigma Aldrich</td>
			<td>H1138-500ml</td>
			<td style="width:261px;">for culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heat-inactivated calf serum</td>
			<td>Sigma Aldrich</td>
			<td>F4135-500ML</td>
			<td style="width:261px;">for culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FGF</td>
			<td>R&#38;D Sytems</td>
			<td>3139-FB-025</td>
			<td style="width:261px;">for culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGF</td>
			<td></td>
			<td></td>
			<td style="width:261px;">for culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10X HBSS</td>
			<td>Life technologies</td>
			<td>14065-056</td>
			<td style="width:261px;">for the dissection of the embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hepes Free Acid</td>
			<td>Sigma Aldrich</td>
			<td>&#160;H4034</td>
			<td style="width:261px;">dilute to 1M (pH7.4)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2.5M D-Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G8769</td>
			<td style="width:261px;">for the dissection of the embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.9M NaHC03</td>
			<td>Life technologies</td>
			<td>25080-094</td>
			<td style="width:261px;">for the dissection of the embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">low-melting agarose</td>
			<td>Fisher&#160;</td>
			<td>BP165-25</td>
			<td style="width:261px;">for generating slices</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Loctite 404 glue</td>
			<td>Loctite 404</td>
			<td>46551</td>
			<td style="width:261px;">keep at 4&#730;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">syto11</td>
			<td>Life technologies</td>
			<td>S7573</td>
			<td style="width:261px;">Make 5&#181;l aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3 mg/ml collagen type I&#160;</td>
			<td>Life technologies</td>
			<td>A1048301</td>
			<td style="width:261px;">for culturing slices</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glass bottom dish</td>
			<td>MatTek</td>
			<td>P35G-1.5-14-C</td>
			<td style="width:261px;">for culturing slices</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">petri dishes</td>
			<td></td>
			<td></td>
			<td style="width:261px;">for dissection of the embryos</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">digital thermometer</td>
			<td></td>
			<td></td>
			<td style="width:261px;">to measure the temperature of the agarose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">spatula</td>
			<td></td>
			<td></td>
			<td style="width:261px;">to transfer brains</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">paintbrush</td>
			<td></td>
			<td></td>
			<td style="width:261px;">alternative to transfer brains</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">vibratome</td>
			<td>Leica</td>
			<td>VT1000s</td>
			<td style="width:261px;">for generating slices</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dissecting microscope</td>
			<td></td>
			<td></td>
			<td style="width:261px;">dissecting out embryos</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">imaging microscope</td>
			<td></td>
			<td></td>
			<td style="width:261px;">A confocal microscope is required, it needs to be equipped with an incubation chamber</td>
		</tr>
	</tbody>
</table>

live imaging of mitosis in the developing mouse embryonic cortex

虽然时间短，有丝分裂是一个复杂和动态的多步骤过程，基本为器官，包括脑的发育。在发展中大脑皮层，神经祖细胞的有丝分裂异常可引起脑容量和功能的缺陷。因此，有迫切需要的工具来理解的神经祖有丝分裂的机制。在啮齿类动物皮质发育是研究这一过程的杰出典范。神经祖细胞有丝分裂通常在检查固定脑切片。该协议将详细描述用于在有丝分裂体外胚胎脑片实时成像的方法。我们将描述的关键步骤，此步骤，其中包括：脑提取，嵌入脑，脑切片vibratome切片，染色和切片的培养，和时间推移成像。然后，我们将演示并详细说明如何执行有丝分裂的收购后分析。我们有代表性的结果神父嗡使用活体染料Syto11，转基因小鼠（组蛋白H2B-EGFP和中心蛋白-EGFP），并在子宫内的电穿孔（的mCherry-α-微管蛋白），此测定法。我们将讨论如何此程序可以最优化，以及它如何可以修改为有丝分裂的基因调控的研究。有丝分裂中的脑切片的实时成像是一种灵活的方法，以评估年龄，解剖，和遗传扰动在一个受控制的环境的影响，并产生大量的具有高时间和空间分辨率的数据。因此，该协议将补充为神经前体有丝分裂分析现有的工具。

这个实验的成功，多有丝分裂细胞中的一个实时成像会议期间的观察很大程度上取决于双方的诚信和地点进行采集，切片解剖层次。如下面讨论的，切片的解剖水平是一个重要因素。 图3A示rostro-尾和中间-侧部，其中我们最成功的位置。对于这个问题的更多讨论，请参阅Noctor 26。切片的完整性可在该切片的不同区域而变化。这可以先于时间推移（使用整个切片的成像与步骤7.6?...

Watch this Scientific Journal Video about Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex at JoVE.com

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

神经祖细胞有丝分裂是神经发生的一个关键参数。很多我们的神经祖有丝分裂的理解是基于固定的组织分析。在胚胎大脑切片实时成像是一种通用的技术，以评估有丝分裂与在受控环境中高时空分辨率。

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 ...

Research

JoVE Journal

Neuroscience

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

慢性成像小鼠的视觉皮层使用减薄，颅骨的制备

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

科研

神经科学

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

多鼠神经解剖学磁共振成像

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

在发展中国家斑马鱼的前脑的神经祖细胞克隆相关的定时实时成像

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

高分辨率实时成像发展中的神经上皮细胞的行为

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

体外实时成像在小鼠神经上皮单细胞分裂

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

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

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

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 ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

室管膜纤毛在小鼠脑侧脑室实时成像

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

同时双光子<em&gt;在体内</em&gt;突触输入的影像和突触后目标中的鼠标Retrosplenial的Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

生活成像的脑皮层细胞使用二维培养系统

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

前子宫内胚胎小鼠脑电穿孔和脊髓切片培养迁移 GABAergic 中间神经元的活体成像

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...