Name: isolation and culture of adult zebrafish brain-derived neurospheres
Uploaded: 2016-02-29T15:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres at JoVE.com

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 Sant&#233; et de la Recherche M&#233;dicale (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).

Zebrafish of the WTCF strain were raised and maintained according to protocols approved by the Yale University Institutional Animal Care and Use Committee (IACUC protocol number 2012-11473). All experiments should first be approved by all relevant governmental and institutional ethics regulating bodies regarding the use of animals for research purposes.
1. Preparations
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	<li>Prepare 10 ml of dissection medium, add 200 &#181;l of 100x penicillin-streptomycin i...

<ol>
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The main aim of this protocol is to isolate and culture adult zebrafish brain-derived neurospheres for studying the cellular and molecular features of neural stem/progenitor cells. Here, we report how to select multipotent neural cells and generate the three neural cell types, i.e., astrocytes, neurons and oligodendrocytes, from the adult zebrafish brain. The protocol is highly significant since a reproducible neurosphere-forming assay had not been established in the zebrafish so far.
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Mammalian neural stem cells (NSCs) have been characterized in vitro by their ability to grow in free-floating cultures as clusters of dividing cells termed neurospheres1. In the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF), NSCs divide either symmetrically to generate self-renewing NSCs, or asymmetrically to generate two different daughter cells, i.e., a differentiating progenitor cell and a novel NSC. Neurosphere cultures are therefore a mixture of neural stem/progenitor cells and more differentiated neural cells2-4. NSCs can, however, be distinguished from other neurosphere cell-types by two specific properties: they display long-term self-renewal in free-floating cultures and they can differentiate into all neural cell lineages (i.e., neurons, astrocytes, and oligodendrocytes) following withdrawal of growth factors and adhesion to extracellular matrix substrates. In mammals, the neurosphere culture system was the first in vitro system used to demonstrate the presence of NSCs in the adult brain and remains the most commonly used tool to analyze proliferation, self-renewal capacity and multipotency of neural stem and progenitor cells. Therefore, even though sphere-forming assays suffer from some disadvantages and limitations4, this culture system is valuable for evaluating the potential of a cell to behave as a stem cell when removed from its in vivo niche4 and has been instrumental in identifying key regulators of NSC self-renewal and cell fate determination5-7.
In contrast to mammals who have limited adult neurogenesis, zebrafish constitutively produce new neurons along the whole brain axis throughout their life. The zebrafish adult brain displays multiple neurogenic niches harboring neural stem/progenitor cells making zebrafish a powerful model organism for understanding the stem cell activity in the brain as well as the molecular programs required for central nervous system regeneration. Over the past 17 years, several research groups developed methodologies for isolating and culturing zebrafish neural cells8,9. These studies were aimed at producing embryonic neuronal and glial cells in vitro, but not at maintaining NSCs and investigating their properties. Although neurospheres have been generated in the adult Apteronotus leptorhynchus (Brown Ghost Knifefish)10, a neurosphere-forming assay in the zebrafish remained to be established.
Here we describe a neurosphere-forming assay to demonstrate the role of miR-107 during zebrafish neurogenesis11. The protocol enables: 1) the collection of adult neural stem/progenitor cells either from zebrafish whole brain or from several dissected brain regions such as the telencephalon, the tectum, and the cerebellum; 2) the generation of floating and self-renewing neurospheres from adult neural stem/progenitor cells; 3) the down- and up-regulation of the expression of coding genes or small non-coding RNAs11 in neurospheres, in order to investigate their roles in the proliferation and differentiation of neural stem/progenitor cells.

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			<td>DPBS 1X</td>
			<td>Life Technologies</td>
			<td>14190-144</td>
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			<td>DMEM/F12 1X</td>
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			<td>11330-032</td>
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			<td>L-Cysteine hydrochloride monohydrate&#160;</td>
			<td>Sigma</td>
			<td>C6852-25g</td>
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			<td>B-27</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
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			<td>N-2</td>
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			<td>N-2 supplement (100x) liquid&#160;</td>
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			<td>HEPES&#160;</td>
			<td>Life Technologies</td>
			<td>15630</td>
			<td>1M</td>
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			<td>D-(+)-Glucose 45%&#160;</td>
			<td>Sigma</td>
			<td>G8769</td>
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			<td>Penicillin-streptomycin&#160;</td>
			<td>Life Technologies</td>
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			<td>Fetal Bovine Serum&#160;</td>
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			<td>16000044</td>
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			<td>&#160;100-18B</td>
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			<td>AF-100-15</td>
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			<td>Insulin&#160;</td>
			<td>Sigma</td>
			<td>I5500-50 mg</td>
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			<td>DNAse</td>
			<td>Sigma</td>
			<td>DN25-10mg</td>
			<td></td>
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			<td>Papain&#160;</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LS003126</td>
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			<td>Matrigel&#160;</td>
			<td>Becton Dickinson</td>
			<td>356234</td>
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			<td>PFA&#160;</td>
			<td>TCI</td>
			<td>P0018</td>
			<td></td>
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			<td>PBS</td>
			<td>AmericanBio</td>
			<td>AB11072-04000</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine MS-222</td>
			<td>Sigma</td>
			<td>A5040</td>
			<td>stock solution of 4 mg/ml.&#160;</td>
		</tr>
		<tr>
			<td>Trycold gel&#160;</td>
			<td>Sigma</td>
			<td>TGP8</td>
			<td>gel pack</td>
		</tr>
		<tr>
			<td>Amaxa Basic Nucleofector Kit</td>
			<td>Lonza</td>
			<td>VPI-1004</td>
			<td></td>
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			<td>Trypan blue stain&#160;</td>
			<td>Life Technologies</td>
			<td>15250061</td>
			<td></td>
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isolation and culture of adult zebrafish brain-derived neurospheres

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.

General Scheme of the Adult Zebrafish Neurosphere Culture
Here we describe all the steps of the protocol of a neurosphere-forming assay performed from the adult zebrafish brain. First, adult neural stem/progenitor cells have been collected either from zebrafish whole brain or from several dissected brain regions such as the telencephalon, the tectum and the cerebellum (Figures 1A-C). Single cell suspension of ad...

Watch this Scientific Journal Video about Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres at JoVE.com

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

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