Name: large-scale reconstructions and independent, unbiased clustering based on morphological metrics to classify neurons in selective populations
Uploaded: 2017-02-15T12:30:00
Description: Watch this Scientific Journal Video about Large-scale Reconstructions and Independent, Unbiased Clustering Based on Morphological Metrics to Classify Neurons in Selective Populations at JoVE.com

我们想感谢博士。埃德卡拉威和马蒂Usrey允许我们使用的预先研究和利比Fairless和柿园刘与神经重建帮助的一部分准备的组织。和白厅基金：这项工作是由美国国立卫生研究院（NEI EY018683）资助。

注意：在此研究中检查的组织制备为单独的研究5的一部分。因此，所有的涉及使用动物的实验方法已经详细在Briggs 等人的实验方法部分中所述。 （2016年）。所有涉及这在现有研究的一部分进行的动物程序进行的机构动物护理和使用委员会的批准。 2 - 注射病毒进入dLGN和脑组织的组织学处理的步骤中的部分1简要地描述如下。 1.立体定位注射...

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神经解剖学研究已经保持神经和连接组学和结构与功能的关系近期备受关注的支柱已经更新了选择性神经元群的详细的形态学特征热情。传统上，神经解剖学研究依赖于神经元的定性分类成形态不同的类专家neuroanatomists定义的神经元。在所述技术用于重建神经元和提取形态学数据的进步，现在可以利用更复杂的和定量的数据分析方法，形态不同的神经元类无偏方式进行分类。在这项研究中，一步?...

神经解剖学处于“连接组学”神经科学1和近期利益的基础之一续签了解神经元群体的形态多样性和特殊的神经元2之间的连接热情。用于标记的方法和重建神经元与最近的创新，包括遗传性和病毒介导的电路跟踪有很大的提高接近3，4，使神经元群体5的更全面的形态的调查。除了在标记单个神经元的改进，定量数据分析技术也已出现，使神经元的独立的和无偏分类成基于形态学数据5，6个不同亚群。这些偏见的技术是在更多的繁体版的改进升定性分类方法已经在该领域的标准了一个多世纪。这项研究的目的是概述，一步一步，选择性群体内神经元的病毒介导的标记的组合，这些神经元的综合样本，和定量数据分析的大规模重建基于与独立聚类统计评估。通过结合这些方法，我们概述朝向收集和神经解剖学数据的分析的新方法，以促进选择性的神经元群体中全面采样和形态学独特神经类型的无偏分类。 由于这些方法的一个例子，我们描述了猕猴的丘脑网状核（TRN）的单个扇区内我国人口众多的神经元进行分析。这些数据是从以前的研究7。用于选择性地标记TRN神经元投射到背latera方法采用手术注射编码EGFP 4,8修改狂犬病毒的丘脑（dLGN）l的膝状体（ 具体见材料/设备 ，第2行的表 ）进行了概述。本变形狂犬病毒缺乏基因编码的基本外壳蛋白，消除了病毒的跨突触运动。一旦病毒在注射部位进入轴突终端，它的作用就像与驱动整个感染神经元5，9，10的全树突分支EGFP表达的重要的益处传统逆行示踪剂。因此，该G-删除狂犬病毒可被用于选择性地感染和标签以下注射和逆行运输任何神经元群体。 为了执行特定的神经元群的综合分析，它从采样是重要神经元的人口中的广泛分布。由于病毒介导的标记技术产生完整的细胞内，“高尔基样”与在病毒注射部位轴突许多神经元的填充，是可能的一个大脑结构的全部范围内重建神经元的一个非常大的样品。此外，由于改性狂犬病毒是在感染和标签大量的神经元，以便有效的，但是可能重建数百每只动物的神经元。为为了生成dLGN投射TRN神经元的全面样品取样160的神经元在整个TRN 11的视觉扇区程序进行了概述。描述重建使用神经重建系统包括显微镜，照相机，和重建软件单个神经元的过程。还描述了方法来确定大脑结构中（TRN在中在这种情况下）单个神经元的位置，并核实病毒注射坐使用体积轮廓重建一个结构（在dLGN内这种情况下）内È体积和位置。步骤导出形态学数据和执行独立的聚类分析的基础上对每个神经元的形态测量指标进行了概述。有对聚类方法的限制，也有可用的多种不同的聚类算法。因此，这些选项和的一些更常用算法的优点进行说明。聚类分析不提供集群的唯一性的统计验证。因此，额外的步骤概述，以验证最佳聚类以及内和跨集群形态学数据之间的关系。用于评价集群的TRN数据集，以确认TRN神经元的统计方法被分组为基于被描述10独立形态度量三个独特簇。 因此，通过概述用于选择性标记步骤，重构，并从一个特定的神经元群体分析形态学数据，我们描述用于定量群体内的神经元之间的形态差异的方法。猕猴TRN视觉部门不同的神经元类型的此前的研究结果证实，独立的统计评价方法。总之，我们希望这些技术将广泛适用于神经解剖数据集，并有助于通过建立大脑的神经元群体的多样性量化分级。

<table border="0" cellpadding="0" cellspacing="0" width="818">
	<tbody>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">SAD&#916;G-EGFP</td>
			<td style="width:171px;">E.M. Callaway Laboratory, Salk Institute</td>
			<td style="width:133px;"></td>
			<td style="width:281px;">Prepared by Dr. F. Osakada. G-deleted rabies virus available through the Salk Institute Viral Core</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Recording electrode: platinum/iridium or tungsten</td>
			<td style="width:171px;">FHC</td>
			<td style="width:133px;">UEPSGGSE1N2M</td>
			<td style="width:281px;">Visit website (www.fh-co.com) for alternative order specifications</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:233px;">Nanoject II</td>
			<td style="width:171px;">Drummod Scientific</td>
			<td style="width:133px;">3-000-204, 110V</td>
			<td style="width:281px;">Alternatives: picospritzer, Hamilton syringe</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:233px;">Freezing microtome</td>
			<td style="width:171px;">Thermo Scientific</td>
			<td style="width:133px;"></td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:233px;">DAB</td>
			<td style="width:171px;">Sigma Aldrich</td>
			<td style="width:133px;">D5905-50TAB</td>
			<td style="width:281px;">3,3&#39;-Diaminobenzidine tetrahydrochloride, tablet, 10 mg substrate per tablet. Caution: carcinogen - must be bleached before discarding</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:233px;">Cytochrome C</td>
			<td style="width:171px;">Sigma Aldrich</td>
			<td style="width:133px;">C2037-100MG</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:233px;">Catalase</td>
			<td style="width:171px;">Sigma-Aldich</td>
			<td style="width:133px;">C9322-5G</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Rabbit anti-GFP</td>
			<td style="width:171px;">Life Technologies/Thermo Fisher</td>
			<td style="width:133px;">#A-11122&#160;</td>
			<td style="width:281px;">Primary antibody</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:35px;width:233px;">Biotinylated goat anti-rabbit</td>
			<td style="width:171px;">Vector Laboratories</td>
			<td style="width:133px;">#BA-1000</td>
			<td style="width:281px;">Secondary antibody</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Neurolucida System&#160;</td>
			<td style="width:171px;">MicroBrightField</td>
			<td style="width:133px;"></td>
			<td style="width:281px;">Software for neuron tracing and analysis. http://www.mbfbioscience.com/neurolucida</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:233px;">Neurolucida Explorer</td>
			<td style="width:171px;">MicroBrightField</td>
			<td style="width:133px;"></td>
			<td style="width:281px;">Data export software</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Microfire Camera&#160;</td>
			<td style="width:171px;">Optronics</td>
			<td style="width:133px;"></td>
			<td style="width:281px;">2-Megapixel true color microscope camera. http://www.simicroscopes.com/pdfs/microfire.pdf</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Nikon E800 Microscope</td>
			<td style="width:171px;">Nikon Instruments Inc.</td>
			<td style="width:133px;"></td>
			<td style="width:281px;">Biological research microscope. http://www.microscopyu.com/museum/eclipseE800.html</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Matlab</td>
			<td style="width:171px;">The MathWorks Inc.&#160;</td>
			<td style="width:133px;"></td>
			<td style="width:281px;">Matrix-based computational mathematics software. http://www.mathworks.com</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Microsoft Office Excel</td>
			<td style="width:171px;">Microsoft</td>
			<td style="width:133px;"></td>
			<td>Spreadsheet program</td>
		</tr>
	</tbody>
</table>

large-scale reconstructions and independent, unbiased clustering based on morphological metrics to classify neurons in selective populations

该协议概述了神经元与使用独立，公正的集群相结合的大规模重建分析创建的选择性神经人口中观察到的形态特征进行全面的调查。这些技术的组合构成用于神经解剖学数据的收集和分析的新方法。总之，这些技术使大规模，和选择性的神经元群体的因此更全面，取样和建立用于说明人口内形态学独特神经类偏定量方法。 该协议概述了采用改性狂犬病毒的选择性标记神经元。 G-删除以下立体定位注射到感兴趣的目标大脑结构的狂犬病毒的作用就像一个逆行示踪剂，并作为EGFP的神经元递送和表达的载体。神经元大量地使用这个感染技术，并表示在他们的树突GFP，生产“高尔基样”单个神经元的完整罢了。因此，病毒介导的逆行追踪方法，通过生产完整的细胞内填充在传统的基于染料逆行追踪技术改进。 个体以及隔离神经元跨越所研究的大脑区域的所有区域，以获得的神经元的一个代表性的样品被选择用于重建。该协议概述了程序，以重建胞体和完成标记细胞跨越多个组织切片的树突分支模式。形态数据，包括脑结构中的每个神经元的位置，提取用于进一步分析。标准编程功能被用于执行独立的聚类分析和基于形态学指标群的评价。为了验证这些分析的聚类分析PERFO的效用，统计评估Rmed指对在猕猴丘脑（TRN）的丘脑网状核重建神经元160制成。无论是原聚类分析和这里执行的统计评价表明TRN神经元被分为三个亚群，每个具有独特形态特征。

我们以前曾表明，选择性群体内神经元的大规模重建是可行的下面喷射改性狂犬病病毒的进入dLGN 5。最近，相同组织被利用来重建160中的神经元TRN的视觉扇区（布拉格等人 ，在审查; 图2A-B），按照上述的详细的方法步骤。在TRN的研究中，TRN神经元的三种独特簇鉴定根据10形态度量独立聚类分析：细胞体区域中，单元主体的圆度，相对于TRN...

Watch this Scientific Journal Video about Large-scale Reconstructions and Independent, Unbiased Clustering Based on Morphological Metrics to Classify Neurons in Selective Populations at JoVE.com

Large-scale Reconstructions and Independent, Unbiased Clustering Based on Morphological Metrics to Classify Neurons in Selective Populations

这个协议描述以下具有修饰的狂犬病毒表达荧光标记物，以及独立的，公正的聚类分析，使不同的神经元亚类之间的形态度量的全面表征逆行感染选择性神经元群体的大规模重建，进行标记。

基于形态学度量大规模重建和独立，公正聚类分类神经元选择性人群

This protocol outlines large-scale reconstructions of neurons combined with the use of independent and unbiased clustering analyses to create a ...

Research

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Neuroscience

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

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漂白后荧光标记蛋白的荧光恢复（FRAP）在培养海马神经元的树突棘

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

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Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

由运动神经元调制生理，形态学和神经化学的表征

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Vibrodissociation从啮齿动物脑切片的神经元，研究突触传递和图像突触前的码头

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Recording Large-scale Neuronal Ensembles with Silicon Probes in the Anesthetized Rat

在麻醉大鼠的硅探头录制的大型神经元合唱

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of ...

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

慢性成人出生的嗅觉神经元的选择性病毒转导在体内 Optogenetic刺激

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

移动硅探针的神经行为鼠害的大型记录

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

隔膜的神经支配膈运动神经元的功能和形态评估

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...