谢谢孙威利对技术援助和克里斯Gellynck博士有益的意见和三个匿名评审的建设性意见。这项工作是由美国的新宁听力重建倡议

小鼠组织从Sox2的EGFP收获-reporter维护和安乐死按照中国的护理和使用实验动物的动物护理准则加拿大议会小鼠12。 1.培养胚胎耳蜗<ol><li>通过混合8.89毫升DMEM中，加入1ml的胎牛血清（FBS），100微升100×N2补充，和10μl的10mg / ml的环丙沙星补充的Dulbecco改良的Eagle培养基（DMEM）。通过混合495毫升的HBSS，用5毫升100％的HEPES补充Hank氏平衡盐溶液（HBSS）中。 </li><li>准备玻璃底培养皿： <ol><li>在层流罩，悬浮200μl的基底膜提取物衬底的5ml DMEM中。准备直径35 mm的玻璃底培养皿10毫米井。吸取150微升底物/ DMEM在每个玻璃中央见底菜。这些菜可以使用后40分钟温育，或可以存储在CO 2 </sub&gt;培养箱中在35℃下进行至少一周的时间。 注：玻璃有底菜肴用于下列原因：以确保盘的底部是透明的并且适于成像，以良好的，其允许外植体，以解决在一个容易找到区和最后的盘的中心创建一个使一个试验后的样品可用于免疫染色及随后的分析处理。 </li></ol></li><li>准备工作站和工具： <ol><li>打开层流洁净工作台，并喷洒下来用70％乙醇，以创造一个干净的工作空间。浸泡镊子，勺子，和一个黑色184硅酮弹性体培养皿（184硅酮弹性体（10份）混合，固化剂（1份）和木炭粉（2.5克））在70％EtOH中。 </li></ol></li><li>在洁净工作台，收获胚胎的实验。收集在冰适当妊娠冷HBSS中补充有1％HEPES的胚胎。 <ol><li>确定外部或可见机关将展示用荧光立体显微镜报告基因活性并检查胚胎。收集表现出报告基因活性在冰冷的HBSS中补充有1％HEPES，因为这些是在实验的受试者的胚胎。 </li></ol></li><li>收集颞骨： <ol><li>工作迅速，使用冷色光源和细镊子，收集幼崽的头。照顾到夹在颈椎和下颚下方，以避免损坏的颞骨。 </li><li>小心地打开头盖骨。取出脑，修剪过的头部的前部，并且所述后颅骨转移到新鲜的冰冷的HBSS在干净的培养皿补充有1％HEPES。仔细剖析出花生形颞骨注意保持前庭系统的机智。 </li></ol></li><li>解剖耳蜗： <ol><li>传送颞骨到在冰黑硅弹性体涂覆的培养皿冷HBSS中补充有1％HEPES，针骨的前庭区域。插入昆虫针在的倾斜角，以稳定的颞骨和创建​​房间的钳子。钉扎是在这个过程中的一个关键步骤，因为如果颞骨被允许移动至太大，很难收获完整的耳蜗。 </li><li>小心取出围绕耳蜗的软骨。插入钳子的一个齿进入软骨在耳蜗的基底的外边缘和夹子的孔进入软骨。 </li><li>夹的翼片向上侧，插入钳子的齿和轻轻分开从软骨管道的屋顶。剪辑水平穿过顶部和倾斜，并小心地取下胶囊的前部。插入镊子的一个叉头中剩余的软骨与管道之间并轻轻夹关闭最后一节。耳蜗的顶端面现露出。 </li><li>开始在基，抓其中所述导管的屋顶满足耳蜗上皮面积和打开耳蜗管。轻轻撕下屋顶，修剪必要时，直到它被完全除去。剪掉膜左内侧上的导管中的任何部分。清理多余的间叶组织的管道，并从分离的前庭系统的风管。 </li></ol></li><li>文化的外植体： <ol><li>将外植体，腔表面了，在一个基板镀膜玻璃底培养皿的中心，精心编制了所有的液体，留下了两分钟。加入150微升补充DMEM落明智的外植体，注意不要打扰它。应的外植体自由漂浮，定位钳，但要小心，外植体沉淀到培养皿的底部，使得它可以连接到基板上。 </li><li>将玻璃底菜在深直径12cm的培养皿中，用一小碟的无菌水来保持湿度。将培养物在一个35℃的培养箱中过夜，以使外植体附着到基片和压平。 </li></ol></li></ol> 2.现场的ImagING <ol><li>选择植样本。用荧光立体显微镜来评估每个外植耳蜗的条件。只有选择的外植体，其中所述管道是完好并附着在玻璃从基部到顶点。 </li><li>设置培养箱中于35℃，5％CO 2下培养耳蜗组织。把文化进入孵化器的显微镜： <ol><li>轻轻吸过的DMEM补充和替换，至少500微升新鲜培养基。用于成像长达6天，1-1.5毫升为好。在其中植体的连接不牢固的情况下，吸取的媒体的环周围的盘的边缘。这个额外的液体将一个微型“加湿室&#39;而不干扰外植体，而它仍然附着于基质。 <ol><li>可选地，使用铰接盘覆盖，打开盖子，而盘停留在其在显微镜嵌合如果试剂需要在图像集合之间的时间间隔交换。铰链盖菜让盖被开不干扰样品或从它们的设置去除菜肴。这样可保持其对后续的图像捕捉精确位置。 </li></ol></li><li>将包含相应的样品放入样品盘架的玻璃底菜。在本实施例的显微镜具有旋转平台，保持835毫米样品菜肴。 </li><li>在层流罩更换塑料盖用玻璃盖并插入餐具放入样品盘支架。将样品盘支架培养箱照顾内不从培养皿的底部去除所述外植体或扰乱介质。 </li></ol></li><li>成立了成像程序： <ol><li>交换机上的显微镜，紫外灯和摄像头，打开图像处理软件。 </li><li>找到样品，挑的成像区域，重点平面和调整曝光时间顺序每道菜。选择焦点不仅取决于外植体在起动时的视图的平面，但人所以铭记如何组织将移动多久的时间过程会运行。 </li><li>设置为Z堆栈围绕所选焦平面定心在荧光通道。设置采样进行实验的次数和时间长度。采样的频率也由它需要采集图像的时间是有限的，所以这应该在选择视场和设置为Z堆叠后确定。在这种情况下，需要采样的地方，每隔30分钟，超过五天。在实验的持续时间可长达14天。 </li></ol></li><li>生成的电影： <ol><li>在时间流逝期结束打开的图像文件。打开连续采集点在这种情况下的Metamorph软件使用。逐帧选出最佳焦平面为示出感兴趣的细胞群。 </li><li>这些图像转换为.avi文件，或者将其导出为一蒙太奇以生成一组可被打开并分析在图像处理软件或转换为多个预估图像利用视频处理软件TS。 </li></ol></li></ol>

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作者什么都没有透露。

有几种技术要点当培养物被建立并在成立的时间推移显微镜为了长期成像来考虑。 我们用基底膜基质作为基质培养耳蜗植体，但在衬底应匹配于细胞类型。例如，将图像的神经元培养物，它可能是更好的是提供一种纤连蛋白涂层。温育温度和气体组成也应当根据组织类型选择。选择外植体的年龄是很重要的。我们开始对E13在最早因为对E12的生长方向是难以预料。因为它是必?...

哺乳动物耳蜗是高度有序的复杂器官。在小鼠中，从上一天E11耳泡和发育程序完成后，将原始内耳的出现在出生后早期阶段之间，细胞信号转导的多种波和协调的基因表达的变化发生。从基本运行到顶点的蜗管的是声音探测感觉上皮尔蒂的，或器官。柯蒂氏器的发展精美控制，使得通过显影结束时，其将包括内毛细胞的单排，三排外毛细胞的散布有五排支持细胞（两列支柱细胞，三行Deiters&#39;细胞）1。偏离这个精确的顺序会导致听力下降，heighlighting studye的国税发起源和感官epithemlium 2的图案化的重要性。 在胚胎小鼠COCH的体外培养LEA是研究Corti器的发展机制的一个重要工具。该技术成立于1974年，并在过去的40年中，一直被用来阐明许多由感官上皮规定和Corti器建立3的机制。耳蜗是一个复杂的器官的动态发育过程;一个操作可能需要长达7天的表现1。例如，添加GSK3β抑制剂在E13耳蜗植体培养时，最佳的潜伏期，观察该化合物的一个健壮效果为6天4。 显影耳蜗的实时成像可以调查变化的柯蒂氏器的形态，基因表达的变化，跟踪迁移的，增殖或死亡的细胞，并允许实时观察药剂的结果的和信号的中断途径。到现在为止，住耳蜗的摄像已经主要使用共聚焦显微镜对柯蒂氏在短时间内5-8的脏器图像的小区域进行的，但该技术具有由于外植体存活的局限性。在较长期的操作慢发育过程的影响成像，成像环境是至关重要的。典型的共焦实时成像系统采用的加湿塑料箱，它位于所述的显微镜。热和湿度，可以通过在孵育箱那里它满足显微镜台，通过接入窗，通过铰链孔，并通过围绕microscope-各部分的间隙的间隙逸出，如目标或光源。这不是最优的用于维持健康植超过两三天。 我们定义“孵化显微镜&#39;为倒置显微镜内部的标准的CO 2培养箱中密封，而不是在显微镜周围建造一个孵化器。孵化器显微镜前趋于实验的寿命，使得而非成像在两三天，样品可以被成像长达两周。培养箱显微镜提供用于细胞生长和分化良好的环境，以最小的干扰外植体培养物和标准控制的条件。在研究，发生在多天里，通常通过从培养箱中取出，并携带它们到一个倒置荧光显微镜诉诸成像样品，每天。虽然这种方法可以工作，从培养箱中取出盘子对其造成的敏感发展组织应力。由于除去从孵化器中的更改培养用培养基和波动的温度的酸度可导致次优的发展和不健康的组织。成象的区域在同一焦平面上，并在相同的取向，在每一个时间点是非常有挑战性的。通过利用恒温箱中的自动化系统，它能够维持健康组织，采集图像，在多个时间点，并确保在同一区域被捕获在每一帧。近年来几个集成显微镜组织培养箱被开发出来，这些不仅在临床实践9，而且在干细胞和癌症研究10,11是有用的。 在这里，我们提出了一个协议，用于胚胎小鼠耳蜗外植体的长期实时成像。我们使用自动显微镜系统内部的标准的CO 2培养箱具有捕获多个样品的图像在设定的时间点的能力。该系统由一个倒置显微镜孵化器内设定的。样品被放置在一个旋转的讲台，允许多个样品的成像在每个时间点。照明，图像采集和讲台的旋转是通过的Metamorph软件操作的自动化系统控制。通过操作软件设置的成像程序，我们可以设置一个实验，运行长达TWØ周以最少的人工干预。在这个例子中我们使用两个亮场和荧光显示大规模增长和耳蜗的重排，并且具体地，prosensory区域。在该实验中，耳蜗将从Sox2的EGFP报告小鼠解剖胚胎天E13。 在体外培养物将被建立，然后成像五天。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle media</td>
			<td>Gibco</td>
			<td>12430</td>
			<td>Multiple brands manufacture this</td>
			<td>http://www.lifetechnologies.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/classical-media/dmem.html</td>
		</tr>
		<tr>
			<td>Basement membrane extract&#160;</td>
			<td>Corning</td>
			<td>354230</td>
			<td>Matrigel. Alternative similar products are available from other suppliers. 
			http://catalog2.corning.com/Lifesciences/en-US/Shopping/Product.aspx?categoryname=Cell+Culture+and+Bioprocess%28Lifesciences%29|Extracellular+Matrix+Proteins+ECMs+and+Attachment+Factors%28Lifesciences%29|Matrigel+Basement+Membrane+Matrix+%28Lifesciences%29</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>16000044</td>
			<td>Multiple brands manufacture this 
			http://www.lifetechnologies.com/search/global/searchAction.action?query=fbs&#38;resultPage=1&#38;results PerPage=15&#38;autocomplete=</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>5630080</td>
			<td>Multiple brands manufacture this 
			http://www.lifetechnologies.com/search/global/searchAction.action?query=hepes&#38;resultPage=1&#38;results PerPage=15&#38;autocomplete=</td>
		</tr>
		<tr>
			<td>100 x N2 supplement</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td>Multiple brands manufacture this 
			http://www.lifetechnologies.com/search/global/searchAction.action?query=n2&#38;resultPage=1&#38;results PerPage=15&#38;autocomplete=</td>
		</tr>
		<tr>
			<td>Ciprofloxacin</td>
			<td>Sigma Aldrich</td>
			<td>17850-5G-F</td>
			<td>Multiple brands manufacture this 
			http://www.sigmaaldrich.com/catalog/product/fluka/17850?lang=en&#38;region=CA</td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution</td>
			<td>Gibco</td>
			<td>14170161</td>
			<td>Multiple brands manufacture this. Should be refidgerated before use. 
			http://www.lifetechnologies.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/reagents/balanced-salt-solutions/hbss-hanks-balanced-salt-solution.html?s_kwcid=AL!3652!3!26107410508!e!!g!!hbss&#38;ef_id=xoFOglw2s UMAAMU8:20140228185720:s</td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>Fine Science tools</td>
			<td>11254-20</td>
			<td>Size number 5 
			http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=350&#38;CategoryId=29</td>
		</tr>
		<tr>
			<td>Curette&#160;</td>
			<td>Fine Science Tools</td>
			<td>10080-05</td>
			<td>size 1 mm&#160; 
			http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=91&#38;CategoryId=118</td>
		</tr>
		<tr>
			<td>Insect Pins</td>
			<td>Fine Science Tools</td>
			<td>26001-35</td>
			<td>Must be stainless Steel 
			http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=124</td>
		</tr>
		<tr>
			<td>50 mm plastic dishes</td>
			<td>Corning/Falcon</td>
			<td>351006</td>
			<td>Multiple brands manufacture this</td>
		</tr>
		<tr>
			<td>charcoal</td>
			<td>Sigma Aldrich</td>
			<td>05105-250G</td>
			<td>Multiple brands manufacture similar items 
			http://www.sigmaaldrich.com/catalog/product/fluka/05105?lang=en&#38;region=CA</td>
		</tr>
		<tr>
			<td>184 silicone elastomer</td>
			<td>Dow/Corning&#160;</td>
			<td>SYLGARD&#174; 184 SILICONE ELASTOMER KIT</td>
			<td>Dishes are home made several weeks in advance.&#160; Silicone elastomer can be from any supplier.<a href="http://www.dowcorning.com/applications/search/products/Details.aspx?prod=01064291">http://www.dowcorning.com/applications/search/products/Details.aspx?prod=01064291</a></td>
		</tr>
		<tr>
			<td>Glass bottom dishes</td>
			<td>MatTek</td>
			<td>P35G-0-10-C</td>
			<td>The dimensions of the dish are determined by the specifications of the imaging system.&#160; 35 mm diameter, 10mm well, number 0 coverslip fits Olympus Vivaview FL. 
			http://glass-bottom-dishes.com/catalog/index.php?main_page=product_info&#38;cPath= 1_4_15&#38;products_id=2</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Zeiss&#160;</td>
			<td>495101-9804-000</td>
			<td>&#160;Stemi 2000 model. multiple brands manufacture similar items 
			http://microscopy.zeiss.com/microscopy/en_de/products/stereo-zoom-microscopes/stemi-2000.html</td>
		</tr>
		<tr>
			<td>Cold Light Source</td>
			<td>Zeiss</td>
			<td>000000-1063-182</td>
			<td>Multiple brands manufacture similar items 
			http://microscopy.zeiss.com/microscopy/en_de/products/microscope-components/lightsources.html</td>
		</tr>
		<tr>
			<td>Fluorescent Stereomicroscope&#160;</td>
			<td>Leica microsystems</td>
			<td>Contact Leica microsystems</td>
			<td>Leica M165-FC. Multiple brands manufacture similar items 
			http://www.leica-microsystems.com/products/stereo-microscopes-macroscopes/fluorescence/details/product/leica-m165-fc/</td>
		</tr>
		<tr>
			<td>Incubator Microscope +imaging software</td>
			<td>Olympus</td>
			<td>Contact Olympus</td>
			<td>Inverted microcope sealed inside a Co2 incubator. Vivaview FL incubator microscope with proprietry Metamorpoh imaging software. 
			http://olympuscanada.com/seg_section/product.asp?product=1055&#38;c=0</td>
		</tr>
		<tr>
			<td>Clean bench</td>
			<td>Thermo Scientific</td>
			<td>51029701</td>
			<td>Multiple brands manufacture similar items 
			http://www.thermoscientific.com/en/product/heraguard-eco-clean-bench.html</td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo Scientific</td>
			<td>3310</td>
			<td>Multiple brands manufacture similar items 
			http://www.thermoscientific.com/en/products/co2-incubators.html</td>
		</tr>
		<tr>
			<td>Laminar Flow hood</td>
			<td>Thermo Scientific</td>
			<td>51026651</td>
			<td>Multiple brands manufacture similar items 
			http://www.thermoscientific.com/en/products/biological-safety-cabinets-clean-benches.html</td>
		</tr>
	</tbody>
</table>

long-term time lapse imaging of mouse cochlear explants

在这里，我们提出了活鼠胚胎的耳蜗植长期时间推移成像的方法。负责建设的哺乳动物耳蜗收益的高度有序的，复杂的结构，大约十天后的发展计划。为了研究在此期间基因表达的变化和它们的响应的药物或基因操作，长期成像是必要的。先前，实时成像已经典型地通过移植组织的存活，在湿润的腔室顶上标准显微镜的限制。难以保持对培养物生长有关的湿度和温度的最佳条件已经放置在成像实验的长度的限制。显微镜集成到修改后的组织培养孵化器提供长期，实时成像的良好环境。在这个方法中，我们演示了如何建立胚胎小鼠耳蜗植，以及如何使用孵化器的显微镜来进行时间的推移意马使用毫微都亮场和荧光显微镜检查的典型胚胎天（E） - 13耳蜗植体和Sox2，耳蜗的prosensory细胞的标记，在5天的行为。

在这里，我们将展示一个蒙太奇（ 图1），看电影（ 图2），这说明一个典型的器官耳蜗植将如何发展，如果镀上E13.5。一个Sox2的记者鼠标采用可视化的prosensory区域。动画示出了耳蜗经历生长和收敛性和扩展，使细胞中的绿色Sox2的域的横向区域似乎没有划分为围绕它展开的组织。这是Corti器的特性;关于E13准感觉上皮退出细胞周期和随后称为防扩散13?...

Watch this Scientific Journal Video about Long-term Time Lapse Imaging of Mouse Cochlear Explants at JoVE.com

Long-term Time Lapse Imaging of Mouse Cochlear Explants

胚胎哺乳动物耳蜗的实时成像是具有挑战性的，因为手头的发育过程超过十天时间梯度进行操作。在这里，我们提出了培养，然后从成像荧光记者鼠标采取了五天的胚胎耳蜗植体组织的方法。

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 ...

long-term-time-lapse-imaging-of-mouse-cochlear-explants

Research

JoVE Journal

Neuroscience

9.5K 次观看.  Sunnybrook Research Institute. 胚胎哺乳动物耳蜗的实时成像是具有挑战性的，因为手头的发育过程超过十天时间梯度进行操作。在这里，我们提出了培养，然后从成像荧光记者鼠标采取了五天的胚胎耳蜗植体组织的方法。 

视频: Long-term Time Lapse Imaging of Mouse Cochlear Explants

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

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 ...

科研

神经科学

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

抛光和钢筋，颅骨变薄窗口，为长期的小鼠脑成像

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

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

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

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 ...

Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish

The nervous system is often described as a hard-wired component of the body even though it is a considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity. Unlike the central nervous system (CNS), the peripheral nervous system (PNS) is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that govern this phenomenon. Using zebrafish as a model system, we have the unprecedented opportunity to couple regenerative studies with in vivo imaging and genetic manipulation. Peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. Axons are ensheathed by myelinating or non-myelinating Schwann cells, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. Following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris and re-innervate targets. To investigate the roles of all peripheral glia in PNS regeneration, we describe here an axon transection assay that uses a commercially available nitrogen-pumped dye laser to axotomize motor nerves in live transgenic zebrafish. We further describe the methods to couple these experiments to time-lapse imaging of injured and control nerves. This experimental paradigm can be used to not only assess the role that glia play in nerve regeneration, but can also be the platform for elucidating the molecular mechanisms that govern nervous system repair.

Although the peripheral nervous system (PNS) is capable of significant repair after injury, little is known about the cellular and molecular mechanisms that govern this phenomenon. Using live, transgenic zebrafish and a reproducible nerve transection assay, we can study dynamic glial cell behaviors during nerve degeneration and regeneration.

电机神经切断和时间推移成像胶质细胞行为在现场斑马鱼

The  nervous system is often described as a hard-wired component of the body even  though it is a considerably fluid organ system that reacts to external ...

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

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

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

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

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 ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

琼脂糖微室的长期钙成像<em&gt;秀丽隐杆线虫</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

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

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 has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

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

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

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 ...

Long-term Sensory Conflict in Freely Behaving Mice

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning in mice. By permanently wearing a device fixed on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages. Therefore, this protocol readily enables the study of the visual system and multisensory interactions over an extended timeframe that would not be accessible otherwise. In addition to lowering the experimental costs of long-term sensory learning in naturally behaving mice, this approach accommodates the combination of in vivo and in vitro experiments. In the reported example, video-oculography is performed to quantify the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) before and after learning. Mice exposed to this long-term sensory conflict between visual and vestibular inputs presented a strong VOR gain decrease but exhibited few OKR changes. Detailed steps of device assembly, animal care, and reflex measurements are hereby reported.

The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages.

自由生育小鼠的长期感觉冲突

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for ...

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding environment and initiate contacts with potential synaptic partners. Although the connection between dendritic branch dynamics and synaptogenesis is well established, how developmental and activity-dependent processes regulate dendritic branch dynamics is not well understood. This is partly due to the technical difficulties associated with the live imaging and quantitative analyses of these fine structures using an in vivo system. We established a method to study dendrite dynamics using Drosophila larval ventral lateral neurons (LNvs), which can be individually labeled using genetic approaches and are accessible for live imaging. Taking advantage of this system, we developed protocols to capture branch dynamics of the whole dendritic arbor of a single labeled LNv through time-lapse live imaging. We then performed post-processing to improve image quality through drift correction and deconvolution, followed by analyzing branch dynamics at the single-branch level by annotating spatial positions of all branch terminals. Lastly, we developed R scripts (Supplementary File) and specific parameters to quantify branch dynamics using the coordinate information generated by the terminal tracing. Collectively, this protocol allows us to achieve a detailed quantitative description of branch dynamics of the neuronal dendritic arbor with high temporal and spatial resolution. The methods we developed are generally applicable to sparsely labeled neurons in both in vitro and in vivo conditions.

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Here, we describe the method we employed to image highly motile dendritic filopodia in a live preparation of the Drosophila larval brain, and the protocol we developed to quantify time-lapse 3D imaging datasets for quantitative assessments of dendrite dynamics in developing neurons.

发展果蝇神经元的快速丹德林分体动力学的延时实时成像和定量

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding ...

Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of the mouse neuroretina&#160;with intact retinal pigment epithelium (RPE). Depending on the research question, retinas can be isolated from wild-type animals or from disease models, to study, for instance, diabetic retinopathy or hereditary retinal degeneration. Eyes from early postnatal day 2&#8211;9 animals are enucleated under aseptic conditions. They are partially digested in proteinase K to allow for a detachment of the choroid from the RPE. Under the stereoscope, a small incision is made in the cornea creating two edges from where the choroid and sclera can be gently peeled off from the RPE and neuroretina. The lens is then removed, and the eyecup is cut in four points to give it a four-wedged shape resembling a clover leaf. The tissue is finally transferred in a hanging drop into a cell culture insert holding a polycarbonate culturing membrane. The cultures are then maintained in R16 medium, without serum or antibiotics, under entirely defined conditions, with a medium change every second day.
The procedure described enables the isolation of the retina and the preservation of its normal physiological and histotypic context for culturing periods of at least 2 weeks. These features make organotypic retinal explant cultures an excellent model with high predictive value, for studies into retinal development, disease mechanisms, and electrophysiology, while also enabling pharmacological screening.

The protocol describes organotypic explants of mouse neuroretina, cultivated together with its retinal pigment epithelium (RPE), in R16 defined medium, free of serum and antibiotics. This method is relatively simple to perform, less expensive, and time-consuming when compared to in vivo experiments, and can be adapted to numerous experimental applications.

长期，无血清培养有机小鼠视网膜外植与完整的视网膜色素上皮

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of ...