Name: organotypic slice cultures to study oligodendrocyte dynamics and myelination
Uploaded: 2014-08-25T15:00:00
Description: Watch this Scientific Journal Video about Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination at JoVE.com

This work was funded by grants from the National Multiple Sclerosis Society (RG4179 to A.N.), the National Institutes of Health (NIH R01NS073425 and R01NS074870 to A.N.) and the National Science Foundation (A.N.). We thank Dr. Frank Kirchhoff (University of Saarland, Homburg Germany) for providing PLPDsRed transgenic mice. We thank Youfen Sun for her assistance in maintaining the transgenic mouse colony.

注：すべての動物の手順は、ガイドラインに従っているとコネチカット大学の施設内動物管理使用委員会（IACUC）によって承認されている。 注：それぞれ用いたYFPレポーター20（JAX＃006148）ライン：これらの実験のために、構成NG2cre 18（JAX番号008533）および誘導性NG2creER 16（JAX番号008538）トランスジェニックマウスは、Z / EG 19（JAX＃003920）またはgtRosa26交配画像NG2細胞と?...

<ol>
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中枢神経系の髄鞘形成は、効率的な神経細胞のコミュニケーションと軸索の生存22に不可欠である。 25 -ほとんどの脳領域16,23における居住人口を維持しながら、NG2細胞は、継続的に大人になってミエリンオリゴデンドロサイトを生成します。これらの細胞の分化を調節するいくつかの遺伝的および分子メカニズムを説明してきたが、多くは発見されていない。器官型?...

4 -中枢神経系のOrganoytpicスライス培養はsemiintactシステム1において、ニューロンおよびグリア細胞生物学を研究するために極めて有用であることが証明されている。 9 -これらの培養物を採用し、そのような細胞外環境の操作を繰り返し、長期生細胞イメージングのための容易なアクセスおよび電気生理学的記録5と、一次解離細胞培養の多くの利点を保持することは比較的簡単である。また、スライス培養は、3次元組織細胞構築、地域の神経接続を維持し、ほとんどの主要な細胞型がシステムに存在している。これらの特性は、これらの培養ユニークで便利なシステムは、セルラーと環境の相互作用を持つ単一細胞の挙動や生理学を調査することにします。 NG2細胞は増殖およびmを生成し続ける、哺乳動物の中枢神経系におけるグリア細胞の集団である胚および生後発育10の間にオリゴデンドロサイトをyelinating。それらは、広範囲に解離した初代細胞培養物において研究されており、蛍光タンパク質のNG2細胞特異的発現を有するトランスジェニックマウス系統の最近の開発は、急性スライス標本におけるインビボでの運命マッピングおよび電気生理学的記録に容易になった。でも、これらの研究で、少しはNG2細胞の増殖およびオリゴデンドロサイト分化の時間的なダイナミクスについてはほとんど知られていない。解離した細胞培養が広く薬理学的および遺伝的操作の相対的機能のために使用されるが、それは細胞の微小環境のコンテキストを維持することが望ましい場合に特に異なる脳領域におけるこれらの細胞の機能的な違いを問い合わせるには適していない。スライス培養は、細胞の薬理学的操作に適しているとオリゴデンドロサイトミエリン形成11,12を調査するために使用されている単純な代替物を提供する（LPC）をリゾレシチンまたは抗体誘発性脱髄13,14、および薬理学的治療15を介して再ミエリンの誘導後ウラル応答。 前脳および小脳の両方から採取した器官型スライス培養におけるNG2細胞増殖およびオリゴデンドロサイト分化の分析をライブイメージングし、固定組織（または後固定）を調査し、実行するための方法が記載されている。これは、分割後に単16 NG2細胞の細胞運命を研究するために、成長因子誘導されるNG2細胞増殖の17地域および年齢依存性の違いを発見するために使用できる強力な方法である。この比較的単純な技術はさらに、これらのグリア細胞の生理学および神経活動またはミエリン損傷への応答を調節する細胞内因性および/または環境のメカニズムを調査する、広くアクセス可能です。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Slice Culture Medium</td>
		</tr>
		<tr>
			<td>Minimum Essential Medium</td>
			<td>Invitrogen</td>
			<td>11090</td>
			<td>50%</td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution</td>
			<td>Invitrogen</td>
			<td>14175</td>
			<td>25%</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H-4034</td>
			<td>25mM</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Invitrogen</td>
			<td>25030</td>
			<td>1mM</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I6634</td>
			<td>1mg/L</td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>A-0278</td>
			<td>0.4mM</td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Hyclone</td>
			<td>SH30074.03</td>
			<td>25%</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Titrate to pH 7.22 with NaOH, filter with bottle top filter, and store at 4 degrees Celsius</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Dissection Buffer</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td>124mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P5405</td>
			<td>3.004mM</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td>1.25mM</td>
		</tr>
		<tr>
			<td>MgSO4 (anhydrous)</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td>4.004mM</td>
		</tr>
		<tr>
			<td>CaCl2 2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>C7902</td>
			<td>2mM</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td>26mM</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td>10mM</td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>A-0278</td>
			<td>2.0mM</td>
		</tr>
		<tr>
			<td>Adenosine</td>
			<td>Sigma-Aldrich</td>
			<td>A4036</td>
			<td>0.075mM</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Filter with bottle top filter and store at 4 degrees Celsius, oxygenate with 95%O2 5%CO2 before use</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Other Reagents and Supplies</td>
		</tr>
		<tr>
			<td>4-hydroxy tamoxifen (4OHT)</td>
			<td>Sigma-Aldrich</td>
			<td>H7904</td>
			<td>100nM</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>EM Sciences</td>
			<td>19200</td>
			<td>4%</td>
		</tr>
		<tr>
			<td>L-Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>L-6027</td>
			<td>0.1M</td>
		</tr>
		<tr>
			<td>Sodium Metaperiodate</td>
			<td>Sigma-Aldrich</td>
			<td>S-1878</td>
			<td>0.01M</td>
		</tr>
		<tr>
			<td>Rabbit anti NG2 antibody</td>
			<td>Chemicon (Millipore)</td>
			<td>AB5320</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Mouse anti CC1 antibody</td>
			<td>Calbiochem (Millipore)</td>
			<td>OP80</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Mouse anti Ki67 antibody</td>
			<td>Vision Biosystems</td>
			<td>NCL-Ki67-MM1</td>
			<td>1:300</td>
		</tr>
		<tr>
			<td>Mouse anti NeuN antibody</td>
			<td>Chemicon (Millipore)</td>
			<td>MAB377</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Species specific secondary antibodies</td>
			<td>Jackson Immunoresearch</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum (NGS)</td>
			<td></td>
			<td></td>
			<td>5%</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td></td>
			<td></td>
			<td>0.10%</td>
		</tr>
		<tr>
			<td>Mounting medium with DAPI</td>
			<td>Vector Labs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell culture membrane inserts 0.45&#956;m pore size</td>
			<td>Fisher Scientific</td>
			<td>PICM03050</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle top filters</td>
			<td>Fisher Scientific</td>
			<td>09-740-25E</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>95% O2 5% CO2 gas mix</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile six well plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting (hippocampal) or weighing spatulas</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Manual or automatic tissue chopper</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable transfer pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>35mm and 60mm sterile Petri dishes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Phase Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar Flow Hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Fluorescence Microcope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

organotypic slice cultures to study oligodendrocyte dynamics and myelination

NG2を発現する細胞（polydendrocytes、オリゴデンドロサイト前駆細胞）は、中枢神経系における第四の主要なグリア細胞集団である。胚および生後発育の間、彼らは積極的に増殖し、ミエリンオリゴデンドロサイトを生成します。これらの細胞は、一般的に、一次解離し文化、ニューロン共培養において、および固定された組織で研究されてきた。新たに利用可能なトランスジェニックマウス系統は、培養系をスライス使用すると、前脳および小脳の両方の灰白質と白質領域においてオリゴデンドロサイト系譜細胞の増殖および分化を調査するために使用することができる。スライス培養は、生後初期のマウスから調製され、最大1ヶ月間培養物中で維持される。これらのスライスは、細胞の挙動および相互作用を調査するために、培養期間にわたって複数回撮像することができる。この方法は、NG2細胞分裂の可視化との詳細な分析を可能にしながら、オリゴデンドロサイト分化を導くステップ地域に依存可能にするカミカゼNG2細胞とオリゴデンドロサイトの機能異質。これは、密接にインビボで見出さことに似ている細胞環境において経時的にこれらの細胞に影響を与える内因性および外因性のシグナルを調査するために使用することができる強力な技術である。

ZEG、NG2creER：YFP及びPLPDsRedトランスジェニックマウス系統の代表的なデータの例は、P8 NG2creの前脳および小脳の両方からスライス培養を用いて得られたその下に示す。 NG2細胞は、前脳および小脳切片における日間（ 図1B-D、ビデオ1）の上に画像化することができ、これらの細胞の表現型は、固定およびNG2およびCC1抗体による免疫染色（ 図1E）後に決定することができ...

Watch this Scientific Journal Video about Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination at JoVE.com

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

オリゴデンドロサイトダイナミクスと髄鞘形成を研究するための器官型スライス培養

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

Research

JoVE Journal

Neuroscience

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

神経損傷を調べるための器官型海馬スライス培養モデル

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

神経科学

Organotypic Hippocampal Slice Cultures

器官型海馬切片培養

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

器官小脳文化：アポトーシス課題と検出

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

新皮質の器官型スライス標本からの全細胞記録

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

器官培養における神経雪崩のマルチ電極アレイの録音

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

出生後のマウス組織から培養オリゴデンドロサイトと共培養Myelinatingニューロン/オリゴデンドロサイトエンリッチドの導出

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

胚腹側中脳の器官型スライス培養：ドーパミン作動性ニューロンの発達を研究するための体制 in vitroで</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

器官切片培養におけるプルキンエ細胞の樹状突起の形態分析

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Entorhino海馬器官型スライス培養における神経血管リモデリングの解析

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

研究保護および腫瘍細胞の侵襲性をモデルとして脊髄海馬スライス培養

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...