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細胞とその子孫。画像成熟したオリゴデンドロサイトには、21を使用したトランスジェニックマウスをPLPDsRed。一貫性の生存のために、スライスは、アップ前脳と小脳の両方から生後10日目にマウスから単離することができる。 1スライスの準備<ol><li>培養フード中で、6ウェルプレート中で組織培養インサートを配置し、1mlのスライス培養培地（試薬セクションのレシピ）を加える。解剖の前に、5％CO 2を少なくとも2時間、37℃で細胞培養インキュベーターでプレートを置きます。 </li><li> 70％エタノールですべてのツールと​​組織チョッパーを滅菌する。 </li><li>解剖の前の開始に少なくとも15分間、氷上で95％O 2、5％CO 2をバブル解剖バッファー（試薬セクションのレシピ）。 </li><li>承認された動物プロトコールに従って5〜10分間氷上に置くことにより仔マウスを麻酔。マウスは尾とつま先のピンチに応答しないことを確認することにより麻酔の適切な深さを確かめる。 </li><li>動物プロトコールに従って鋭いハサミを使って動物を首を切る。すぐに滅菌ツールを使用して、第一横切開に続いて矢状カットを行うことで、前脳および小脳の上の頭蓋骨を削除します。慎重に側に組織を圧延することにより、脳と小脳の腹側表面から脳神経をカットします。 </li><li>氷のように冷たい酸素添加解剖バッファーを含む滅菌35mmの皿に組織を置きます。 </li><li>カミソリの刃を使用して、小脳および前脳を切断。その後半ば矢状カットのthroを作る前脳および小脳ぐふ、半球を分離する。 </li><li>手動の組織チョッパーで前脳と小脳の300μmの厚さの冠状および矢状断面をカットします。注：手動の組織チョッパーをアガロース包埋を必要とせずに培養インキュベーション解剖からの迅速な処理を可能にする。以前に説明したように9ビブラトームを使用することもできる。 </li><li>氷の上に新鮮に泡立て冷たい解剖バッファーに分けスライスを転送します。 </li><li>各部を分離し、培養インサートで6ウェル皿をプレインキュベートするためにそれらを転送するために、小さな解剖や計量へらを使用してください。 </li><li>使い捨てトランスファーピペットまたはP 200ピペットチップを用いて培養インサートの表面から余分な解剖バッファを削除します。 2〜3前脳または三小脳スライスが1つの培養インサート上に配置することができる。 注：前脳文化と一貫性のある結果のためには、脳梁の前三分の一にまたがるスライスを取ることが理想的である（ 図1A）は、同じスライス内の両方の灰白質と白質の領域を研究するためである。各動物は、通常6前脳スライスと6小脳スライスが得られます。 </li><li>インキュベーター中で6ウェルプレートを置き、スライス固定されるまで一日おきに、次にスライス標本後にスライス培地1ml 1日とスライス培地を交換し。 </li><li>実験によっては、培養中の5-7日後にスライスを使用。最初の数日後に透明になるだけのスライスを使用し、不均一な不透明領域を有するものを捨てる​​。注：スライス内の不透明な領域は、目で表示され、位相差の下で暗い細胞の塊として表示されます。技術が習得された後、死んで不透明なスライスはスライス未満の1〜5％で、めったに起こらない。 </li></ol> 2 NG2細胞分裂のタイムラプスイメージングおよびオリゴデンドロサイト分化注：私たちはNG2creを使用NG2細胞の増殖および分化のタイムラプスイメージングを実行するには：ZEGマウス16NG2細胞およびそれらの子孫（ 図1C-E）でEGFPを発現する。この行のレポーター発現は、直ちに少なくとも4週間の期間にセクショニングの後のスライスでGFP +細胞のライブ画像を得るために十分に堅牢である。鮮明な画像を、培養の最初の3〜5日間で発生したスライスの初期間伐期間後に得られる。 <ol><li>実験の間、37℃で細胞培養インキュベーター内のスライスを維持し、時間の短い期間だけ除去（スライスあたり15分未満）、6ウェルプレートに蓋をしながら撮影を維持する。 </li><li>デジタルカメラを備えた倒立蛍光顕微鏡を用いて、10×対物レンズを用いて第1の時点の画像を取り込む。注記：ファーストタイムポイントは、実験によって異なりますし、後の切片を調製した日またはどの時点であってもよい。 </li><li>スライスの両方グレーと白質領域に時点1での複数の領域から画像をキャプチャします。目に注意してくださいE領域は、スライス内の特定のランドマークによって、後続のイメージングセッションのために使用することができ、回路図に画像の位置をマッピングすることによって画像化した。有用なランドマークは白質路のスライスおよび/または方向のエッジを含むことができる。画像各時点における各スライス上の四から八箇所。 </li><li> NG2細胞の分裂とオリゴデンドロサイト分化を調べる場合には、理想的には上の5-7日、4〜6時間の間隔で次の画像をキャプチャします。 </li><li>すべての地域の最終的な画像をキャプチャし、すぐにスライスを修正します。その後のスライスの上に別の1ミリリットルを追加し、カルチャーインサートの底まで（0.1M L-リジン0.01Mのメタ過ヨウ素酸ナトリウムを用いて4％PFA）固定液1ミリリットルを加える。それが膜から切り離すことができますように、スライスの上に直接固定液を噴出しないように注意してください。 30分間、組織を固定し、4章で説明したように発達段階特異的マーカーを用いた免疫組織化学を進める。 </li><li> 0.2 Mリン酸ナトリウム緩衝液3×10 mを有する洗浄スライスインチ1-3日間、0.2 Mリン酸ナトリウム緩衝液中に4℃で必要に応じて、店舗のスライス24時間以内に染色を行う理想的に免疫染色が、前に。 4章で後述のように、オリゴデンドロサイト（ 図2D-F）に分化した細胞の割合を決定するために、免疫組織化学を続行します。 </li></ol>スライス培養で誘導レポータートランスジェニックマウスを使用したNG2細胞子孫の3運命のマッピング注：培養物中の特定の時点からNG2細胞の運命を追跡するために、誘導性NG2creER：YFPトランスジェニックマウスを使用することができる。 <ol><li>培養培地に、エタノールに溶解した100nMの4OHTを追加することによって、Cre組換えおよびレポーター発現を誘導する。注：レポーター陽性細胞は〜20〜25％YFP + NG2 + / NG2 +細胞の誘導効率で1〜2日以内に表示されますと、この時点ですべてのNG2細胞段階における細胞（ 図2A〜C）になります</li><li>スライスを修正し、洗う異なる時点での、さまざまな操作をした後に（セクション2.5および2.6に記載されている）。 </li></ol> 4。スライス免疫組織化学<ol><li>メスを用いて、インサートの外に取り付けられたスライスを有する膜をカット。 </li><li>膜をピックアップする際のスライスの組成を破壊しないように注意しながら、ピンセットのセットを用いてリン酸緩衝生理食塩水（PBS）を含有する24ウェルプレートの各ウェルにスライスを転送する。 </li><li> 1時間PBS中の5％正常ヤギ血清（NGS）および0.1％トリトン-X100を含有するブロッキング溶液で24ウェルプレートにスライスを転送する。 </li><li> 4℃で、PBS中の5％NGS中の一次抗体でO / N内のスライスをインキュベートする。 NG2細胞段階における細胞を同定するために、NG2およびPDGFRαに対する抗体を使用しています。分化したオリゴデンドロサイトを識別するための、大腸腺腫性ポリポーシス抗原に対する抗体を使用する（APC;クローンCC1）を。 </li><li> PBS 3×15分で二日目の洗浄スライス上で、その後二次antiboでインキュベート室温で1時間、PBS中の5％NGSで死ぬ。 PBS 3×15分でスライスを洗浄し、スライド上にマウントします。膜は客観的かつ組織との間にされないようにスライドが直面しているスライスアップと膜とのマウント。 </li></ol>

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+third+dimension+bridges+the+gap+between+cell+culture+and+live+tissue.">The third dimension bridges the gap between cell culture and live tissue.</a> Nature Reviews. Molecular Cell Biology. 8 (10), 839-845 (2007).</li><li>Lubetzki, C., Demerens, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Even+in+culture,+oligodendrocytes+myelinate+solely+axons.">Even in culture, oligodendrocytes myelinate solely axons.</a> Proceedings of the National Academy of Sciences of the United States of America. 90 (14), 6820-6824 (1993).</li><li>Demerens, C., Stankoff, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+myelination+in+the+central+nervous+system+by+electrical+activity.">Induction of myelination in the central nervous system by electrical activity.</a> Proceedings of the National Academy of Sciences of the United States of America. 93 (18), 9887-9892 (1996).</li><li>Wood, P. M., Bunge, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+sensory+axons+are+mitogenic+for+Schwann+cells.">Evidence that sensory axons are mitogenic for Schwann cells.</a> Nature. 256 (5519), 662-664 (1975).</li><li>Stevens, B., Tanner, S., Fields, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+myelination+by+specific+patterns+of+neural+impulses.">Control of myelination by specific patterns of neural impulses.</a> The Journal of Neuroscience. 18 (22), 9303-9311 (1998).</li><li>Stevens, B., Porta, S., Haak, L. L., Gallo, V., Fields, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenosine:+a+neuron-glial+transmitter+promoting+myelination+in+the+CNS+in+response+to+action+potentials.">Adenosine: a neuron-glial transmitter promoting myelination in the CNS in response to action potentials.</a> Neuron. 36 (5), 855-868 (2002).</li><li>Wake, H., Lee, P. R., Fields, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+local+protein+synthesis+and+initial+events+in+myelination+by+action+potentials.">Control of local protein synthesis and initial events in myelination by action potentials.</a> Science. 333 (6049), 1647-1651 (2011).</li><li>Rosenberg, S. S., Kelland, E. E., Tokar, E., De la Torre, A. R., Chan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+geometric+and+spatial+constraints+of+the+microenvironment+induce+oligodendrocyte+differentiation.">The geometric and spatial constraints of the microenvironment induce oligodendrocyte differentiation.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (38), 14662-14667 (2008).</li></ol>

The authors declare that they have no competing financial interests

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 ...

organotypic-slice-cultures-to-study-oligodendrocyte-dynamics

Research

JoVE Journal

Neuroscience

18.2K ビュー.  University of Connecticut. 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.

ビデオ: Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying 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

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

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

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. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

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

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

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

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

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

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

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

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

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

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

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

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

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

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

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

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior 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. ...