Name: three-dimensional quantification of dendritic spines from pyramidal neurons derived from human induced pluripotent stem cells
Uploaded: 2015-10-10T14:00:00.000+00:00
Duration: 10 min 18 s
Description: Watch this Scientific Journal Video about Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells at JoVE.com

This work was funded by the Institut Pasteur, the Bettencourt-Schueller foundation, Centre National de la Recherche Scientifique, University Paris Diderot, Agence Nationale de la Recherche (ANR-13-SAMA-0006; SynDivAutism), the Conny-Maeva Charitable Foundation, the Cognacq Jay Foundation, the Orange Foundation, and the Fondamental Foundation. L.G. is supported by an undergraduate fellowship from the Health Ministry. We acknowledge the help of BitPlane in particular Georgia Golfis, in the early stage of this work.

1.神经文化注意:在多能干细胞成纤维细胞重编程，承诺向背侧端脑谱系，推导，放大，和银行的晚期皮质祖细胞（LCP）在Boissart 等人 4进行了描述。还根据Boissart 等人 4在稍微修改执行LCP样细胞的神经元分化。其他程序已经开发了用于成纤维细胞的成诱导多能干细胞，接着通过分化成神经元的直接重编程。该协议被保留下来，因为它允许选择性生产锥体谷氨酸能神经元的。 <ol><li>治疗6孔培养板用玻璃盖玻片用聚鸟氨酸（在DPBS中稀释至1/6，原料浓度0.01％）O / N，随后洗涤三次在DPBS中。然后在至少10小时的流罩下添加层粘连蛋白（储备浓度为1mg / ml，稀释500倍的DPBS）<html> 。 </li><li>板并调度NSC以低密度N2添加剂的（50,000细胞/ cm 2）在6孔培养板用在3毫升由DMEM / F12的培养基盖玻片（500毫升），2瓶（5 ml的每种）， 2瓶B27补充剂（每次10毫升），10毫升笔，链霉素（青霉素= 10,000单位/毫升，链霉素= 10000单位/毫升），1毫升2-巯基乙醇（原液:50毫米）和层粘连蛋白（1 / 500），无生长因子。关键的一步:认真执行这一步骤，以减少细胞集群增加细胞的缓慢旋转的动作。 </li><li>取出培养基。添加含有新鲜的层粘连蛋白溶液2微克/毫升，以保持附着在玻璃盖玻片的神经元，并避免结块新鲜N2B27网上平台。改变介质每3天。为了防止电池从干燥加入新鲜培养基之前保持一些剩余的培养基（200微升）的。可替代地，进行快速改变总体积（3毫升）中。 </li></ol>e_title"&gt; 2。慢病毒转导注意:GFP的慢病毒载体承蒙以下乌韦Maskos实验室博士在巴斯德研究所（巴黎）提供及根据公布的协议5制备。 GFP表达由鼠标磷酸甘油酸激酶（PGK）启动子驱动。在这项研究中，病毒滴度为400纳克/μL（原液在PBS 1X）的。 <ol><li>加入1微升含有40纳克每培养的GFP的慢病毒载体以及（6孔板）的原液转导在成熟通过病毒颗粒的任何步骤人类源自iPSC的神经元，并培育48小时在新鲜培养基中。注意:对于这个协议，孵化时间允许脊柱结构的一个很好的标志。 </li></ol> 3.免疫注意:为了提高整个棘形态的标签，使用抗GFP抗体在透化条件下进行免疫荧光标记。 </p&gt; <ol><li>盖玻片上在4％多聚甲醛除去培养基并固定转导的细胞在室温10分钟，然后清洗3次的1×PBS（每次10分钟）。 </li><li>在PBS浸入盖玻片补充有0.05％的Triton（100×）和10％马血清的1小时在室温下，然后洗3次在1×PBS中。 </li><li>加入100微升的1×PBS的补充与针对GFP和由1000上每个盖玻片的因子稀释4％马血清和初级抗体（1/1000）。在孵育暗箱O / N在4°C，然后洗3次，1×PBS。 </li><li>稀释的Alexa Fluor 488缀合的抗体（1/200）的PBS补充有吐温20的0.5％，并孵育1小时，在室温。然后洗3次，1×PBS中并安装在载玻片盖玻片安装介质荧光显微镜。 </li></ol> 4.树突棘成像<ol><li>通过激光共聚焦扫描显微镜进行共聚焦成像。 </li><li>选择健康的神经元与锥体形态的次全树突分支，并从独立的实验量化每种条件至少10的神经元。量化60 - 每枝晶100微米。 </li><li>获得使用40X油NA = 1.3的物镜和488nm的激光线的GFP激励图像，具有典型的峰值功率在大约20微瓦样本水平。大约80纳米组像素大小适当样树突棘。 注:在随后的分析要求的图像，其噪声不影响树突棘和的正确分割。与上述空间采样和功率设置，我们观察到3.15微秒的像素停留时间是足以将收集足够的光子来构建这样的图像。为暗淡的样品，图像质量可以通过平均2至4个扫描得到改善。可能需要购置若干XY切片以覆盖感兴趣的区域，然后处理前一起必须缝合。 </li><li>品尝整个神经元的体积，获得一个Z堆栈，用为Z间距从150纳米到300纳米，得20至30ž切片。 注意:实现了与这些设置的横向空间分辨率为234 nm和轴向分辨率为591纳米。这里所选择的采样是足够的，但作为轴向分辨率比要被成像的棘的最小尺寸大，则分析有利于该横向延伸从树突棘。 </li></ol>树突棘5.三维定量注意:以下部分具体介绍了使用了Imaris软件进行分析的。备选实现存在，包括NeuronStudio 15或的Metamorph 8，它可以提供类似的结果。 使用下列键设置: <ol><li>作为一个预处理级，通过由软件提供的图像处理设备利用高斯滤波。通过图像处理&gt; Smoo执行高斯滤波事&gt;高斯滤波器。设置过滤器宽度为等于在XY像素大小。 </li><li>执行树突的半自动跟踪，使用该软件的灯丝示踪模块。 <ol><li>首先，估计枝晶直径，通过使用该软件的切片标签的距离的工具。 </li><li>在超越选项卡中，单击长丝工具。为了更好的稳健性，该协议依赖于半自动化跟踪;点击跳过自动创建 。该模块接口现在显示绘图选项卡。这里，选择AutoPath为方法， 枝蔓作为一种类型，并输入所估计的枝晶的直径。 </li><li>使用指针的选择模式 ，把光标移动到一个盒。对树突起点按住Shift键并单击鼠标右键。注:该软件进行初始计算。 </li><li>将沿枝晶的指针。从起点（代表编作为蓝色球体），黄线表示最可能的树突路径​​被示出。对树突端点按住Shift键并单击鼠标左键。 </li></ol></li><li>执行自动化的刺分割。注意:在跟踪枝晶的棘将由模块接口自动找到。 <ol><li>在模块界面中，单击创建选项卡上。 在重建下拉列表中，挑选重建枝蔓径及检查保持数据框中 。 然后单击重建。 </li><li>设置阈值 ，使得所分割的体积对应于实际树枝状结晶体积。作为一个算法 ， 然后从距离图最短距离。单击下一步按钮。 </li><li>确定最小棘头直径和最大长度，再通过使用软件的片标签的距离的工具，然后再回来了超越选项卡并输入参数。 F或者该协议，约200个值 - 300纳米的最小直径4微米的最大长度是很好的出发点。不要选中允许分局棘框。 单击下一步按钮。 </li><li>调整种子点阈值 ，使代表刺蓝色点定位到实际的脊椎头。 单击下一步按钮。注:现在的核心计算完成，可能会很长。 </li></ol></li><li>通过将模块接口的工具选项卡分类刺。点击分类刺。在提示用户界面时，请确保有四大类，由它们的形态定义如下:末节:长度&lt;1微米;蘑菇:长度（脊椎）&gt; 3和最大宽度（头）&gt;平均宽度（颈）×2;细细长长的:平均宽度（头）≥平均宽度（颈部）;丝状伪足样:长度≤4微米（无头）。 注:莫独乐界面生成包含分类的结果四个新的灯丝的对象。 </li><li>出口统计数据:在所有这四个对象接口的，去统计选项卡。 点击导出所有统计到文件按钮 。 注:其他统计值可导出为树突（如，长度，面积，平均直径，分支深度，分枝角度，体积等 ），以及用于棘（ 例如，直线度，的附着，长度和不同的卷区域脊骨零件，脊柱直径，密度等 ）。 </li></ol>

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The authors have nothing to disclose.

的锥体神经元的形态学特征的量化依靠软件。灯丝示踪接口用于神经元和刺的分割，以及XT模块用于自己的分析。 分析我们技术的准确性，我们首先比较所测量的形态学参数（长度，面积和总体积脊柱适用时），与那些使用大鼠成熟锥体神经元在培养6,7和人脑组织3出版。密度是相当的所有情况。没有量 ​​的数据是在大鼠神经元描述了使用自己的协议为容积...

皮质锥体神经元的树突棘是它们沿着这些神经元亚型的啮齿动物，灵长类动物和人类的大脑基底和顶树突分布小而薄的突起。他们最兴奋性突触的位点，并显示在学习和认知过程的关键功能。人树突棘的详细结构已经从技术上研究了电子显微镜2。然而，这种方法是费时和表示繁重的工作量。最近，使用组合的大型人工脊椎分析3特定软件的三维（3D）重建树突棘的形态的已报道在人类大脑皮质。 绿色荧光蛋白（GFP）的技术联接到免疫表示由荧光显微镜的精确工具脊柱识别和形状测定。这种方法可以容易地应用于培养的神经元。豪版本，没有数据已被报道脊柱成熟和形态上从诱导的多能干细胞（IPSC）衍生的人神经元分析。 本研究的目的是描述一种协议，它允许从体外培养的人神经元树突棘成像。绿色荧光蛋白标记，激光共聚焦显微镜和3D分析了Imaris软件的灯丝示踪模块进行了本协议中使用。培养的步骤所必需的获得层的皮层谷氨酸能神经元二至四从神经干细胞（NS​​C）也在这里简单说明。整个协议为人类NSC生产已经在别处4出版。

<table><tbody><tr><td>D-PBS (1x), sans Calcium, Magnesium et Phenol Red</td><td>Gibco/ Life Technologies</td><td>14190169</td><td></td></tr><tr><td>Poly-L-Ornithine Solution Bioreagent</td><td>Sigma Aldrich</td><td>P4957</td><td></td></tr><tr><td>Mouse laminin</td><td>Dutscher Dominique</td><td>354232</td><td></td></tr><tr><td>N&lt;sub&gt;2&lt;/sub&gt; Supplement</td><td>Gibco/ Life Technologies</td><td>17502048</td><td></td></tr><tr><td>B-27 Supplement w/o Vitamin A (50x)</td><td>Gibco/ Life Technologies</td><td>12587010</td><td></td></tr><tr><td>DMEM/NUT.MIX F-12 W/GLUT-I</td><td>Gibco/ Life Technologies</td><td>31331028</td><td></td></tr><tr><td>Neurobasal Med SFM</td><td>Gibco/ Life Technologies</td><td>21103049</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Gibco/ Life Technologies</td><td>31350-010</td><td></td></tr><tr><td>Penicillin-Steptomycin</td><td>Gibco/ Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>GFP Rabbit Serum Polyclonal Antibody</td><td>Gibco/ Life Technologies</td><td>A-6455</td><td></td></tr><tr><td>Horse serum</td><td>Gibco/ Life Technologies</td><td>16050130</td><td></td></tr><tr><td>Alexa Fluor 488 Goat Anti-Rabbit&amp;nbsp;</td><td>Gibco/ Life Technologies</td><td>A11034</td><td></td></tr><tr><td>Polyclonal Anti-betaIII tubulin Antibody</td><td>Millipore</td><td>AB9354</td><td></td></tr><tr><td>Coverglass 13 mm</td><td>VWR</td><td>631-0150</td><td></td></tr><tr><td>Prolong Gold Antifade Reagent with DAPI</td><td>Gibco/ Life Technologies</td><td>P36931</td><td></td></tr><tr><td>Tween(R) 20 Bioextra, Viscous Liquid</td><td>Sigma Aldrich Chimie</td><td>P7949</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich Chimie</td><td>X100-100ML</td><td></td></tr><tr><td>Human Fibroblasts</td><td>Coriell Cell Line Biorepository</td><td>GM 4603 and GM 1869</td><td>Coriell Institute for Medical Research, Camden, NJ, USA</td></tr><tr><td>Confocal laser scanning microscope</td><td>Zeiss (Germany)</td><td>LSM 700</td><td></td></tr><tr><td>Imaris Software</td><td>Bitplane AG, Zurich</td><td>6.4.0 version</td><td>Filament Tracer and Imaris XT modules are necessary</td></tr><tr><td>Huygens Software</td><td>Huygens software, SVI, Netherlands</td><td>Pro version</td><td>Optional (for deconvolution testing)</td></tr></tbody></table>

three-dimensional quantification of dendritic spines from pyramidal neurons derived from human induced pluripotent stem cells

树突棘是对应于兴奋性突触的中枢神经系统的突触后隔室的小突起。它们沿树突分布。它们的形态，在很大程度上取决于神经元活动，它们是动态的。树突棘在其表面表达并在突触后密度的水平谷氨酸受体（AMPA和NMDA受体）。每个脊椎使神经元能够独立控制其国家和地方的活动。脊柱形态已被广泛研究，在大脑皮质的谷氨酸锥体细胞，同时使用在体内的方法和从啮齿动物的组织得到的神经元培养物。神经病理症状可以关联到改变的脊柱感应和成熟，如图啮齿动物培养的神经元和一维定量分析1。本研究描述了一个协议，用于脊椎形态的三维定量分析使用人类cortic人神经元的神经干细胞（后期皮质祖细胞）获得。这些细胞最初从诱导的多能干细胞获得的。该协议允许脊柱形态的不同培养时间的分析，并与来自对照个体与那些从患者的精神疾病得到得到诱导多能干细胞之间的可能的比较。

<html>本研究描述了一种标准化协议，用于从iPSC的衍生锥体神经元的树突培养脊柱量化。该协议允许对人类神经元脊柱成熟及其与在标准啮齿动物神经元培养棘的成熟以及体内动物模型可以比较的分析。 图1A表示的不同步骤培养的，其允许生产皮层锥体神经元的方案。这样示意表示，以便更好地了解生产赋予不同类别刺锥体神经元的全球时间缩放提供。 Repr...

Watch this Scientific Journal Video about Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells at JoVE.com

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

锥体神经元的树突棘是哺乳动物大脑皮质最兴奋性突触的部位。此方法描述在从诱导的多能干细胞衍生的人皮质锥体谷氨酸能神经元脊柱形态的三维定量分析。

树突棘从锥体神经元的三维定量从人类诱导多能干细胞衍生

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

three-dimensional-quantification-dendritic-spines-from-pyramidal

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

11.4K 次观看.  Institut Pasteur. 锥体神经元的树突棘是哺乳动物大脑皮质最兴奋性突触的部位。此方法描述在从诱导的多能干细胞衍生的人皮质锥体谷氨酸能神经元脊柱形态的三维定量分析。 

视频: Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

集成的无人类诱导多能干细胞的生成方法头发衍生角质形成细胞

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

科研

发育生物学

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

从人多能干细胞的黑素细胞的无饲养推导

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

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 ...

神经科学

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

成像大鼠原代海马神经元树突棘使用结构照明显微镜

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

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

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

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 ...

3D Modeling of Dendritic Spines with Synaptic Plasticity

Computational modeling of diffusion and reaction of chemical species in a three-dimensional (3D) geometry is a fundamental method to understand the mechanisms of synaptic plasticity in dendritic spines. In this protocol, the detailed 3D structure of the dendrites and dendritic spines is modeled with meshes on the software Blender with CellBlender. The synaptic and extrasynaptic regions are defined on the mesh. Next, the synaptic receptor and synaptic anchor molecules are defined with their diffusion constants. Finally, the chemical reactions between synaptic receptors and synaptic anchors are included and the computational model is solved numerically with the software MCell. This method describes the spatiotemporal path of every single molecule in a 3D geometrical structure. Thus, it is very useful to study the trafficking of synaptic receptors in and out of the dendritic spines during the occurrence of synaptic plasticity. A limitation of this method is that the high number of molecules slows the speed of the simulations. Modeling of dendritic spines with this method allows the study of homosynaptic potentiation and depression within single spines and heterosynaptic plasticity between neighbor dendritic spines.

The protocol develops a three-dimensional (3D) model of a dendritic segment with dendritic spines for modeling synaptic plasticity. The constructed mesh can be used for computational modeling of AMPA receptor trafficking in the long-term synaptic plasticity using the software program Blender with CellBlender and MCell.

具有突触可塑性树突脊柱的三维建模

Computational modeling of diffusion and reaction of chemical species in a three-dimensional (3D) geometry is a fundamental method to understand the ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

脑切片和原细胞培养中金字塔神经元的弹道标记

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

Rapid Golgi Stain for Dendritic Spine Visualization in Hippocampus and Prefrontal Cortex

Golgi impregnation, using the Golgi staining kit with minor adaptations, is used to impregnate dendritic spines in the rat hippocampus and medial prefrontal cortex. This technique is a marked improvement over previous methods of Golgi impregnation because the premixed chemicals are safer to use, neurons are consistently well impregnated, there is far less background debris, and for a given region, there are extremely small deviations in spine density between experiments. Moreover, brains can be accumulated after a certain point and kept frozen until further processing. Using this method any brain region of interest can be studied. Once stained and cover slipped, dendritic spine density is determined by counting the number of spines for a length of dendrite and expressed as spine density per 10 &#181;m dendrite.

The protocol describes a modification of the rapid Golgi method, which can be adapted to any part of the nervous system, for staining neurons in the hippocampus and medial prefrontal cortex of the rat.

快速高尔基染色用于海马体和前额叶皮层树突状脊柱可视化

Golgi impregnation, using the Golgi staining kit with minor adaptations, is used to impregnate dendritic spines in the rat hippocampus and medial ...

<meta charset="utf8"></head><body>使用 Neurolucida 360 进行树突状脊柱分析的方案

Dendritic Spine Quantification Using an Automatic Three-Dimensional Neuron Reconstruction Software

Synaptic connections allow for the exchange and processing of information between neurons. The post-synaptic site of excitatory synapses is often formed on dendritic spines. Dendritic spines are structures of great interest in research centered around synaptic plasticity, neurodevelopment, and neurological and psychiatric disorders. Dendritic spines undergo structural modifications during their lifespan, with properties such as total spine number, dendritic spine size, and morphologically defined subtype altering in response to different processes. Delineating the molecular mechanisms regulating these structural alterations of dendritic spines relies on morphological measurement. This mandates accurate and reproducible dendritic spine analysis to provide experimental evidence. The present study outlines a detailed protocol for dendritic spine quantification and classification using Neurolucida 360 (automatic three-dimensional neuron reconstruction software). This protocol allows for the determination of key dendritic spine properties such as total spine density, spine head volume, and classification into spine subtypes thus enabling effective analysis of dendritic spine structural phenotypes.

<meta charset="utf8"></head><body>作者聚焦:优化树突状棘分析，实现海马 CA1 顶端树突的平衡手动和自动评估

Dendritic spines are post-synaptic compartments of most excitatory synapses. Alterations to dendritic spine morphology occur during neurodevelopment, aging, learning, and many neurological and psychiatric disorders, underscoring the importance of reliable dendritic spine analysis. This protocol describes quantifying dendritic spine morphology accurately and reproducibly using automatic three-dimensional neuron reconstruction software.

使用自动三维神经元重建软件进行树突状脊柱定量

Synaptic connections allow for the exchange and processing of information between neurons. The post-synaptic site of excitatory synapses is often formed ...

树突棘从锥体神经元的三维定量从人类诱导多能干细胞衍生

摘要

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此视频中的章节

集成的无人类诱导多能干细胞的生成方法头发衍生角质形成细胞

从人多能干细胞的黑素细胞的无饲养推导

在培养的中枢神经系统的神经元树突棘形态分析

成像大鼠原代海马神经元树突棘使用结构照明显微镜

树突分支中的海马区对小鼠的齿状回评

具有突触可塑性树突脊柱的三维建模

脑切片和原细胞培养中金字塔神经元的弹道标记

快速高尔基染色用于海马体和前额叶皮层树突状脊柱可视化

使用自动三维神经元重建软件进行树突状脊柱定量

使用自动三维神经元重建软件进行树突状脊柱定量