この作品は、バーゼル大学、生物医学科、スイス国立科学財団（31003A_141007）によってサポートされていました。マルクスSaxerは、技術的な支援を行いました。

動物実験は、2010年9月22日の欧州共同体理事会指令に準拠して実施した（63分の2010 / EU）、レビューとスイス当局によって許された。 1器官Entorhino海馬スライス培養を設定する<ol><li>静的インキュベーション法4,11を使用して、生後4日目（P4）仔マウスからentorhino海馬器官型スライス培養（EHOSCs）を準備します。ここでは、のC57Bl6及びCB6F1マウスを使用しています。 <ol><li> すなわちスライス培養の調製のためのマウスの仔あたり約30分、6仔マウスの同腹のための3時間を取る。滅菌手術器具との層流ベンチで滅菌条件下ですべての手順を実行します。 </li><li> septo時空軸に沿ったスライスの位置からの干渉の可能性を回避するためには、中隔の半分しか海馬スライスを使用しています。特に明記しない場合は、室温ですべてのステップを実行する。 </li></ol></li><li>トンカットオフ彼は外科ハサミでP4マウスの子犬の頭。無麻酔は、このステップは必要ありません。 <ol><li>脳の周りの頭蓋骨を外し、大脳半球と中脳の尾の端部との間に亀裂を識別します。慎重に頭蓋骨から脳への脳の吻側の一部を除去して、L-グルタミンの安定化された形を添加したMEMからなる氷のように冷たい準備培地に入れてください。 </li></ol></li><li>氷冷準備媒体中に浸漬された脳を持つ実体顕微鏡下で解剖を続行します。皮質半球から残りの髄膜を除去します。半球の内側尾表面で海馬​​を特定し、脳の残りの部分から隣接嗅内皮質と一緒に分離する。 <ol><li>組織チョッパーに組織を移し、海馬のsepto - 時間軸に無菌状態の横方向の下で厚さ400μmのスライスをカット。 </li></ol></li><li> CE上のスライスを置きllの培養インサート（インサートあたり約6スライス）とインキュベーション培地のウェルあたり1mlで6ウェルプレートでそれらをインキュベートする（47ミリリットルMEMを25mlイーグル培地25mlのウマ血清1mlのグルタマックスIは、1mlの37℃、5％CO 2を含む加湿雰囲気中の滅菌10％グルコース溶液、pH7.3）で。メディア翌日、その後1週間に一日おきにアップを変更します。 </li></ol>再灌流を用いたin vitro低酸素及び酸素グルコース欠乏で 2 <ol><li>手順の正確な時刻表については、表1を参照してください。低酸素症またはOGDへの曝露の前に5日間（DIV5）の培養EHOSCs。 2％のB27サプリメント、1％のグルタマックス、追加の0.1％のグルコースを含むNeurobasal培地：この時点で、以下の組成を有する無血清培地、25％ウマ血清を含む培養培地から培地に切り替える。 </li><li>文献7でコンポーネントの.theの最終濃度を詳述したように、OGD媒体を準備すると、次のようになります。0.3のCaCl 2、70mMの塩化ナトリウム、5.25ミリモルのNaHCO 3、70mMのKClを、pH6.8の1.25ミリモルのNaH 2 PO 4、2mMのMgSO 4を、および10mMスクロース。 2個の6ウェル組織培養プレートの各ウェルにOGD培地1mlを加える。 <ol><li>低酸素チャンバ中で1時間N 2でOGD培地を灌流した後、37℃のインキュベーター中でO / Nを密封し続ける。 </li><li>低酸素指標として嫌気ストリップを使用してください。ストリップの色は白にピンク色に変化する際に酸素を含まないことがメディアを考えてみましょう。 </li><li>低酸素症の誘導のために、グルコースの代わりにOGD培地と無血清培地を使用しています。使用前に、OGD媒体について上記で詳述したように、低酸素室でN 2で培地を灌流。 </li></ol></li><li>スライスはOGDに曝露される前に、30分間N 2で再度OGD培地を灌流する。 30分間のスライスに1 mg / mlのヨウ2μlのヨウ化物（PI）（2μg/ mlの最終濃度）ウェル当たりのソリューションを追加し、選択し実験のための唯一の健康なスライスは、PI陽性細胞の数が少ないことによって示されるように。 </li><li> 図1に示したOGD曝露と再灌流のスケジュールを使用します。15分間のいずれかの酸素のみ（低酸素）または酸素とグルコース（OGD）のスライスを奪う。 <ol><li>低酸素症またはOGDの誘導のために、OGD培地にスライスを転送し、低酸素室で15分間保つ。培養物をOGD媒体とバックへの定期的な媒体から切り替えたとき、インサートの両側に残っている全ての流体が離れて吸引されていることを確認します。 </li><li>低酸素症またはOGD後、含酸素無血清培地でOGD培地を交換し、培養液を5％CO 2、37℃での正規インキュベーター中で無血清培地中で3,24または48時間回復させる。細胞死の決定のために固定を再び前に、PIを追加します。 </li></ol></li></ol> Entorhino海馬器官型スライス培養の3薬理学的治療<ol><li> Cultur薬理学的治療への曝露の前に培養培地中で5日間（DIV5）の電子のEHOSCs。薬理学的治療OGD中または直後にスライスが無血清培地に移される際にOGD後のいずれかを起動します。 </li><li> OGDから保護するための治療法。 <ol><li> （15分間）OGD誘導中または直後に30分間のOGD誘導後培養培地に100μMの6-シアノ-7ニトロキノキサリン-2,3 - ジオン（CNQX）または1μMテトロドトキシン（TTX）を追加します。次に、固定の前48時間正常酸素無血清培地中でインキュベートする。 </li></ol></li><li>興奮毒性死の誘導のための治療。 <ol><li>インキュベーション培地スイッチ培養における5日後、30分間100μM（RS）-αアミノ-3 - ヒドロキシ-5 - メチル-4 - イソキサゾールカルボン酸（AMPA）を含む無血清培地である。 </li><li>吸引によりAMPAを含む培地を取り外します（インサート上の任意の残留培地を除去するために注意してください）​​、通常の無血清培地で新鮮なプレートにスライスを転送し、固定されるまで48時間保管してください。 </li></ol></li></ol> 4の固定および免疫染色Entorhino海馬器官型スライス培養<ol><li>ヨウ化プロピジウム（PI）染色。 <ol><li> 2μg/ mlの濃度で生きているスライスの培養培地への30分間のPIを追加する。蛍光光学系を備えた倒立顕微鏡で生きた文化を表示し、さらなる処理のためのスライスを選択します。免疫組織化学と組み合わせたPI染色のために、前の文化の定着にPI溶液に30分を追加します。 </li></ol></li><li>スライスの固定。 <ol><li> 6ウェルプレートから培地を除去し、各ウェルに、新たに調製した4％パラホルムアルデヒド（PFA）を3mlを加える。その後、冷蔵庫に6ウェルプレートを配置し、4℃でO / Nを修正。 </li><li>次の日、PFA溶液を除去し、リン酸緩衝生理食塩水（PBS）でスライス3-4回洗浄する。 </li></ol></li><li>細胞培養i番目からスライスの除去フリーフローティングセクションの免疫組織化学のために手順をnsertブロッキング。 <ol><li>慎重に切り離し、サイズ0以下のアートペイントブラシを用いて培養インサートからスライスを削除します。 （10％正常ヤギ血清（NGS）および2％のTriton-X100を含むPBS）をブロッキング緩衝液で満たされた96ウェルプレートのウェルに各個別のスライスを転送する。オービタルシェーカーで一定の攪拌下、室温で2時間ブロッキングバッファー内のスライスにしてください。 </li></ol></li><li>スライス培養の免疫染色<ol><li>標識のため血管は1の希釈でポリクローナル抗ラミニンを使用します。200をPBSで1％NGSおよび0.5％トリトン-X100を含有する。 </li><li>ラベリングニューロンのために1の希釈で抗微小管結合タンパク質2（MAP2）を使用する：PBS中500は、1％NGSおよび0.5％トリトン-X100を含む。 </li><li>一定の攪拌しながら4℃で48時間、一次抗体とのスライスをインキュベートする。インキュベーション期間の後、3〜4回、0.5％トリトン-X100を含むPBS中でそれらを洗浄。 </li&gt; <li>二次抗体を希釈1：500のPBS、1％NGSおよび0.5％トリトン-X100を含有する。 Alexaの蛍光団結合二次抗体を用いた暗闇の中で、室温で2時間のスライスをインキュベートする。アレクサ350または488に結合させ、適切なヤギ抗ウサギまたはヤギ抗マウス二次抗体を使用してください。 </li><li>二次抗体溶液を除去し、トリス緩衝生理食塩水（TBS）で切片を3回洗浄する;スライドガラス上にマウントし、モヴィオールでスライドをカバースリップ。 </li></ol></li><li>染色されたスライスの顕微鏡観察</li><li>落射蛍光装置と標準的な顕微鏡や共焦点顕微鏡のいずれかで染色されたスライスを表示します。適切なカメラを入れて撮影する。いくつかの画像では、明るい背景で暗く染色された血管を得るために、蛍光のコントラスト（ 図5および6）を反転させる。 </li></ol>培養における地域血管密度の5。決定<ol><li> vesseを定量化文献8に記載されているように、血管の交差に基づいて、Lの密度。 </li><li>これらの測定のためのラミニンのために染色したスライスを使用してください。各グリッドは、平方当たり100×100μmで、0.25ミリメートル2の全分野に等しく、6×6のグリッドで10倍の倍率の撮影した画像を重ね合わせる。 </li><li>オーバーレイCA1、CA3、DG、およびEC地域の3つのこのようなグリッド（ 図2）。グリッドの線と交差血管をカウントします。 </li><li>良い統計的関連性を持つ結果を得るために、各マウスの仔あたり3スライスを持つ3仔マウスからのデータを含む、少なくとも3つの独立した実験で測定を行う。 </li><li>各地域のグリッドからの血管交差の平均値を決定し、適切な統計ソフトウェアを使用して事後検定（p &lt;有意であると定義されている0.05）のようにボンフェローニ補正をANOVAにより統計分析を行う。 </li></ol>

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動物実験は1986年11月24日（609分の86 / EEC）の欧州共同体理事会指令に従って行われたとスイス当局によって審査し、許可された。著者らは、開示することは何もない。

ここに提示される方法で、海馬器官型切片培養は、損傷後の神経組織におけるプラスチックの変化を研究するための多目的ツールとして使用することができる。器官型スライス培養7、虚血6の後のニューロンの反応を研究するために過去に使用されてきた血管の変化の同時研究の新たな局面が大幅この方法の潜在的な用途を高める。単独のOGDまたは低酸素症の誘導は、器官型?...

中枢神経系、血管系による酸素とグルコース供給の喪失または減少に特に敏感である。脳への血液供給のさえかなり短い中断は、一般的な脳卒中症候群につながる、関連する脳領域の機能が永久に失わを誘導することができる。原発被災地のニューロンの喪失に加えて、一般的に二次被害を通じて追加の遅延した神経細胞の損失がある。残念ながら、これまで、二次神経細胞死の低減のための神経保護治療は1入手できなかった。二次的損傷のメカニズムを研究するための研究努力は、中大脳動脈閉塞および血栓性閉塞、さまざまな技法のような脳虚血の動物モデルの使用に依存している（最近のレビューのために2を参照）。並行して、またにより制限および動物モデルの使用で倫理的な問題のために、さまざまなCNS組織の器官型切片培養は、STUを可能にするように使用されている負傷3-5のさまざまなタイプに神経細胞の反応のDY。 虚血性脳損傷を模倣する条件下でニューロンの反応を研究するために、酸素グルコース欠乏（OGD）のモデルシステムが開発されている。このモデルでは、スライス培養物を一時的にグルコースを欠いており、酸素の非存在下で、窒素ガスで平衡化した培地に曝露される。このような治療では、 インビボ 6,7 における虚血性傷害後に観察されたものとかなり類似している神経細胞の損傷および損失を誘導することが可能である。海 ​​馬では、このような治療はCA1ではなく、特異的に神経細胞の喪失を誘導するCA3領域または海馬の歯状回。対照的に、これまでのスライス培養における血管反応の研究が広く行われていない。明白な理由は、スライス培養モデルにおける循環および血管灌流の欠如である。しかし、それはbと維持することが可能であることが以前に示されている数日8,9用のCNS ​​スライス培養における100dは血管。 この方法の全体的な目標は、OGD後の神経細胞の運命を監視するが、傷害応答の重要な部分である血管系の運命と改造のために研究を拡張することだけではありません。今までこのような研究は、動物実験（;カヴァグリアら、2001。デ·ヨングら 、1999）の使用を必要とした。プロトコルがここに提示、当社は、そのような研究は、低酸素症、または両方ニューロンの生存と血管反応の分析に続いて興奮毒性病変のいずれかによって攻撃さentorhino海馬器官型スライス培養で詳細に行うことができる方法を。このプロトコルは、この主題10日に以前に発表された研究に基づいており、CNSにおける神経血管の相互作用に興味を持ってあらゆる研究室のために役立ちます。

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the analysis of neurovascular remodeling in entorhino-hippocampal organotypic slice cultures

虚血性脳損傷は、適切な脳機能を損なう最も一般的かつ破壊的な条件の中で、多くの場合、影響を受けた患者の機能欠損を永続化につながる。集中的な研究努力にもかかわらず、神経損傷を軽減し、遅延した二次死から虚血性の領域でニューロンを保護する利用できる有効な治療選択肢は、まだありません。この分野の研究は、典型的には、精巧で、問題の動物モデルの使用を含む。酸素およびグルコース欠乏（OGD）でチャレンジEntorhino海馬器官型切片培養物は、脳虚血を模倣するインビトロモデルにおいて確立されている。この研究の新規な態様は、脳血管の変化を比較し、相関させることができるニューロンの変化およびニューロンコンパートメント及び血管コンパートメントの両方の反応に加えて、研究されていることである。このプロトコルで提示される方法は、実質的にまたは潜在的用途を広げるganotypicスライス培養法。単独のOGDまたは低酸素症の誘導は、器官型スライス培養ではなく単純な手段によって適用され、神経組織における信頼性と再現性の損傷につながることができます。これは、生体内での脳卒中および虚血を誘導し、複雑で、問題の動物実験と全く対照的である。血管系の反応の研究を含めるように分析を広げることにより、脳の機能を維持し、復元する方法の新たな方法を提供することができます。ここで紹介するスライス培養法は、虚血性脳損傷の研究のための魅力的で重要なツールに発展する可能性があり、神経保護を目的とした潜在的な治療措置をテストするのに便利かもしれません。

酸素グルコース欠乏及び低酸素症は、海馬CA1領域において特異的に神経細胞死及び血管の減少を誘導する 。 以前7の記載と同様の海馬（ 図3）のCA1領域に特異的にヨウ化プロピジウム染色によって見られるようにOGDまたは酸素欠乏は、単独で15分間、細胞死の強力な誘導を誘導した。マーカーは、血管のネットワークを可視化すると、それは血管密度及び組織?...

Watch this Scientific Journal Video about The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures at JoVE.com

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

虚血性脳損傷の多くの局面を再現することができentorhino海馬器官型切片培養のためのプロトコルは、提示される。神経細胞の変化に加えて、神経脈管構造の変化を研究することによって、このプロトコルは、損傷後の神経組織内のプラスチック製の変化を研究するための汎用性の高いツールです。

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 ...

the-analysis-neurovascular-remodeling-entorhino-hippocampal

Research

JoVE Journal

Neuroscience

9.7K ビュー.  University of Basel. 虚血性脳損傷の多くの局面を再現することができentorhino海馬器官型切片培養のためのプロトコルは、提示される。神経細胞の変化に加えて、神経脈管構造の変化を研究することによって、このプロトコルは、損傷後の神経組織内のプラスチック製の変化を研究するための汎用性の高いツールです。 

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

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

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the anterior hypothalamus. The clock is directly synchronized by light via the retina and optic nerve. Circadian oscillations are generated by interacting negative feedback loops of a number of so called "clock genes" and their protein products, including the Period (Per) genes. The core clock is also dependent on membrane depolarization, calcium and cAMP 1. The SCN shows daily oscillations in clock gene expression, metabolic activity and spontaneous electrical activity. Remarkably, this endogenous cyclic activity persists in adult tissue slices of the SCN 2-4. In this way, the biological clock can easily be studied in vitro, allowing molecular, electrophysiological and metabolic investigations of the pacemaker function.
The SCN is a small, well-defined bilateral structure located right above the optic chiasm 5. In the rat it contains ~8.000 neurons in each nucleus and has dimensions of approximately 947 &mu;m (length, rostrocaudal axis) x 424 &mu;m (width) x 390 &mu;m (height) 6. To dissect out the SCN it is necessary to cut a brain slice at the specific level of the brain where the SCN can be identified. Here, we describe the dissecting and slicing procedure of the SCN, which is similar for mouse and rat brains. Further, we show how to culture the dissected tissue organotypically on a membrane 7, a technique developed for SCN tissue culture by Yamazaki et al. 8. Finally, we demonstrate how transgenic tissue can be used for measuring expression of clock genes/proteins using dynamic luciferase reporter technology, a method that originally was used for circadian measurements by Geusz et al. 9. We here use SCN tissues from the transgenic knock-in PERIOD2::LUCIFERASE mice produced by Yoo et al. 10. The mice contain a fusion protein of PERIOD (PER) 2 and the firefly enzyme LUCIFERASE. When PER2 is translated in the presence of the substrate for luciferase, i.e. luciferin, the PER2 expression can be monitored as bioluminescence when luciferase catalyzes the oxidation of luciferin. The number of emitted photons positively correlates to the amount of produced PER2 protein, and the bioluminescence rhythms match the PER2 protein rhythm in vivo 10. In this way the cyclic variation in PER2 expression can be continuously monitored real time during many days. The protocol we follow for tissue culturing and real-time bioluminescence recording has been thoroughly described by Yamazaki and Takahashi 11.

The procedure of preparing slices containing the adult mouse hypothalamic suprachiasmatic nucleus (SCN), and a rapid way to culture the SCN tissue in organotypic culture condition, are reported. Further, the measurement of oscillatory clock gene protein expression using dynamic luciferase reporter technology is described.

スライスの準備、器官組織培養と視交叉上核における時計遺伝子の活動のルシフェラーゼ記録

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

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 ...

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 ...

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. ...