分离培养成年斑马鱼脑源性神经球
Summary
在这里，我们提供一个可重复的方法，使用从整个脑或从任一端脑，顶盖或成年斑马鱼脑的小脑区域衍生的神经球测定以检查成人神经发生。此外，我们描述来操纵在斑马鱼神经球的基因表达的步骤。
Abstract
The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.
Introduction
哺乳动物神经干细胞（NSCs）都被他们在自由浮动的文化成长为划分称为神经球1细胞群的能力，其特征体外 。在表皮生长因子（EGF）和成纤维细胞生长因子（FGF）的存在下，神经干细胞分化或对称，产生自我更新的神经干细胞，或不对称，以产生两个不同的子细胞， 即 ，微分祖细胞和新颖NSC。因此，神经干文化是神经干/祖细胞多分化的神经细胞2-4的混合物的神经干细胞可以，但是，从其他神经干细胞类型由两个特定的属性区别：他们自由显示屏长期自我更新浮培养物，它们可以分化成以下的生长因子撤出和粘附到细胞外基质底物的所有神经细胞谱系（ 即 ，神经元，星形胶质细胞和少突胶质）。在mammALS的神经球培养系统是用来证明神经干细胞在成人大脑中存在的第一个体外系统和保持来分析增殖，自我更新能力和神经干细胞和祖细胞的多能的最常用的工具。因此，即使球形成测定法从一些缺点和局限性4苦，该培养系统，是从它的体内利基4中取出时，并一直工具在确定关键调节评估细胞的表现为干细胞的潜在有价值NSC的自我更新和细胞命运决定5-7。
相较于哺乳动物谁限制了成年神经，斑马鱼组成产生沿着整个生命全脑轴新的神经细胞。斑马鱼的成人脑显示多个神经源性龛窝藏神经干/祖细胞使斑马鱼强大的模式生物的的理解了他干在脑细胞活动，以及中枢神经系统的再生所需要的分子的程序。在过去的17年里，几个研究小组开发的方法进行分离和培养斑马鱼的神经细胞8,9。这些研究的目的是生产胚胎神经元和在体外神经胶质细胞，而不是在保持NSCs的并调查它们的属性。虽然神经球已在成年Apteronotus leptorhynchus（布朗鬼西刀鱼）10产生的，在斑马鱼神经球形成测定法仍有待建立。
这里，我们描述一个神经球形成测定法来证明斑马鱼神经11中的miR-107的作用。该协议使得：1）无论是从斑马鱼全脑或从几个解剖大脑区域，如端脑，所述顶盖和小脑成人神经干/祖细胞的收集; 2）FL的代浮动和成人神经干/祖细胞的自我更新的神经球; 3）向下和上调编码基因或小的非编码RNA 11在神经球，的表达为了研究它们在神经干/祖细胞的增殖和分化的作用。
Protocol
Representative Results
Discussion
该协议的主要目的是分离和培养成年斑马鱼脑源性神经球用于研究神经干/祖细胞的细胞和分子的特性。在这里，我们报告如何选择多能神经细胞并产生三个神经细胞类型， 即 ，星形胶质细胞，神经元和少突胶质细胞，从成年斑马鱼大脑。因为可重现神经球形成测定法尚未在斑马鱼建立迄今为止协议是非常显著。
在协议中的几个关键步骤，需要得到尊重，并在故障排?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors thank Guillermina Hill-Teran and Marie-Elise Schwartz for assistance. This work was supported by grants from the National Institutes of Health (5R00HL105791 to S.N.) and from the Alzheimer (NIRP 12-259162). This work was also supported by Institut National de la Santé et de la Recherche Médicale (CFC and JLT), Agence Nationale de la Recherche (13-BSV4-0002-01 (JLT), NIH (1R01EB016629-01A1 (JLT), Connecticut Stem Cell Research Fund (13-SCA-Yale-04 (JLT).
Materials
| DPBS 1X | Life Technologies | 14190-144 | |
| DMEM/F12 1X | Life Technologies | 11330-032 | |
| L-Cysteine hydrochloride monohydrate | Sigma | C6852-25g | |
| B-27 | Life Technologies | 17504-044 | |
| N-2 | Life Technologies | 17502-048 | N-2 supplement (100x) liquid |
| HEPES | Life Technologies | 15630 | 1M |
| D-(+)-Glucose 45% | Sigma | G8769 | |
| Penicillin-streptomycin | Life Technologies | 15140-122 | |
| Fetal Bovine Serum | Life Technologies | 16000044 | |
| Human FGF-basic | Peprotech | 100-18B | |
| Human EGF | Peprotech | AF-100-15 | |
| Insulin | Sigma | I5500-50 mg | |
| DNAse | Sigma | DN25-10mg | |
| Papain | Worthington Biochemical Corporation | LS003126 | |
| Matrigel | Becton Dickinson | 356234 | |
| PFA | TCI | P0018 | |
| PBS | AmericanBio | AB11072-04000 | |
| Tricaine MS-222 | Sigma | A5040 | stock solution of 4 mg/ml. |
| Trycold gel | Sigma | TGP8 | gel pack |
| Amaxa Basic Nucleofector Kit | Lonza | VPI-1004 | |
| Trypan blue stain | Life Technologies | 15250061 |
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