本研究では、イプセン財団によって一部で賄われていた。 IMVは、分子生物学のアメリカの癌協会の教授であり、そして典型的なライフサイエンスのアーウィンとジョーンジェイコブスチェアを保持しています。この作品は、NIH、Leducq財団、Meriaux財団、エリソン医学財団、イプセン/ Biomeasure、サノフィアベンティス、前立腺がん財団、およびHNとフランシスC. Bergerの財団からの補助金によって部分的にサポートされていました。 PASは、神経科学のためのマックナイト基金基金と薬物乱用（R01 DA019022）の国民の協会によって資金を供給される。著者はマークシュミットに感謝します。グラフィックアートはジェイミーT.サイモンによって設計されました。スポンサーが親切にInvitrogen社によって提供された。

1。器官小脳文化： <ol><li>が正常に生成するために器官小脳培養は、P0〜P3やP8 - P12の間にマウスを使用してください。この発達段階は、我々の研究のためのより適切だったので私たちの実験のために我々は、P8 - P12の範囲でマウスを使用していました。 </li><li>解剖と培養液を準備します。 <ol><li>脳の解剖やスライス標本を1mMの塩化カルシウムを含む低ナトリウム人工脳脊髄液（ACSF）、10mMのD -グルコース、4mmの塩化カリウム、5mMの塩化マグネシウム、26ミリメートル、重炭酸ナトリウム、246mMスクロースおよびフェノールレッド溶液（1:1000）で実施されています、pHは7.3。ソリューションを殺菌するために、4で0.22μmのフィルターとストアを介してフィルタ℃に</li><li>小脳スライスを75％最小必須培地イーグル（MEM）、25％熱不活化ウマ血清、25mMのHEPES、1mMのグルタミン、5mgの/ mLのグルコース、ペニシリン（100 U / mL）およびストレプトマイシン（100 U / mL）に培養されています。二週間、最大で4で0.22μmフィルターとストア° Cを介してメディアをフィルタします。 </li></ol></li><li>それが赤からオレンジ色にシフトするので、以前にCarbogen（95％酸素、5％二酸化炭素）（注）でバブリング氷冷ACSF培地に脳を解剖し、Carbogenとその飽和は、溶液のpHを変更します。解剖は、脳の構造を損傷しないように注意しながら可能な限り最小限の時間で実行する必要があります。 </li><li>ビブラトームを用いて400μmの矢セクションで脳をスライスして。 <ol><li>小脳の可能な限り少ないカットしようとして、一方の半球にカミソリの刃と矢状切断してください、これはスライスのために平坦な表面を提供します。皮質は、スライスを簡単にするためにサポートのためにのみ使用されますが、時間が重要な問題であるため、それはかみそりの刃で皮質の吻側半分をカットする方が便利です。 </li><li>金属のビブラトームプレートに脳の位置を固定する（スーパーグルー）シアノアクリレートを使用します。接着剤を追加する前に氷の上でプレートを冷却する為、薄い接着剤層を形成するためにチューブの先端を使用しても、それを広めることが重要です。スーパー接着剤の過剰がある場合はそれはACSFに接触し、サンプルが失われるとき、それはプレートから分離させます。 </li><li>一度脳は、脳をカバーし、それが冷たい保つためにプレートの下に氷を置くのに十分なACSFのメディアを追加し、ビブラトームプレートに取り付けられています。 </li><li> 400μmのスライスをカットし、氷上でACSFを含む6ウェル細胞培養プレートにプラスチック製パスツールピペット（それを広げ、スライスへの損傷を避けるために、先端をカット）とそれらを転送する。 </li><li>解剖顕微鏡未満のインスリンの注射器（小口径28ゲージの針付き注射器）の助けを借りて脳の残りの部分から小脳を区切ります。いくつかのスライスは、スーパー接着剤が端に接続されている必要があります：これは、スライスの存続に影響を与えることができるとして、それを削除しようとする瞬間です。小脳の構造を損なうことなく、慎重にそれをすることが重要です。 </li></ol></li><li> 0.4μmの細孔と30ミリメートル培養プレートのインサートを含む6ウェルプレートに小脳スライスを転送する。 ℃、5％CO 2（細胞培養インキュベーター内）で37少なくとも2時間インキュベーションすることにより事前に調整されたウェル当たりの培地、1 mLを追加します。に気泡が下敷きにしないし、彼らが完全に引き出されているとは、液体がそれらをカバーしていないことを確認してインサートの上に小脳スライス（インサートあたり1-3）に配置されていることを確認してメディアの上に挿入して置きます。必要に応じて、メディアの過剰を吸引除去する。 37 ° C、5％CO 2培養液ごとに2日または3日を変更する。使用される培地は、細胞の生存にとって重要なスライス由来栄養因子を含んでいるので、それは、一度に培地の一部のみ（例えば½ボリューム）に置き換えるのがベストです。 </li></ol> 2。小脳スライスのFas処理と染色： <ol><li>アポトーシスチャレンジ実験のため、我々第一文化試験管 3（DIV3） で一日でメディアを変更する5日間のスライス。 DIV5文化にメディアの変化は文化のいくつかの死亡を誘発することができるので、細胞死のきれいな背景を持つためにメディアに直接追加することによって、Fas抗体のアゴニスト抗体のJo2のの0.5μg/ mLで扱われます。井戸の半分は、コントロールとして未処理のままされています。 </li><li> 24時間後に吸引でインサートの下にメディアを取り外します。スライスを修正するには、下に氷冷4％PFAを1mLとインサート上に1 mLを加える。室温で10分間インキュベートする。 </li><li> 10分後にPFAを削除し、冷PBSでサッと洗う。 </li><li>次のPBSを除去し、室温で5分間、PBSとインキュベートの20％氷冷メタノールのインサートの上に1mLの下に、1 mLを加える。 </li><li>再びPBSで洗浄し、下に1 mlを添加し、透過性のためのPBS中の0.5％のインサートトリトン- X - 100上記の1 mLを。 4℃で12時間の最小インキュベート℃に</li><li>透過した後、最小のためにPBSで20％BSAでブロックする4室の温度で時間または4℃で一晩この段階でスライスを4で少なくとも3日間℃でブロッキング溶液で保存することができます</li><li>スライスを染色するために、慎重に小脳スライスに接続されている挿入膜の一部をカットし、PBSを含む24ウェルプレートに移す。 </li><li>免疫染色は一晩1時間TUNEL試薬で最初にしてCalbidinの抗体を用いて、順番に行われます。 <ol><li> PBSを吸引除去し、各ウェルは、スライスの表面全体を確実に覆うことにTUNEL試薬から250μLを加える。暗所で一時間、37℃でインキュベートする。 </li><li>一時間はTUNELミックスを削除し、10分間PBSで3回洗浄した後。 </li><li>最後の洗浄後、PBSを除去し、PBSでカルビンジン抗体の1 / 1000希釈、250μLを追加し、4℃で一晩インキュベートします暗所で。 </li><li>一次抗体溶液を除去し、10分間PBSで3回洗浄する。 </li><li>暗所で室温で少なくとも3時間のためにPBSとインキュベートで1 / 500希釈した二次抗体250μLを加える。我々が使用TUNELキットはTMR赤で標識、従ってカルビンジンのための私たちの二次抗体はAlexa - 488に結合されるている。 </li><li> PBSで10分間3回洗浄し、DAPIを含むマウントメディアを使用してガラスの顕微鏡スライド上にスライスをマウントします。 </li><li>それぞれカルビンジンとTUNELのために488 nmの、561 nm励起wavelenghtを使用して共焦点顕微鏡と画像のスライスを。 </li></ol></li></ol> 3。代表的な結果： このプロトコルの主な課題は、私たちの最終的な読み出しが細胞死となる、特に以来、健康な小脳スライス培養を生成できるようにすることです。健全な文化は、細胞体層は半透明なものとして表示され、顕微鏡（図1）下小脳の葉状構造を見ることができます。文化の数日後のスライスが薄いと非神経細胞に開始するが、スライスのマージンからの移行を観察することができます。文化の数日後のスライスをカバー暗い円形の細胞は（おそらくマクロファージ）、最終的にin vitroで 10〜14日（図2）5の後で数に減少します。スライスの生存のための究極の証拠は、このようなプルキンエ細胞のためのカルビンジンなど、さまざまな細胞マーカー、のために染色することです。図3は、いくつかのTUNEL陽性の核とプルキンエ細胞層の代表的な共焦点顕微鏡画像を示しています。 いくつかのスライスは、アポトーシス刺激を受けた場合であっても、それらは唯一の24時間暴露されたことを考慮することが重要です。これは、TUNEL染色による死細胞のDNA断片化を観察するのに十分な時間を使用できますが、定義されたプルキンエ細胞層は依然として存在していた。 <img src="/files/ftp_upload/2564/2564fig1.jpg" alt="図1" /> 図1。 DIV2で健康な小脳スライス。この画像半透明の細胞体層と健全なスライスの葉状小脳の構造を示しています。 <img src="/files/ftp_upload/2564/2564fig2.jpg" alt="図2" /> 図2。 DIV 7の健康な小脳スライス。この画像では、我々はまだ小脳と暗い細胞体の外観の葉状構造を観察することができます。 <img src="/files/ftp_upload/2564/2564fig3.jpg" alt="図3" /> 図3。 Fas抗体処理した小脳スライスの代表的なイメージが。この共焦点画像は、カルビンジン（緑）とTUNEL染色（赤）の陽性アポトーシス細胞で染色したプルキンエ細胞を示しています。プルキンエ細胞は、単一の行に表示されませんが、薄層のような構造を形成するクラスタ化されています。

<ol>
	<li>Hogue, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+fetal+ependymal+cells+in+tissue+cultures.">Human fetal ependymal cells in tissue cultures.</a> Anat Rec. 99, 523-529 (1947).</li><li>Hogue, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+studies+of+human+fetal+brain+cells+in+tissue+cultures.">Review of studies of human fetal brain cells in tissue cultures.</a> Etudes Neonatales. 1, 1-13 (1952).</li><li>Gogolla, N., Galimberti, I., DePaola, V., Caroni, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staining+protocol+for+organotypic+hippocampal+slice+cultures.">Staining protocol for organotypic hippocampal slice cultures.</a> Nat Protoc. 1, 2452-2456 (2006).</li><li>Stoppini, L., Buchs, P. A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> J Neurosci Methods. 37, 173-182 (1991).</li><li>Beat, H., G&#228;hwiler, S. M. T., McKinney, R. A., Debanne, D., Robertson, R. T., Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Organotypic Slice Cultures of Neural Tissue in Culturing Nerve Cells. , 461-498 (1998).</li></ol>

実施したすべての動物実験は、IACUCによって確立された方針に従って行われていた。ソーク研究所は、AAALAC認定施設です。

このメソッドは、神経器官の文化の多くの可能なアプリケーションの一つを説明し、それは、私たちは野生型と変異型脳切片におけるアポトーシス刺激の効果を検討することができました。 この実験の最も重要な部分は一貫して健康な小脳スライス培養を生成できるようにすることです。主な要因は、解剖やスライシング手法と可能な限り最短時間でプロトコルを実行する機能ですが、同時にスライスを傷つけないように気をつけながら。別の重大な要素は、培地の調製と組成物である、これらの文化は非常に敏感ですので、メディアのいくつかのレシピやブランドをテストしなければならなかった。血清またはメディアの他のコンポーネントのであっても別のバッチは、スライスの生存に影響を与える可能性があります。我々は、Invitrogenのロット＃480116からの馬の血清と最良の結果を得た。 小脳スライスが生成されると一小脳開発、電気生理学、単独でまたはアポトーシス刺激、興奮または酸素欠乏に応答して細胞の生存率を検討することができます。野生型と変異型小脳スライスを用いて比較研究は、小脳の機能と開発の多くの面で非常に洞察されている。

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11090</td>
<td>&nbsp;</td>
<tr>
<td>Horse Serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16050</td>
<td>&nbsp;</td>
<tr>
<td>Hepes</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15630</td>
<td>&nbsp;</td>
<tr>
<td>Glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/ Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Superglue</td>
<td>&nbsp;</td>
<td>Krazy Glue</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>6 well / 24 well cell culture plates</td>
<td>&nbsp;</td>
<td>Corning Incorporated</td>
<td>3506/ 3527</td>
<td>&nbsp;</td>
<tr>
<td>30mm culture plate inserts (0.4-&mu;m pore)</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>PICM03050</td>
<td>&nbsp;</td>
<tr>
<td>Purified NA/LE Hamster anti-mouse CD95 (Jo2)</td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>554254</td>
<td>&nbsp;</td>
<tr>
<td>In situ cell death detection kit, TMR red</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>12 156 792 910</td>
<td>&nbsp;</td>
<tr>
<td>Rabbit anti-Calbindin D-28k antibody</td>
<td>&nbsp;</td>
<td>Swant</td>
<td>CB-38a</td>
<td>&nbsp;</td>
<tr>
<td>Alexa 488-goat anti-rabbit IgG secondary antibody</td>
<td>&nbsp;</td>
<td>Invitrogen (Molecular probes)</td>
<td>A-11008</td>
<td>&nbsp;</td>
<tr>
<td>Vectashield Mounting Medium with DAPI</td>
<td>&nbsp;</td>
<td>Vector labs</td>
<td>H-1200</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting Microscope</td>
<td>&nbsp;</td>
<td>Zeiss Stemi SV 11</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vibratome</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Confocal Microscope</td>
<td>&nbsp;</td>
<td>Leica TCS SP2 AOBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
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organotypic cerebellar cultures: apoptotic challenges and detection

神経組織の器官型培養物は、1947年1,2でホギュが導入され、神経科学の分野で大きなブレークスルーを構成している。それ以来、技術は、器官培養を準備するために多くの異なる方法があるさらに、現在は開発されました。ここで紹介する方法はStoppini らによって記述される1つから翻案されました。スライスの準備のためとGogolla らから。染色手順3,4のために。 このテクニックのユニークな特徴は、すべての細胞組織と神経回路網が中断されているの解離の文化上の大きな利点を提供し、あなたが彼らの元の構造の海馬や小脳などの脳の異なる部分を勉強できることです。それは解離初代培養として取得するにはプルキンエ細胞の研究は、極めて困難なことができるので小脳の場合には、さらに有利である。このメソッドは、in vitroで小脳の特定の発達の特徴を学ぶだけでなく、野生型と変異マウスの両方における電気生理学的および薬理学的実験のために使用することができます。 方法は、アポトーシス細胞死を測定するTUNEL染色を用いて、このような発展途上小脳におけるFasリガンドとしてのアポトーシス刺激の効果を研究するために設計されてここで説明する。 TUNEL染色は、プルキンエ細胞のためのようなカルビンジンような細胞型特異的なマーカーと組み合わせている場合、それは細胞集団特異的に細胞死を評価することが可能である。カルビンジン染色はまた小脳の文化の質を評価の目的を果たします。

Watch this Scientific Journal Video about Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection at JoVE.com

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

このメソッドは、器官小脳文化の世代と異なる小脳の細胞型の生存率に一定のアポトーシス刺激の効果を説明します。

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. ...

organotypic-cerebellar-cultures-apoptotic-challenges-and-detection

Research

JoVE Journal

Neuroscience

18.1K ビュー.  The Salk Institute for Biological Studies. このメソッドは、器官小脳文化の世代と異なる小脳の細胞型の生存率に一定のアポトーシス刺激の効果を説明します。

ビデオ: Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

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 ...

神経科学

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 ...

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 ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

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 ...

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 ...

Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating innate immunity. Primary rodent cell culture models have greatly advanced our understanding of neuronal-glial interactions, but only recently have methods to specifically eliminate microglia from mixed cultures been utilized. One such technique &#8211; described here &#8211; is the use of L-leucine methyl ester, a lysomotropic agent that is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis13,14. Experiments using L-leucine methyl ester have the power to identify the contribution of microglia to the surrounding cellular environment under diverse culture conditions. Using a protocol optimized in our laboratory, we describe how to eliminate microglia from P5 rodent cerebellar granule cell culture. This approach allows one to assess the relative impact of microglia on experimental data, as well as determine whether microglia are playing a neuroprotective or neurotoxic role in culture models of neurological conditions, such as stroke, Alzheimer&#8217;s or Parkinson&#8217;s disease.

Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature.

L-ロイシンメチルエステルを使用した小脳顆粒細胞培養物からのミクログリアの選択的枯渇

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

マルチ電極アレイ上に成長させた器官脊髄共培養における機能的再生の調査

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

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. ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

Ex 子宮エレクトロポレーションと gaba 作動性介在ニューロンの移行のライブ イメージング用マウス萌芽期の脳の脊髄スライス培養

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...