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
Summary
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。
Abstract
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。
Introduction
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（NSC）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。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| PD-PBS (1X), sans Calcium, Magnesium et Phenol Red | Gibco/ Life Technologies | 14190169 | |
| Poly-L-Ornithine Solution Bioreagent | Sigma Aldrich | P4957 | |
| Mouse laminin | Dutscher Dominique | 354232 | |
| N2 Supplement | Gibco/ Life Technologies | 17502048 | |
| B-27 Supplement w/o vit A (50X) | Gibco/ Life Technologies | 12587010 | |
| DMEM/NUT.MIX F-12 W/GLUT-I | Gibco/ Life Technologies | 31331028 | |
| Neurobasal Med SFM | Gibco/ Life Technologies | 21103049 | |
| 2-mercaptoethanol | Gibco/ Life Technologies | 31350-010 | |
| Pen-Steptomycin | Gibco/ Life Technologies | 15140-122 | |
| GFP Rabbit Serum Polyclonal Antibody | Gibco/ Life Technologies | A-6455 | |
| Horse serum | Gibco/ Life Technologies | 16050130 | |
| Alexa Fluor 488 Goat Anti-Rabbit | Gibco/ Life Technologies | A11034 | |
| Polyclonal Anti-betaIII tubulin antibody | Millipore | AB9354 | |
| Coverglass 13 mm | VWR | 631-0150 | |
| Prolong Gold Antifade Reagent avec DAPI | Gibco/ Life Technologies | P36931 | |
| Tween(R) 20 Bioextra, Viscous Liquid | Sigma Aldrich Chimie | P7949 | |
| Triton X-100 | Sigma Aldrich Chimie | X100-100ML | |
| Human Fibroblasts | Coriell Cell Line Biorepository | GM 4603 and GM 1869 | Coriell Institute for Medical Research, Camden, NJ, USA |
| Confocal laser scanning microscope | Zeiss (Germany) | LSM 700 | |
| Imaris Software | Bitplane AG, Zurich | 6.4.0 version | Filament Tracer and Imaris XT modules are necessary |
| Huygens Software | Huygens software, SVI, Netherlands | Pro version | Optional (for deconvolution testing) |
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