1。菜的制备<ol><li>用于片文化的菜玻璃底船（作为基地盖玻片）（WillCo菜）。这些都是摆在镜头上组织了6厘米的组织培养皿，保持干净的玻璃底部。 </li><li>在实验前的一天，加2毫升0.1％的聚-L-赖氨酸的解决方案，以平底的玻璃盘在室温下孵育5分钟，让聚-L-赖氨酸涂层的玻璃底部。 </li><li>删除聚-L-赖氨酸的解决方案，并在去离子水冲洗三次，再一次在70％的乙醇。 </li><li>离开干通宵。菜也可以干，轻轻在低功率微波加热30-45秒。 </li></ol> 2。胚胎的电穿孔<ol><li> 37鸡蛋在孵化°C间汉堡 - 哈密尔顿（HH）阶段10（〜36小时）（或其他所需的阶段）。 </li><li>开始之前准备玻璃针（我们使用火焰/布朗模型P87微毛细管拔），打破了尖端的needlE使用在解剖显微镜下细的镊子。针到底应该足够清晰地刺穿，而不是如此狭窄，阻碍DNA溶液注射的胚胎。 </li><li>胚胎两侧的窗口鸡蛋和地方电极（5毫米外） </li><li>成神经管，注入的DNA（0.025 - 0.5微克/μL去离子水与少量的快速绿色着色）。 </li><li>适用于电流 - V THREE 12-17倍，与脉冲之间的950毫秒，50毫秒脉冲长度。 </li><li>我们使用低浓度的DNA和低电电压来实现马赛克，使我们可以按照单个细胞的表达。 </li><li>覆盖在蛋壳的窗口cellotape，并确保它是密封的。 </li><li>允许胚胎为3-4小时或隔夜恢复在37°C。 </li></ol> 3。胶原蛋白和切片培养基制备<ol><li>前准备切片胶原蛋白组合和片约一个小时的培养基。 </li><li>到300微升ţYPE 1胶原蛋白加入100微升0.1％乙酸溶液和100μL5×L15培养基。每次加入后彻底涡。该解决方案应该变黄。 </li><li>现在加入15-20微升7.5％碳酸氢钠，彻底旋涡。解决方案应该成为略带粉色的，这需要碳酸氢钠量可能会有所不同。保持在冰上，并作出新的每一次。 </li><li>到10毫升的Neurobasal培养基中添加的B-27补充和Glutamax的，1×终浓度为10μL庆大霉素溶液。 </li><li>地方在37℃孵化介质缓冲5％的CO 2。离开容器的顶部松动，使其能够平衡的CO 2。 </li></ol> 4。片文化<ol><li>从鸡蛋中取出胚胎和L15培养基洗。 </li><li>在组织培养皿中，在底层和sylgard引脚通过周围的多余胚胎膜出来，使他们都拉直的地方。 </li><li>使用一个麦克风roknife，通过地区的利益，尽可能地直片胚胎。脊髓，切片厚1-2体节之间。离开胚胎切片，当你切其他的胚胎，这样你就不会失去他们。 </li><li>准备切断〜1毫米的小费和附加到P2或P10 200μL微量尖。这篇文章将被用来传输脊髓片玻璃底菜。 10μl的秘诀是不广泛，足以拿起片。 </li><li>大衣内的胶原吸取1μL胶原混合，提前准备的一角。离开1-2分钟，然后用L15培养基冲洗。这将防止从坚持以尖端的内部组织。 </li><li>分离使用microknife的胚胎切片和删除使用装有200μl的尖端，并设置为1μL的P2或P10的菜。尝试以尽可能少的介质。 </li><li>涡胶原蛋白的结构和降5-8μL这对聚-L-赖氨酸涂层菜。 </li><li> immediately到位胚胎切片一双细镊子将胶原蛋白和位置。片的位置应使成像的一面是用盖玻片冲洗。组织应坚持以聚-L-赖氨酸涂层盖玻片。 </li><li>重复此盖玻片上，直到你有几片。我们通常可以把一盘6-9片。 </li><li>一旦所有的片到位，盖菜，让胶原蛋白为20分钟。最早放置的胶原蛋白，有些人可能已经开始干涸。为了防止这种情况添加少量的L15这些前覆盖。 </li><li>一旦设置，小心加入2ml片培养基一直平衡在37°C在5％CO 2，至少一个小时。必须小心，不要接触从盖玻片的胶原蛋白。 </li><li> 1 37°/ 5％CO2培养箱的地方，并允许片恢复至少3个小时前成像。如果是没有湿度，在有机玻璃盒子机智的地方菜公顷的湿纸巾位在一个角落里。 </li></ol> 5。成像胚胎切片<ol><li>我们使用1 DeltaVision核心的宽视场显微镜与环境WeatherStation室安装图像片。室经常保持在37°C，与CO 2灌流装置维持在5％CO 2/95％空气显微镜阶段。 </li><li>通常进行成像采​​用40×/ 1.30 NA油浸镜头和图像捕获与CoolSnap HQ2冷却CCD相机。 </li><li> Z轴部分被捕获每1.5微米至45微米的组织。应保持尽可能低的曝光时间。我们通常会为每个Z-节公开为5-50毫秒。拍摄的图像，每7分钟，并使用该DeltaVision系统的精确点访问功能，可以参观到9片。 </li></ol> 6。代表结果脊髓祖细胞的一个例子时间推移顺序是如图。 2a和相应的电影1。影像开始在为期两天的岁（时时阶段12）胚胎的脊髓切片。这种细胞转染与表达GFPαTubulin结构。在这个早期阶段，神经祖细胞经历主要是在此期间，细胞分裂产生两个进一步的循环内皮祖细胞的祖祖模式师图。 2b和相应的电影2显示了一个细胞转染绿色荧光蛋白，磷脂（GPI锚，GFP）标记细胞膜。这种细胞经过表决，在此期间，基底的过程分成两个子细胞同样继承。 <img alt="图1" src="/files/ftp_upload/3920/3920fig1.jpg" /> 图1。时间推移成像片文化协议。 <img alt="图2" src="/files/ftp_upload/3920/3920fig2.jpg" /> 图2。单元Behavior在正常脊髓开发。（一）选定的帧从电影1。 GFP-微管蛋白表达细胞分裂与切割面是垂直的根尖表面（2小时20分钟），产生两个子细胞再次分裂（24小时23分钟和25小时54分钟）。 （二）从电影2选定的帧。与GFP的GPI转染细胞进行细胞分裂期间，基底过程被分割（白色箭头）和同样由子细胞继承。 电影1。 <a href="http://www.jove.com/files/ftp_upload/3920/3920movie1.mov" target="_blank">点击这里观看电影</a> 。 电影2： <a href="http://www.jove.com/files/ftp_upload/3920/3920movie1.mov" target="_blank">点击这里观看电影</a> 。

<ol>
	<li>Ulloa, F., Briscoe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogens+and+the+Control+of+Cell+Proliferation+and+Patterning+in+the+Spinal+Cord.">Morphogens and the Control of Cell Proliferation and Patterning in the Spinal Cord.</a> Cell Cycle. 6, 2640-2649 (2007).</li><li>Briscoe, J., Novitch, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+pathways+linking+progenitor+patterning,+cell+fates+and+neurogenesis+in+the+ventral+neural+tube.">Regulatory pathways linking progenitor patterning, cell fates and neurogenesis in the ventral neural tube.</a> Philosophical Transactions of the Royal Society B: Biological Sciences. 363, 57-70 (2008).</li><li>Shiau, C., Das, R., Storey, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+effective+assay+for+high+cellular+resolution+time-lapse+imaging+of+sensory+placode+formation+and+morphogenesis.">An effective assay for high cellular resolution time-lapse imaging of sensory placode formation and morphogenesis.</a> BMC Neuroscience. 12, 37 (2011).</li><li>Wilcock, A. C., Swedlow, J. R., Storey, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+spindle+orientation+distinguishes+stem+cell+and+terminal+modes+of+neuron+production+in+the+early+spinal+cord.">Mitotic spindle orientation distinguishes stem cell and terminal modes of neuron production in the early spinal cord.</a> Development. 134, 1943-1954 (2007).</li><li>Vilas-Boas, F., Fior, R., Swedlow, J. R., Storey, K. G., Henrique, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+reporter+of+notch+signalling+indicates+regulated+and+random+notch+activation+during+Vertebrate+neurogenesis.">A novel reporter of notch signalling indicates regulated and random notch activation during Vertebrate neurogenesis.</a> BMC Biology. 9, 58 (2011).</li><li>Herman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+microscopy.">Fluorescence microscopy.</a> Bios Scientific Publishers. , (1998).</li><li>Conchello, J. -. A., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+sectioning+microscopy.">Optical sectioning microscopy.</a> Nat. Meth. 2, 920-931 (2005).</li><li>Pawley, J. B., Pawley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fundamental Limits in Confocal Microscopy Handbook Of Biological Confocal. , (2006).</li><li>Swedlow, J. R., Hu, K., Andrews, P. D., Roos, D. S., Murray, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+tubulin+content+in+Toxoplasma+gondii:+A+comparison+of+laser-scanning+confocal+and+wide-field+fluorescence+microscopy.">Measuring tubulin content in Toxoplasma gondii: A comparison of laser-scanning confocal and wide-field fluorescence microscopy.</a> Proceedings of the National Academy of Sciences. 99, 2014-2019 (2002).</li><li>Murray, J. M., Appleton, P. L., Swedlow, J. R., Waters, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+performance+in+three-dimensional+fluorescence+microscopy.">Evaluating performance in three-dimensional fluorescence microscopy.</a> Journal of Microscopy. 228, 390-405 (2007).</li><li>Biggs, D. S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+Deconvolution+Microscopy.">3D Deconvolution Microscopy.</a> Current Protocols in Cytometry. , (2001).</li><li>Chalfie, M., Tu, Y., Euskirchen, G., Ward, W., Prasher, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+fluorescent+protein+as+a+marker+for+gene+expression.">Green fluorescent protein as a marker for gene expression.</a> Science. 263, 802-805 (1994).</li><li>Wiedenmann, J., Oswald, F., Nienhaus, G. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+proteins+for+live+cell+imaging:+Opportunities,+limitations,+and+challenges.">Fluorescent proteins for live cell imaging: Opportunities, limitations, and challenges.</a> IUBMB Life. 61, 1029-1042 (2009).</li></ol>

没有利益冲突的声明。

我们在座的一种新型的时间推移成像法监测细胞在鸡胚胎切片文化行为。此法可长达70个小时的活组织高分辨率成像，虽然24-48小时之间的时间框架是更容易捕捉。使用高NA石油的目标和时间点之间的相对短的时间间隔，使图像采集分辨率高，以及允许我们监视细胞的行为，常常迅速发生，并可以很容易地错过了在常规时间推移，利用共聚焦成像显微镜。 此法的主要优点是通过长时间的图像细胞的能力。宽视场6 7显微镜激光共焦扫描，而不是使用，这是至关重要的。共聚焦显微镜历来提供了宽视场显微镜的诸多优势，包括采取的光学部分直接消除重点信息的能力<sup&gt; 7，但使用针孔导致亏损的光探测器8，不包括相当数量的信息，需要更长的曝光时间。另一方面，宽视场显微镜，使用全域照明，通过客观的光通过发送的探测器。这具有很高的量子效率的耦合冷却电荷耦合器件（CCD）相机相比，共聚焦显微镜，具有非常快的曝光时间，信噪比高信号，确保成像。在我们的应用程序中，我们必须长时间纪录的3D堆栈和最终解决小近衍射有限结构的图像。虽然共聚焦显微镜防止焦永远到达探测器的光，从而显着降低的背景中厚，密集的标记样品7，8，它只是一个可用的信号噪声比达到更大的光输入比宽10场显微镜。对于非常敏感的光sparsel像我们的电穿孔的脊髓切片Y-标记的样本，因此，宽视场显微镜的性能优于共聚焦显微镜。当由卷积相结合的图像恢复，消除了重点信息，提高对比度，宽领域，然后是特别小，11物昏花的检测。虽然并不适合每个组织成像实验，这种方法已经为我们的活细胞成像中的应用非常有效。 荧光蛋白的选择是一个重要因素，可以影响细胞的存活。我们发现，获得最好的结果是从结构作为标记使用绿色荧光蛋白（GFP）12。目前，我们正在评估一系列的红色荧光蛋白GFP双通道时的失误，可以有效地结合使用。有多种可用这种蛋白质13。虽然许多蛋白质​​稳定与荧光蛋白融合，一些蛋白质可能呈现不稳定的融合，从而导致细胞活力下降。在这种情况下，使用包含一个内部核糖体进入位点（IRES）的结构是非常有用的荧光蛋白分离蛋白。 当成像脊髓切片，必须小心，以尽量减少暴露于荧光灯。必须使用显微镜目镜透射光（明）片定位和位置。荧光图像应始终获得使用显微镜软件，使用最小的曝光时间。这些应保持在5-50毫秒范围内，应尝试使用中性密度过滤器。虽然更长的曝光时间，最初可能会产生更清晰的图像，荧光灯光线照射的光毒作用，可能导致细胞死亡。第一5-10组织最接近盖玻片微米的不应成像作为本地区包含组织可能已损坏切片。 虽然这里给出的例子是在农户阶段10和影像6-7小时后电穿孔胚胎，这种方法可用于对胚胎HH阶段18。在稍后阶段，脊髓组织大得多，因此很难通过手工切片。在这些情况下，嵌入在低熔点琼脂糖胚胎与vibratome切片可能会产生更好的效果。即使我们提出此法作为一个发展中的脊髓成像细胞的方法，这种方法也被修改图像等胚胎组织，包括感官placodes 3。

<table border="1"><tbody><tr><td> 试剂 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> WillCo美食</td><td> WillCo井</td><td> GWst-3522 </td><td></td></tr><tr><td>聚-L-赖氨酸</td><td>西格玛</td><td> P8970 </td><td>在Tris 0.1％ </td></tr><tr><td>硼硅玻璃毛细管</td><td>哈佛仪器</td><td> GC100-10 </td><td> 1.0毫米外直径， 0.58毫米内直径</td></tr><tr><td>电极（Genetrodes， 大毛，L形</td><td> A型肉毒毒素</td><td> 10-001850-01 </td><td> 5毫米镀金</td></tr><tr><td>流脑830平方脉冲发生器</td><td> A型肉毒毒素</td><td> 45-0002 </td><td> 在OVO electroopration， </td></tr><tr><td>快绿</td><td>西格玛</td><td> F7258 </td><td></td></tr><tr><td> I型大鼠胶原</td><td>西格玛</td><td> C3857 </td><td></td></tr><tr><td> L-15的介质（粉） </td><td>西格玛</td><td></td><td> 2.76克/ l在蒸馏水 5倍的解决方案</td></tr><tr><td>碳酸氢钠</td><td>西格玛</td><td> S8761 </td><td> 7.5％的解决方案</td></tr><tr><td>的Neurobasal培养基</td><td> Invitrogen公司</td><td> 12348-017 </td><td>无酚红</td></tr><tr><td> glutamax </td><td> Invitrogen公司</td><td> A1286001 </td><td> 100X的解决方案</td></tr><tr><td>的B-27的补充</td><td> Invitrogen公司</td><td> 17504-044 </td><td></td></tr><tr><td>庆大霉素</td><td> Invitrogen公司</td><td> 15710049 </td><td> 10毫克/毫升</td></tr><tr><td> L15培养基（液体1X） </td><td> Invitrogen公司</td><td> 11415-049 </td><td></td></tr><tr><td> microknife </td><td> Altomed </td><td> A10102 </tD&gt; <td> 15度倾斜</td></tr><tr><td>不锈钢无头管脚</td><td>沃特金斯和唐卡斯特</td><td> E6871 </td><td> A1尺寸</td></tr><tr><td> sylgard 184弹性体</td><td>厂商VWR </td><td> 634165S </td><td></td></tr><tr><td> DeltaVision核心显微镜系统</td><td>应用精密</td><td></td><td></td></tr><tr><td> coolsnap HQ2 CCD相机</td><td> photometrics </td><td></td><td></td></tr></tbody></table>

high-resolution live imaging of cell behavior in the developing neuroepithelium

胚胎脊髓由骑自行车引起的中枢神经系统（CNS）的神经元和神经胶质细胞的很大比例的神经祖细胞。虽然被称为格局脊髓的分子机制，并引起神经细胞的分化1，2，我们还缺乏深刻的理解了这些细胞的行为水平的早期事件。为他们进行神经再生研究实时的神经祖细胞的行为，因此，这是关键。 在过去，实时成像早期胚胎组织细胞/组织的可行性以及在文化荧光成像的光毒作用受到限制。在这里，我们提出一种新的分析，长时间成像等组织利用一种新型的体外片文化协议和宽视场荧光显微镜（ 图1）。这种方法实现长期鸡胚S的时间推移监测高时空分辨率的的皮纳尔线与祖细胞。 此法可修改一系列胚胎组织3，4，除了观察细胞和亚细胞的行为，发展新型基因活性和高度敏感的记者形象（例如，Notch信号5）使此法了解如何信令与强大的工具，调节胚胎发育期间细胞的行为。

Watch this Scientific Journal Video about High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium at JoVE.com

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

实时成像胚胎组织挑战过很长一段时间。在这里，我们提出了一个小鸡脊髓细胞和亚细胞的变化，具有高时空分辨率的长期监测分析。这种技术可以适应其他地区的神经系统和胚胎发育。

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 ...

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JoVE Journal

Neuroscience

13.2K 次观看.  University of Dundee, Dundee, UK. 实时成像胚胎组织挑战过很长一段时间。在这里，我们提出了一个小鸡脊髓细胞和亚细胞的变化，具有高时空分辨率的长期监测分析。这种技术可以适应其他地区的神经系统和胚胎发育。

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

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

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

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 ...

科研

神经科学

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

高分辨率人类中脑功能磁共振成像方法

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 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 ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

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 ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

高分辨率<em&gt;在体内</em&gt;使用3T磁共振成像手动分割协议为人类海马子字段

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

一个程序，植入脊柱商会纵<em&gt;在体内</em&gt;鼠标脊髓成像

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

尼古丁诱导的钙信号和神经递质的释放了沿着腹侧海马轴突实时成像

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

高分辨率结构磁共振人类皮质下的成像<I&gt;在体内</I&gt;与死亡

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.
To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.
Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal&#180;s dynamic behavioral reaction.

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

The automated tracking system Flywalk is used for high-resolution quantification of odor-guided behavior in Drosophila melanogaster.

在气味引导行为的高分辨率定量<em&gt;果蝇</em&gt;使用<em&gt; Flywalk</em&gt;范式

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

高分辨率定量免疫膜受体在视网膜带状突触分析

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...