Name: in vivo postnatal electroporation and time-lapse imaging of neuroblast migration in mouse acute brain slices
Uploaded: 2013-11-25T21:45:00
Description: Watch this Scientific Journal Video about In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices at JoVE.com

MS和YZ是由KCL和KCL-中国博士学位奖学金支持。 MO是由一个生物技术和生物科学研究理事会博士助学金。我们感谢冈部胜和淳一宫崎的PCX-EGFP质粒和Alain Chedotal和雅典娜伊普西兰蒂上电宝贵意见。

这个程序是按照英国内政部规定（动物科学程序法，1986）。科学家应该遵循建立并获当地机构和国家动物监管机构的指引。 1。产后电穿孔 1.1。玻璃毛细管，DNA解决方案和电穿孔仪的研制<ol><li>用于DNA注射准备拉玻璃毛细管（0.86 mm外径：1.5毫米，ID）。 （对于萨特的P-97毛细管拉马指示设置是：热火283;拉50，速度90，时间50）。使在对应于约2微升体积的毛?...

<ol>
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(67), e4061 (2012).</li><li>Snapyan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vasculature+guides+migrating+neuronal+precursors+in+the+adult+mammalian+forebrain+via+brain-derived+neurotrophic+factor+signaling.">Vasculature guides migrating neuronal precursors in the adult mammalian forebrain via brain-derived neurotrophic factor signaling.</a> J. Neurosci. 29, 4172-4188 (2009).</li><li>Platel, J. C., Heintz, T., Young, S., Gordon, V., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tonic+activation+of+GLUK5+kainate+receptors+decreases+neuroblast+migration+in+whole-mounts+of+the+subventricular+zone.">Tonic activation of GLUK5 kainate receptors decreases neuroblast migration in whole-mounts of the subventricular zone.</a> J. Physiol. 586, 3783-3793 (2008).</li><li>Schaar, B. 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神经祖细胞沿RMS有效的迁移，确保他们的后续成熟为功能性的神经元10。神经祖细胞的朝向突出的OB流是可见的人类婴儿期，并有可能在出生后早期人类大脑发育27起到重要的作用。此外，这些细胞能够靶向脑受损伤和神经退行性变4,28的位点。能够实时监控基因操作对成神经细胞动力学的影响就显得至关重要，充分了解神经细胞的运动引导和规范。 在?...

在哺乳动物的大脑，新的神经元（神经）的一代出生后主要发生在两个区域，海马1的齿状回侧脑室和颗粒下层区的脑室下区（SVZ）。聚集在近年来相当多的证据支持对出生后神经发生在海马和嗅球记忆功能1-3的关键作用。重要的是，出生后的神经也持治疗的潜力，因为它与神经退行性疾病的关系，以及神经母细胞迁移到损伤部位在大脑4-6的能力。 脑室下区（SVZ）最近成为一个重要的神经利基。 SVZ来源的神经母细胞对嗅球（OB）通过喙迁移流（RMS）的迁移，使这个在产后大脑1,7,8最长的迁移过程。哺乳动物SVZ / RMS / OB系统已成为一个研究在神经发生不同的步骤，如增殖，迁移和分化1,8有用的模型。许多生长因子和细胞外调节线索SVZ神经发生和迁移沿RMS，但细胞内的分子机制还远远没有得到充分的理解1,9。沿着正确的RMS迁移是新生神经元10的后续成熟至关重要。此外，一些研究表明，SVZ来源的神经母细胞能出的RMS迁移到脑损伤部位4-6,11-13。因此，调查的信令机制，调节神经细胞迁移的根本不仅要了解神经同时也为潜在的治疗应用。 在这里，我们描述了一个详细的协议通过体内电穿孔产后标记SVZ神经祖细胞和使用时间推移旋转盘共聚焦microsco监控他们沿着急性脑切片培养的RMS迁移PY。电穿孔技术被广泛应用于从胚胎到成人阶段14-18发育研究。它是一个强大的工具，目标和操作SVZ神经祖细胞，代表了更便宜和相当快的替代立体定向注射病毒载体转基因模型1,15,19,20或生成。这是一个相对简单的过程，这并不需要手术，并具有高的存活率。电穿孔或shRNA的小鼠遗传模型采用的LoxP系统可用于靶向感兴趣的基因或实现SVZ祖细胞的永久标记，从而表示对成年神经发生的研究21,22的有用工具Cre重组酶表达质粒的构建。 在完整的脑成像RMS的神经细胞迁移仍是由于目前的技术限制的挑战。然而，这个过程可以使用急性脑切片，这提供了合适的共聚焦系统·旋转盘时间推移显微镜进行监测EM相像的体内条件也适合进行药物处理23,24。耦合在产后体内电穿孔随着时间推移成像将有助于控制神经细胞活力的分子机制的理解，并有助于为新的方法来促进大脑修复的发展。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:344px;">Millicell</td>
			<td style="width:195px;">Millipore</td>
			<td style="width:149px;">PICM0RG50</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 mm Glass bottom culture dish</td>
			<td>MatTek</td>
			<td style="width:149px;">P35G-0-14-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gey&#39;s Balanced media</td>
			<td style="width:195px;">Sigma</td>
			<td>G9779-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glucose, 45%</td>
			<td style="width:195px;">Sigma</td>
			<td>G8769-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td style="width:195px;">Sigma</td>
			<td>H3375-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pen/Strep</td>
			<td style="width:195px;">GIBCO</td>
			<td style="width:149px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FCS</td>
			<td style="width:195px;">GIBCO</td>
			<td style="width:149px;">10109-163</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">B27 supplement</td>
			<td style="width:195px;"><a>Invitrogen Life Technologies</a></td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">L-Glutamine</td>
			<td style="width:195px;"><a>Invitrogen Life Technologies</a></td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM (phenol red-free)</td>
			<td style="width:195px;">GIBCO</td>
			<td style="width:149px;">31053-028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fast Green</td>
			<td style="width:195px;">Sigma</td>
			<td>F7252-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:344px;">Glass capillaries for injection</td>
			<td style="width:195px;">Harvard Apparatus</td>
			<td style="width:149px;">30-0057</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:344px;">Aspirator tube</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:149px;">A5177</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sutter P-97 capillary puller</td>
			<td style="width:195px;">Sutter Instrument</td>
			<td style="width:149px;">P-97</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:344px;">ECM830 Square Wave Electroporator</td>
			<td style="width:195px;">Harvard Apparatus</td>
			<td style="width:149px;">45-0052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:344px;">Platinum Tweezertrodes 7 mm</td>
			<td style="width:195px;">Harvard Apparatus</td>
			<td style="width:149px;">45-0488</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:344px;">Footswitch Model 1250F</td>
			<td style="width:195px;">Harvard Apparatus</td>
			<td style="width:149px;">45-0211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel for electrodes</td>
			<td style="width:195px;">Cefar Compex</td>
			<td style="width:149px;">6602048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane</td>
			<td style="width:195px;">Merial</td>
			<td style="width:149px;">AP/DRUGS/220/96</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome</td>
			<td style="width:195px;">Leica</td>
			<td style="width:149px;">VT1000S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glue</td>
			<td>Roti coll</td>
			<td>Roti coll 1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UltraViEW VoX spinning disk system</td>
			<td style="width:195px;">Perkin Elmer</td>
			<td style="width:149px;"></td>
			<td>Customized setup (multiple laser sources can be used) equipped with Hamamatsu ORCA R2 C10600-10B CCD camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:344px;">Volocity software</td>
			<td style="width:195px;">Perkin Elmer</td>
			<td style="width:149px;"></td>
			<td>Acquisition, Quantitation, Visualization Modules</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:344px;">Environmental chamber for microscopy</td>
			<td style="width:195px;">Solent Scientific</td>
			<td style="width:149px;"></td>
			<td>Custom-made</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:344px;">Ti-E inverted microscope</td>
			<td style="width:195px;">Nikon</td>
			<td style="width:149px;"></td>
			<td>CFI Super Plan Fluor ELWD 20X/0.45 NA objective is recommended for the application described in this paper</td>
		</tr>
	</tbody>
</table>

in vivo postnatal electroporation and time-lapse imaging of neuroblast migration in mouse acute brain slices

脑室下区（SVZ）是在出生后大脑中的主神经利基之一。在这里，神经祖细胞增殖和神经母细胞能够沿着喙迁移流（RMS）对嗅球（OB）的移动引起。这种长距离的迁移需要在OB的新生神经元的后续成熟，但调控这一过程的分子机制仍不清楚。调查信号通路控制神经细胞活动力可能不仅有助于了解神经发生的基本步骤，同时也有治疗再生潜能，给了这些神经母细胞的目标受到损伤，中风或脑退化站点的能力。 在这个手稿，我们描述了在体内产后电穿孔，并在小鼠RMS的成神经细胞迁移的随后的时间推移成像的详细协议。产后电穿孔能有效转染SVZ祖细胞，从而产生神经母细胞沿RMS迁移。利用共聚焦旋转盘时间推移显微镜对急性脑切片培养，成神经细胞迁移可以在非常类似于在体内条件下的环境进行监测。此外，成神经细胞活力可以跟踪和定量分析。作为一个例子，我们描述了如何使用表达GFP的质粒在体内电穿孔产后标记和可视化的神经母细胞沿RMS迁移。在条件性敲除小鼠用人的loxP系统或shRNA的Cre重组酶表达质粒电穿孔也可用于靶向感兴趣的基因。药理操纵急性脑片培养可以进行调查不同的信号分子在神经细胞迁移中的作用。通过耦合在体内电穿孔随着时间推移成像，我们希望了解控制神经细胞活力的分子机制，并有助于开发小说换货方法来促进大脑修复。

的SVZ来源的成神经细胞迁移性标签可以沿着RMS可以观察到，一个成功的电穿孔（ 图1B）之后，通常4-8天。更长的时间点，也可以选择，但较少的细胞会在RMS中找到，因为他们大多会已经进入了转播。成神经细胞开始采集成熟粒细胞中的OB的典型形态学特征和电穿孔后约2-3周（未示出）。 〜1小时培养后，从电穿孔的乳鼠脑切片能够可靠地拍摄长达3-4小时。据此前报道23,26，神经...

Watch this Scientific Journal Video about In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices at JoVE.com

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

成神经细胞迁移是在出生后的神经发生一个根本性的事件。我们描述了一个协议，用于使用急性脑片时间推移成像在产后体内电穿孔和随后的迁移可视化的神经母细胞的有效标记。我们包括用于通过视频跟踪神经细胞动力学的定量分析的描述。

成神经细胞迁移的小鼠急性脑片在体内电穿孔产后和时间推移成像

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Research

JoVE Journal

Neuroscience

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

对于高分辨率延时成像神经细胞迁移的一个器官型产后脑切片检测

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

科研

神经科学

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 ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

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 ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

急性片的成年小鼠前脑的神经细胞迁移的延时成像

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

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

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 ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

老鼠耳蜗外植体的长期时间推移成像

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

产后的小脑颗粒细胞迁移利用共聚焦肉眼离体成像

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

延期共聚焦显像移植神经元在器官性切片培养胚胎小鼠脑使用<em&gt;在Utero</em&gt;电穿孔

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices

微胶质过程对急性脑片 atp 或血清素的双光子成像

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of ...