Name: quantitative analysis of neuronal dendritic arborization complexity in drosophila
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila at JoVE.com

我们要感谢威廉·艾默尔的成像技术援助。这项工作得到了治疗阿尔茨海默氏症基金 [r. e. t]、国家卫生研究所 [r01ag014713 和 r01mh60009 至 r. e. t。r03ar063271 和 r15eb019704 至 a. l.] 和国家科学基金会 [nsf1455613 至 a. l.]。

1. 实验准备
<ol><li>制备以下试剂: dulbecco 的磷酸盐缓冲盐水 (pbs);triton x-100;0.2% pbst (pbs + 0.2% triton x-100);32% 的甲醛 (pfa), 稀释成 4%, 使用前;有机硅弹性体基座及固化剂;防褪色安装介质 (例如, prolong gold);和指甲油。</li>
	<li>准备以下设备: 解剖显微镜、两个锋利的钳子和一把用于显微解剖的剪刀、一些用于显微解剖的针脚、制作解剖盘的培养皿、显微镜幻灯片和覆盖物、共聚焦仪和计算机与斐?...

<ol>
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支配表皮的树突是神经元的输入区域, 它们的形态决定了单个神经元如何接收和处理信息。枝晶形态的发展反映了枝晶组织的基因调控。外周神经系统的山龙幼虫是研究树突发育的重要模型, 因为: 1) 与哺乳动物功能相似 11,12;2) 基于枝晶结构11、12 的四个类区别;3) 调节形态发生的遗传因素。在该协议中, ...

树突是神经元的分枝突起, 它覆盖的领域包括神经元从其他神经元1,2的感觉和突触输入。树突是突触形成的重要组成部分, 在整合突触输入以及在神经元中传播电化学刺激方面发挥着至关重要的作用。树突状树状树状树状化 (da) 是一个过程, 神经元通过这个过程形成新的树突树和分支, 形成新的突触。da 的发育和形态, 如分支密度和分组模式, 是多步骤生物过程的结果, 与神经元功能高度相关。该方案的目的是为五味子中神经元树突菌的复杂性的定量分析提供一种方法。
树突的复杂性决定了伙伴神经元的突触类型、连通性和输入。分枝模式和树突密度参与处理收敛到树突场 3,4的信号。树突在发展中具有调整的灵活性。例如, 突触信号对发育阶段和成熟神经系统的体感神经元的树突组织有影响.神经元连接的建立依赖于树突的形态发生和成熟。树突畸形与神经元功能受损有关。研究表明, da 神经元形态发生的异常可能会导致多种神经退行性疾病的病因, 包括阿尔茨海默氏症 (ad)、帕金森病 (pd)、亨廷顿病 (hd) 和卢格里格病/肌萎缩侧索硬化症 (als)6,7,8。突触改变出现在 ad 的早期阶段, 与神经元功能7,8的下降和损伤。然而, 枝晶病理学如何促进这些神经退行性疾病的发病机制的具体细节仍然是难以捉摸的。
树突的发育受编码复杂的调节剂网络的基因的调控, 例如 wnt系列的蛋白质 9、10、转录因子和细胞表面受体上的配体11,12.五丁子由四类组成 (i 类、ii 类、iv 类), 其中 iv 类神经元具有最复杂的分支模式, 并已被用作一个强大的实验系统, 以更好地理解形态发生 13, 14岁在早期形态发生过程中, iv 型神经元中基因的过度表达和 rnai 沉默导致分枝模式的改变和13节枝的修剪.开发一种实用的神经元树突状树状树状树状树状树状树状树状树状化定量分析方法具有重要意义。
我们以前已经证明, 沉默的 sox5, sox102f的嗜酸性粒细胞正畸, 导致短树突的 da 神经元和降低复杂性的 iv-da 15 类神经元。在这里, 我们提出了定量分析的程序, 神经树突树树突状树突状的复杂性 (ndac) 在五味子。该协议, 根据前面描述的方法, 提供了一个简短的方法来分析发展的大感觉神经元。它说明了在第三幼虫体壁 16、17、18、19 中的鲁棒图像标记和 da 神经元。这是一个有价值的协议, 研究 ndac 和发展差异的研究人员希望在体内。

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			<td height="21" style="height:21px;width:363px;">TritonX-100</td>
			<td style="width:223px;">Fisher Scientific</td>
			<td style="width:197px;">9002-93-1</td>
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			<td height="21" style="height:22px;width:363px;">Paraformaldehyde(PFA)</td>
			<td style="width:223px;">Electron Microscopy Sciences</td>
			<td style="width:197px;">15714-S</td>
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			<td height="28" style="height:29px;width:363px;">Sylgard 184 silicone elastomer base and curing agent</td>
			<td style="width:223px;">Dow Corning Corportation</td>
			<td style="width:197px;">3097366-0516;3097358-1004</td>
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			<td height="24" style="height:25px;width:363px;">ProLong Gold Antifade Mountant</td>
			<td style="width:223px;">Thermo Fisher Scientific</td>
			<td style="width:197px;">P36931</td>
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			<td height="21" style="height:21px;width:363px;">Fingernail polish&#160;</td>
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			<td height="21" style="height:21px;width:363px;">Stereo microscope</td>
			<td style="width:223px;">Nikon</td>
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			<td height="21" style="height:21px;width:363px;">Confocal microscope</td>
			<td style="width:223px;">Nikon</td>
			<td style="width:197px;">Eclipse Ti-E</td>
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			<td height="21" style="height:21px;width:363px;">Petri dish</td>
			<td style="width:223px;">Falcon</td>
			<td style="width:197px;">353001</td>
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			<td height="21" style="height:21px;width:363px;">Forceps</td>
			<td style="width:223px;">Dumont</td>
			<td style="width:197px;">11255-20</td>
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			<td height="21" style="height:21px;width:363px;">Scissors&#160;</td>
			<td style="width:223px;">Roboz Surgical Instrument Co</td>
			<td style="width:197px;">RS-5611</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Insect Pins&#160;</td>
			<td style="width:223px;">Roboz Surgical Instrument Co</td>
			<td style="width:197px;">RS-6082-25</td>
			<td></td>
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			<td height="18" style="height:19px;width:363px;">Microscope slides and cover slips</td>
			<td style="width:223px;">Fisher Scientific</td>
			<td style="width:197px;">15-188-52</td>
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quantitative analysis of neuronal dendritic arborization complexity in drosophila

树突是神经元的分枝投射, 树突形态反映了神经系统发育过程中的突触组织。嗜酸菌幼虫神经元树突树化 (da) 是研究神经系统发育过程中神经树突形态发生和基因功能的理想模型。有四类大神经元。第四类是最复杂的, 有分支图案, 几乎覆盖幼虫体壁的整个区域。我们以前用四个参数描述了sox5的嗜酸性粒正无量表对 iv 类神经元树突树树状树突状树突状树突状复杂性 (ndac) 的影响: 树突长度、树突覆盖的表面积、分支的总数, 和分支结构。该协议介绍了 ndac 定量分析的工作流程, 包括幼虫解剖、共聚焦显微镜和利用 imagej 软件进行图像分析的过程。进一步洞察 da 神经元的发育及其潜在机制将提高对神经元功能的理解, 并提供有关神经和神经发育障碍的根本原因的线索。

在 gfp 荧光成像分析中, 用共过度表达的 gfp (uas-gfp; ppk-gal4) 标记了 da 神经元的树突, 用于 gfp 荧光成像分析。用倒置共聚焦显微镜对达神经元树突的形态进行了成像 (图 2)。
利用斐济 imagej 软件追踪了 da 神经元的树突。该文件用于估计枝晶长度 (图 3)。sox102f在 da 神经元 (nuct21) ...

Watch this Scientific Journal Video about Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila at JoVE.com

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

该方案的重点是定量分析嗜德维拉中的神经元树突状树突状树突状树突状的复杂性 (ndac), 可用于树突状形态发生的研究。

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. ...

该程序的总体目标是观察 SOX5 基因在果蝇的神经元发育过程中对树突状状神经元复杂性的影响。该方法有助于研究神经性变性的形态，以及神经系统发育中的基因功能，更好地理解神经退行性疾病基因。最终，该技术提供了树突复杂性的定量分析，可用于许多不同类型的神经树突。这种技术的影响延伸到神经退行性疾病的遗传机制，因为突变是理解基因功能的关键。使用 UAS-Sox102F-RNAi 应变苍蝇或 W118 控制装置设置 UAS-GFP;ppk-GAL4 的设置交叉。在25摄氏度的标准条件下培养苍蝇。大约五到六天，用钳子仔细收集第三个星体幼虫进行解剖。将幼虫放在由硅弹性体基座制成的解剖盘中，以及组织培养的培养培养皿中。然后把盘子放在解剖显微镜下。现在固定幼虫的尾巴露出中线。然后固定幼虫嘴钩，使幼虫的后侧向上。现在，在菜中加入200微升PBS，将幼虫浸入溶液中。接下来，用小切口切开幼虫的尾巴和嘴。然后沿着后线和两个气管之间切开幼虫，从考达尔到罗斯特拉。然后，在身体的四个角各放置一个针，使身体处于平坦的位置。现在，在室温下将幼虫体壁固定为 4% PFA，25 分钟。然后用PBS洗三次，每次洗五分钟。洗涤后，取出针脚，将组织转移到玻璃滑梯上。然后将组织浸入防淡安装介质中，然后用盖滑来安装。让安装的组织风干一小时，然后用指甲抛光密封在盖上。这些幻灯片存放在四摄氏度的黑暗中。使用 20 倍目标用共和显微镜捕获 z 系列图像。首先，设置 z 步数的范围，以包括幼虫体壁中的所有 DA 神经元，并设置步长为半微米。然后将图像存储为 TIF 或 ND2 文件进行处理。接下来，评估 DA 神经元上树突的数量和形态。从评估它们的长度开始。首先，在斐济 ImageJ 中，如有必要，将图像拆分为单独的通道。只需要评估 GFP 通道。接下来，进行 z 投影。在新窗口中，为投影类型选择最大强度，然后选择开始和停止切片。然后单击"确定"以生成具有清晰树突投影的 z 投影图像。现在使用插件工具跟踪中性。首先，转到感兴趣的神经元的 soma，然后单击从细胞体中出现一个树突的地方。然后点击此树突的尖端。如果连接两个点的蓝线运行 neurite 的长度，请按 Y 表示"是"。然后，如果此行适合神经石的完整路径，请单击"完整路径"。如果线不适合路径，则沿此 neurite 进一步单击点，将路径折弯成适合 neurite 长度的多个线段。然后，单击"完成路径"，继续标记下一个 neurite 的路径。完成所有跟踪路径后，选择"分析、测量路径"选项以将路径导出到 CSV 文件。使用此数据可计算用于分析的路径长度和平均值。接下来，计算神经元的表面面积。首先，从斐济 ImageJ 窗口中选择免费手绘图工具。然后，连接感兴趣的神经元的端点。然后，从"分析"窗口中选择"度量值"选项。结果将显示在新框中，该框中具有所选区域的值。将此值复制到数据分析软件。最后，使用"分析骨架路径"插件计算分支总数。DA神经元的树突通过共同表达GP在它们的索玛和树突树干中标记，用于GGFP荧光成像分析。树突的形态是使用倒置共体显微镜成像的。DA神经元的树突被跟踪，如所述。该文件用于估计树突长度。在第三个星体幼虫中，在DA神经元中沉默的Sox102F导致树突总数显著减少，其分支长度约为一半，结构更简单，仅显示一半的分支数量。从逻辑上讲，这导致了树干表面积的减少。该协议提供了一种评估DA感觉神经元发展的快速方法。在尝试此过程时，请务必获取大量 DA 神经元的图像，以获得良好的覆盖范围。按照此过程，可以执行其他方法，如树突分析，以便回答有关树突发育的其他问题。自从它的发展，这项技术已经帮助神经科学领域的研究人员通过模型系统推断神经退行性疾病，如阿尔茨海默氏症。

Research

JoVE Journal

Neuroscience

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

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 ...

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 ...

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

光遗传学的扰动激光照射半完好的神经活动<em&gt;果蝇</em&gt;幼虫运动

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the ...

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 ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

树突分支中的海马区对小鼠的齿状回评

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

攀登缺陷的神经退行性疾病的果蝇模型定量分析

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

Analyzing Dendritic Morphology in Columns and Layers

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 ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

使用高尔基Cox方法评估老年小鼠海马树突状复杂性

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

荧光标记蛋白 ImageJ 的无偏分析在果蝇变性定量细胞生物学中的应用

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...