Name: analyzing dendritic morphology in columns and layers
Uploaded: 2017-03-23T15:00:00
Description: Watch this Scientific Journal Video about Analyzing Dendritic Morphology in Columns and Layers at JoVE.com

这项工作是由美国国立卫生研究院院内研究计划的支持，儿童健康和人类发展（授予HD008913到C-HL）的尤尼斯·肯尼迪·施莱佛国立研究所和信息技术中心（PGM，NP，ESM ，和MM）。

注意：该协议包含三个部分：双视图成像（部分1 - 3），树突跟踪和登记（第4 - 6），和树突分析（第7 - 9）（ 图1）。在材料/设备的表中提供的代码和示例文件。 1.双图像采集注意：此步骤是为了获得所关注的神经元的两个图像栈在两个正交（水平和额叶）取向。 <ol><li>制备该含有疏标记髓质神经元飞大脑（〜10个细胞/脑叶）?...

<ol>
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Y., 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=Tiling+of+R7+axons+in+the+Drosophila+visual+system+is+mediated+both+by+transduction+of+an+activin+signal+to+the+nucleus+and+by+mutual+repulsion.">Tiling of R7 axons in the Drosophila visual system is mediated both by transduction of an activin signal to the nucleus and by mutual repulsion.</a> Neuron. 56 (5), 793-806 (2007).</li><li>Peng, H., Ruan, Z., Long, F., Simpson, J. H., Myers, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=V3D+enables+real-time+3D+visualization+and+quantitative+analysis+of+large-scale+biological+image+data+sets.">V3D enables real-time 3D visualization and quantitative analysis of large-scale biological image data sets.</a> Nat Biotechnol. 28 (4), 348-353 (2010).</li><li>Takemura, S. Y., Lu, Z., Meinertzhagen, I. 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在这里，我们将展示如何图像和分析果蝇神经髓质树突乔木。第一部分，双视图成像，描述了去卷积和两个图像栈的组合成一个高分辨率的图像栈。第二部分，枝晶追踪和登记，介绍髓质神经元的参考列数组的树突的追查​​和登记。第三部分，树突状分析，介绍了使用自定义脚本来分析树突状图案。总之这些协议提供了一个完整的工作流中提取树突状模式的信息相对于图层和列，并确定?...

在开发过程中，神经元在复杂的，但千篇一律的分支图案精致的树突形成与突触前伙伴突触。树突分枝模式相关与神经元的身份和功能。树突乔木的位置确定的，他们收到的突触前输入的类型，而树枝状分枝的复杂性和字段大小支配输入号码。因此，树突形态属性是突触连接和神经元计算的关键因素。在复杂的大脑，诸如苍蝇视叶和脊椎动物视网膜许多地区，突触电路中的列和层组织，以促进信息处理1,2。在这样的列和层的组织，一个独特的形态项目轴突突触前神经元在一个特定层（所谓的特定层的定位）终止，并形成一个有序的两维阵列（所谓的卡勒ð地形图），而突触后神经元延长特定层适当大小的树突接收到正确的类型和数量的突触前输入。而轴突靶向层和列已经得到很好的研究3，4，较少被知道关于树突如何路由到特定层和扩大适当大小感受域，以形成具有正确的突触前伙伴5突触连接。成像和量化树突靶向层和列的困难阻碍了柱状和夹层的大脑结构树突发育的研究。 果蝇神经髓质是研究在列和树突状层路由和电路组件的理想模型。苍蝇髓质神经纤维组织为10层的3D格与约750列。每列由一组传入，的p支配hotoreceptors R7 / R 8和薄层神经元L1 - L5，其终末形成层特定的方式6的地形图。约38种髓质的神经元存在于每髓质列和特定层和具有适当字段大小精细树突从这些传入7接收输入。在延髓的突触电路，在电子显微镜水平得到重建;因此，突触的伙伴关系已经非常成熟7,8。此外，用于标记各种类型髓质神经元的遗传工具可用9，10，11。通过检查三种transmedulla（Tm）为神经元（TM2，TM9和TM20），我们先前已经确定两种细胞类型特异性树突属性：（一）以旧换新神经元投射无论是在向前或向后方向树突（平面PROJ挠度方向），这取决于细胞类型和（ii）延髓神经元的树突终止于特定髓质层中的细胞类型特异性的方式（层特定的终端）12。平面投影方向和特定层的终止足以区分这三种类型的Tm的神经元，而扰乱的Tm反应层和列线索突变影响这些属性的不同的方面。 这里，我们提出用于检查果蝇髓质神经元的列和层（ 图1）的树枝状图案形成一个完整的工作流程。首先，我们展示了双景成像方法，它采用定制的软件合并两个共焦图像叠加产生高品质的图像各向同性。此方法只需要常规共焦显微镜，以产生高质量的图像，其允许树枝状分枝的可靠跟踪，而不诉诸超分辨率显微镜，这样的小号STED（受激发射损耗）或结构照明。第二，我们提出了跟踪树突乔木和用于登记所得突起痕迹到参考列阵列的方法。第三，我们显示了用于推导估计树枝状分支和终止频率提取的平面投影方向和树突的特定层的终端信息，以及作为计算方法。总之，这些方法允许在3D，基于树枝状形态的细胞类型的分类树枝状型态的表征，和潜在缺陷的突变体的鉴定。

<table border="0" cellpadding="0" cellspacing="0" width="1632">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Software</td>
			<td style="width:301px;"></td>
			<td style="width:227px;"></td>
			<td style="width:640px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Huygens Professional&#160;</td>
			<td>Scientific Volume Imaging</td>
			<td>version 16.05</td>
			<td>for image deconvolution (https://svi.nl).&#160; commercial software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MIPAV</td>
			<td></td>
			<td>version 7.3.0</td>
			<td>for image recombination and registration (http://mipav.cit.nih.gov/.).&#160; freeware</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MIPAV plugin: PlugInDrosophilaRetinalRegistration.class</td>
			<td></td>
			<td></td>
			<td>freeware</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MIPAV plugin: PlugInDrosophilaStandardColumnRegistration.class</td>
			<td></td>
			<td></td>
			<td>freeware</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaris</td>
			<td>Bitplane</td>
			<td></td>
			<td>for tracing neurites and assigning reference points for image registration (http://www.bitplane.com). commercial software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vaa3D</td>
			<td></td>
			<td></td>
			<td>for visualizing swc files (https://github.com/Vaa3D/release/releases/).&#160; freeware</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matlab</td>
			<td>Mathworks</td>
			<td>R2014b</td>
			<td>for morphometric analysis of dendrites (http://www.mathworks.com).&#160; commercial software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matlab toolbox: TREES1.14</td>
			<td></td>
			<td>v1.14</td>
			<td>for analyzing dendritic morphometric parameters (http://www.treestoolbox.org/download.html).&#160; freeware</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matlab toolbox: Dendritic_Tree_Toolbox</td>
			<td></td>
			<td>v1.0</td>
			<td>for calculating morphometric parameters (https://science.nichd.nih.gov/confluence/display/snc/Data+collections+for+imagines+combination+and+standardize+column+registration). Freeware</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sample files</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SWC file definition</td>
			<td></td>
			<td></td>
			<td>http://www.neuronland.org/NLMorphologyConverter/MorphologyFormats/SWC/Spec.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">The codes and sample files for image combination and registration</td>
			<td></td>
			<td></td>
			<td>https://science.nichd.nih.gov/confluence/display/snc/Data+collections+for+imagines+combination+and+standardize+column+registration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reference point example&#160;</td>
			<td></td>
			<td></td>
			<td>https://science.nichd.nih.gov/confluence/download/attachments/117216914/points.csv?version=1&#38;modificationDate=1471880596000&#38;api=v2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Computer system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MS Windows Windows 7 x64 or Macintosh OS X 10.7 or later</td>
			<td></td>
			<td></td>
			<td>3GHz 64-bit quad-core processor, 16G RAM (minimal)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optional: Quadro4000&#160; (or above) graphic card</td>
			<td>Nvidia</td>
			<td></td>
			<td>for stereographic visualization of dendrites.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optional: NVIDIA 3D vision2</td>
			<td>Nvidia</td>
			<td></td>
			<td>http://www.nvidia.com/object/3d-vision-main.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optional: 120 Hz LCD display for NVIDIA 3D vision2</td>
			<td></td>
			<td></td>
			<td>http://www.nvidia.com/object/3d-vision-system-requirements.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reagents for imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24B10 antibody</td>
			<td>The Developmental Studies Hybridoma Bank</td>
			<td>24B10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GFP Tag Antibody</td>
			<td>Thermofisher Scientific</td>
			<td>G10362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-Rabbit (H+L), Alexa Fluor 488</td>
			<td>Thermofisher Scientific</td>
			<td>A11034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-Mouse (H+L), Alexa Fluor 568</td>
			<td>Thermofisher Scientific</td>
			<td>A21124</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VECTASHIELD Antifade Mounting Medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mounting&#160;Clay&#160;</td>
			<td>Fisher</td>
			<td>S04179</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% glycerol in 1X PBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover glasses, high performance, D=0.17mm</td>
			<td>Zeiss</td>
			<td>474030-9000-000</td>
			<td></td>
		</tr>
	</tbody>
</table>

analyzing dendritic morphology in columns and layers

在中枢神经系统中，如苍蝇视叶和脊椎动物皮质许多地区，突触电路中的层和列组织发达的动物发展和信息处理中，以促进脑布线。突触后神经元精心树突在特定层的型特异性模式与适当的突触前末梢到突触。飞髓质神经毡是由10层到约750列;每一列是通过在38种髓质的神经元，配合部分7种类型的传入的在一个特定类型的方式终末其中树突支配。该报告详细介绍了程序，图像和分析延髓神经元树突。该工作流包括三个部分：（i）该双视图成像部分结合在正交取向成树突的高分辨率三维图像收集整理二共焦图像堆栈; （二）树突追踪和登记部分树突状痕迹在3D乔木和树突注册痕迹，参考列数组; （ⅲ）该树枝状分析部分分析的树突状图案相对于列和层，包括树突乔木层特定的终止和平面投影的方向，并导出树枝状分支和终止频率的估计。该协议利用建立在开源MIPAV（医学影像处理，分析和可视化）平台和自定义工具箱中的矩阵实验室语言自定义插件。总之，这些协议提供一个完整的工作流来分析层和列果蝇髓质神经元的树突的路由，以确定细胞类型，并确定在突变体的缺陷。

利用这里提出的双视图成像过程，含有疏标记TM20神经元蝇脑在两个正交的方向被成像。成像之前，将脑与用于可视化膜 - 拴系的GFP和感光体轴突适当初级和次级抗体染色。用于成像，大脑首次安装在水平方向（ 图2A，B）。一个GFP标记TM20神经和周围感光体轴突使用共聚焦显微镜（ 图3A）成像。大脑，然后重新对准（ 图2A，B）和在正面...

Watch this Scientific Journal Video about Analyzing Dendritic Morphology in Columns and Layers at JoVE.com

Analyzing Dendritic Morphology in Columns and Layers

在这里，我们将展示如何分析列和层果蝇髓质神经元树突路由。该工作流包括一个双视图成像技术，以改善跟踪，登记树突乔木于基准列数组以及用于分析在三维空间中的树枝状结构的图像质量和计算工具。

分析树突状形态的列和图层

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...

Research

JoVE Journal

Neuroscience

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

肖尔自动数字化的神经元形态分析，多尺度

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

科研

神经科学

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

在培养的中枢神经系统的神经元树突棘形态分析

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

Inducing Dendritic Growth in Cultured Sympathetic Neurons

诱导培养的交感神经元的树突状增长

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

浦肯野细胞树突形态的器官切片培养分析

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining

可视化的一个积极的早期经验，触觉刺激的影响，对树突状形态和突触连接与高尔基染色考克斯

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

间接神经元的星形胶质细胞共培养试验：一个<em&gt;体外</em&gt;设置 - 为神经元的神经胶质细胞相互作用的详细调查

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

嗜德维人神经元树突状树突状复杂度的定量分析

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

可视化和分析星形细胞中细胞内器官和其他货物的细胞内传输

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in ...