この作品は、国立神経疾患研究所およびストローク（NS - 19108）、片頭痛研究財団、そしてリチャードP.クレイグへホワイト財団からの補助金によって賄われていた。さんマーシャP.クレイグは、培地およびスライス培養のメンテナンスの準備に協力した。我々は、この記事の最終版の彼女のコメントやリビジョンのためエレーナグリンベルクに感謝。

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コー​​ルド - プレコンディショニング神経保護に関与サイトカインシグナル伝達系の描写に重要な二つの根本的に重要な概念は、図6と図7に示されています。最初に、サイトカインは通常の脳内シグナル伝達分子が非常に低濃度です。それにもかかわらず、生理的サイトカイン濃度の変化は、遺伝子の発現を変化させる能力（図6）の脳の構造と機能（すなわち、表現型）を変更するために計り知れない可能性を秘めている。さらに、サイトカインは、同様の効果と、単一のサイトカインが変数の効果（図7）を持つことができますを持つことが非常に冗長とその複数のサイトカインの多面的です。従って、正確に冷間前処理演算子（または他の生理的前処理の刺激）から神経保護作用のための生得的なサイトカイン拠点を確立するために、関連するシグナリング変数の複合解析を判定する必要があります。これは、多重化アッセイ戦略によって実現されます。これは、コールドプレコンディショニング神経保護のサイトカイン&quot;署名&quot;を設立します。 <img src="/files/ftp_upload/2192/2192fig6.jpg" alt="図6" /> 図6。脳内サイトカインのシグナル伝達のパワー。イラストは他のよく知ら対応の濃度に比べて脳のサイトカインの生理的濃度の巨大なシグナル伝達力を伝える。濃度は、基準点として、単一の猫のひげをはじめ、距離の逆数として表されます。ナトリウム（コショウの粒で示される10 -1 M）およびカリウム（トマトで示される10 -3 M）は、内の役割はそれぞれ約150及び3mMの、のレベルで間質脳空間に存在し、十分に認識している神経細胞の電気生理学的機能。同様に、pH（すなわち、〜10 -7 Mの水素イオンの濃度とシカゴの湖畔に沿って780メートルとして図示）とカルシウム（すなわち、7.8キロマコーミックからシカゴの湖畔に沿って衛星Googleの画像から見て示すように、〜10 -8 Mの濃度）シカゴ大学の近くにハイドパークでのプロモントリーポイントに置きます。さらに、これらの濃度以上の間隙に放出された神経伝達物質は、局所脳領域の活性に影響を与える。対照的に、サイトカインは、（地球から火星までの距離として示されている）十数万倍未満の濃度で脳の機能を変更することができます。 <img src="/files/ftp_upload/2192/2192fig7.jpg" alt="図7" /> 図7。生得的なサイトカイン経路のインタラクティブなシグナリング。サイトカインは、非常に冗長とその複数のサイトカインの多面的なものは、同様の効果を持つことができ、単一のサイトカインは、変数の効果を持つことができます。そのような多様性は、リガンド、受容体、およびリン酸化のレベルで発生する複雑な対話型のシグナリングに由来する。さらに複雑さは、セル固有の生得的なサイトカイン、受容体、および関連するリン酸化タンパク質の変化の各能力で、脳が別の細胞型から構成されているという事実から生じている。ここに例示を目的として、唯一の生得的なサイトカインシグナル伝達経路は、（免疫細胞の研究から派生）が表示されます。単純にするために、脳は生得的なサイトカインの潜在的な相互作用を示す単セル（白い線）（（IL -1α/βとしてここに呼ばれる）、IL -1αおよびIL -1β、TNF -α、IL - 6、として描かれているIFN -γとIL - 10）、受容体（IL - 1R1、TNFR1、IL-6/gp130、IFNγR、およびIL - 10R）、およびリン酸化タンパク質（すなわち、キナーゼERK1 / 2、P38（P38 - MAPK）やJNK）と転写因子（ATF - 2、NFκB、およびSTAT3）。 TNFR1を介したATF - 2を介して遺伝子発現を誘発するJNK、p38の- MAPKおよびERK1 / 2、に例えば、TNF -αシグナル。 TNF -αはまた、NFκB活性化を介して直接遺伝子発現を変化させる。示されるように一緒に、これらの転写因子の活性化は、サイトカインとその受容体の増加（矢印）と減少した（平滑末端）式を呼び起こす。重要なことは、これらの経路は、他の（例えば、IL -1β）サイトカインの一つサイトカインの変化（例えば、TNF -α）の影響の生産を示している。従って、正確に確立するために関連するシグナリング変数の冷間前処理演算子、複合解​​析から神経保護のためのサイトカインの塩基は決定されなければならない。これは、コールドプレコンディショニングの生得的なサイトカイン&quot;署名&quot;を設立します。 （画像を参照＃25のデータから、コンパイルされています。）

スライス培養の準備<table border="1"><tbody><tr><td> 試薬/装置の名前 </td><td> 会社 </td><td> カタログ/モデル番号 </td><td> コメント </td></tr><tr><td> Millicell - CM組織培養インサート（30 mm）の</td><td>サーモフィッシャー</td><td> PICM0 - 3050 </td><td></td></tr><tr><td>基礎培地イーグル</td><td>インビトロジェン</td><td> 21010 </td><td></td></tr><tr><td>アールの平衡塩類溶液</td><td>シグマ</td><td> E2888 </td><td></td></tr><tr><td>馬血清</td><td>インビトロジェン</td><td> 26050-088 </td><td></td></tr><tr><td>グルタミン</td><td>インビトロジェン</td><td> 35050 </td><td></td></tr><tr><td>ゲンタマイシン</td><td>インビトロジェン</td><td> 15710-064 </td><td></td></tr><tr><td> Fungizone </td><td>インビトロジェン</td><td> 15295 </td><td></td></tr><tr><td> D -グルコース</td><td>シグマ</td><td> G8769 </td><td></td></tr><tr><td> BSL - 1ヒュームフード</td><td>ベイカー</td><td></td><td></td></tr><tr><td>マッキルウェーン組織チョッパー</td><td>ブリンクマン</td><td></td><td></td></tr><tr><td>テフロンディスク</td><td>ブリンクマン</td><td> 023-49-310-1 </td><td></td></tr><tr><td>へら</td><td>サーモフィッシャー</td><td> 14から373 - 25A </td><td></td></tr><tr><td>湾曲した虹彩鋏</td><td>ファイン科学ツール</td><td> 14091〜09 </td><td></td></tr><tr><td>ロングストレートはさみ</td><td>ファイン科学ツール</td><td> 14002〜14 </td><td></td></tr><tr><td>長い鉗子</td><td>サーモフィッシャー</td><td> 13-812-40 </td><td></td></tr><tr><td>角度付き鉗子</td><td>ファイン科学ツール</td><td> 11808〜02 </td><td></td></tr><tr><td>ショート鉗子</td><td>サーモフィッシャー</td><td> 13-812-38 </td><td></td></tr><tr><td> ＃5イノックス鉗子</td><td>ファイン科学ツール</td><td> 11254〜20 </td><td></td></tr><tr><td> 2アイリスspatulae </td><td>ファイン科学ツール</td><td> 10094〜13 </td><td></td></tr><tr><td> 150 × 15ミリメートルのペトリ皿</td><td>サーモフィッシャー</td><td> 08-757-148 </td><td></td></tr><tr><td> 60 × 15ミリメートルのペトリ皿</td><td>サーモフィッシャー</td><td> 08から757 - 100B </td><td></td></tr><tr><td>ワイルドM8実体顕微鏡</td><td>ワイルドHeerbrugg </td><td></td><td></td></tr><tr><td> Geyのバランス塩溶液</td><td>シグマ</td><td> G9779 </td><td></td></tr><tr><td> Blak - Rayの短波長紫外線測定メーター</td><td> UVP </td><td> J - 225 </td><td></td></tr><tr><td> 6ウェルファルコン培養ディッシュ</td><td>サーモフィッシャー</td><td> 08から772 - 1B </td><td></td></tr><tr><td> Neurobasal </td><td>インビトロジェン</td><td> 21103</td><td></td></tr><tr><td> B27サプリメント</td><td>インビトロジェン</td><td> 17504 </td><td></td></tr><tr><td> Gem21 Neuroplex </td><td>ジェミニのバイオ製品</td><td> 400-160-010 </td><td></td></tr><tr><td>アスコルビン酸</td><td>シグマ</td><td> A4544 </td><td></td></tr><tr><td> CO 2アナライザー</td><td>バカラック</td><td> ＃2820 </td><td></td></tr></tbody></table>スライス培養のバイタリティでの事前スクリーニング<table border="1"><tbody><tr><td> 試薬/装置の名前 </td><td> 会社 </td><td> カタログ/モデル番号 </td><td> コメント </td></tr><tr><td> SYTOXグリーン</td><td>インビトロジェン</td><td> S7020 </td><td></td></tr><tr><td> DMIBRE倒立顕微鏡</td><td>ライカ</td><td></td><td></td></tr></tbody></table>冷間前処理<table border="1"><tbody><tr><td> 試薬/装置の名前 </td><td> 会社 </td><td> カタログ/モデル番号 </td><td> コメント </td></tr><tr><td> BSL - 2の換気フード</td><td> NuAire </td><td></td><td></td></tr></tbody></table>興奮毒性損傷<table border="1"><tbody><tr><td> 試薬/装置の名前 </td><td> 会社 </td><td> カタログ/モデル番号 </td><td> コメント </td></tr><tr><td> CoolSnap FX CCDカメラ</td><td> Photometrics </td><td></td><td></td></tr><tr><td>フルオレセイン</td><td>インビトロジェン</td><td> F36915 </td><td></td></tr><tr><td> MetaMorphソフトウェア</td><td>分子デバイス</td><td> V. 7.5.04 </td><td></td></tr><tr><td> 100μmの深さ血球計</td><td> Hausserサイエンティフィック</td><td></td><td></td></tr><tr><td> N -メチル- D -アスパラギン酸</td><td>カルビオケム</td><td> 454575 </td><td></td></tr><tr><td> SigmaStatソフトウェア</td><td> SYSTATソフトウェア株式会社</td><td> V. 3.5 </td><td></td></tr></tbody></table>コー​​ルド - プレコンディショニングを適用した共同治療<table border="1"><tbody><tr><td> 試薬/装置の名前 </td><td> 会社 </td><td> カタログ/モデル番号 </td><td> コメント </td></tr><tr><td> sTNFR1 </td><td> R＆Dシステムズ</td><td> 425 - R1 - 050 </td><td></td></tr><tr><td>ウシ血清アルブミン</td><td>シグマ</td><td> - 4503 </td><td></td></tr></tbody></table> RNA分離<table border="1"><tbody><tr><td> 試薬/装置の名前 </td><td> 会社 </td><td> カタログ/モデル番号 </td><td> コメント </td></tr><tr><td> RNAl ater </td><td>アンビオン</td><td> AM7021 </td><td></td></tr><tr><td>マイクロRNeasyキット</td><td>キアゲン</td><td> 74004 </td><td></td></tr><tr><td> β-メルカプトエタノール</td><td>フィッシャー</td><td> 03446-100 </td><td></td></tr><tr><td> Diposable 1.5 RNaseフリーの杵</td><td>サウスジャージーの精密工具や金型、（株） </td><td> 749521-1590 </td><td></td></tr><tr><td> RNasin </td><td>プロメガ</td><td> N261A </td><td></td></tr><tr><td>トリス[ヒドロキシメチル]アミノメタン</td><td>シグマ</td><td> T3069 </td><td></td></tr><tr><td> EDTA（エチレンジアミン四酢酸二ナトリウム塩水和物</td><td>シグマ</td><td> ＃E7889 </td><td></td></tr></tbody></table> RNAの定量<table border="1"><tbody><tr><td> 試薬/装置の名前 </td><td> 会社 </td><td> カタログ/モデル番号 </td><td> コメント </td></tr><tr><td> RiboGreenアッセイ</td><td>インビトロジェン</td><td> R11491 </td><td></td></tr><tr><td>酵母tRNA </td><td>インビトロジェン</td><td> 15401-011 </td><td></td></tr><tr><td> 1.5mLの遠心チューブ</td><td>米国サイエンティフィック</td><td> 16155500 </td><td></td></tr><tr><td> 96ウェル蛍光アッセイプレート</td><td>ヌンク</td><td> DK - 4000 </td><td></td></tr><tr><td> 96ウェル蛍光プレートリーダー</td><td>テカン</td><td>サファイアII </td><td></td></tr></tbody></table>定量PCR <table border="1"><tbody><tr><td> 試薬/装置の名前 </td><td> 会社 </td><td> カタログ/モデル番号 </td><td> コメント </td></tr><tr><td> iScript逆転写キット</td><td> Bio - Rad社</td><td> 170-8891 </td><td></td></tr><tr><td> SYBRグリーン</td><td>インビトロジェン</td><td> S7563 </td><td></td></tr><tr><td>プラチナTaqポリメラーゼ</td><td>インビトロジェン</td><td> RNA Ultrasense 11732-927 </td><td></td></tr><tr><td> iCyclerサーマルサイクラー</td><td> Bio - Rad社</td><td> iCycler光モジュール</td><td></td></tr><tr><td> iCyclerのシステムソフトウェア</td><td> Bio - Rad社</td><td> V. 3.1 </td><td></td></tr></tbody></table>マイクロアレイのための定量PCR <table border="1"><tbody><tr><td> 試薬/装置の名前 </td><td> 会社 </td><td> カタログ/モデル番号 </td><td> コメント </td></tr><tr><td> アウェイ RNaseの</td><td>分子バイオ製品</td><td> ＃7000 </td><td></td></tr><tr><td> RT 2最初ストランドキット</td><td> SABiosciences </td><td> C - 03 </td><td></td></tr><tr><td> SYBRグリーンベースのPCRミックス</td><td> SABiosciences </td><td> PA - 011 </td><td> iCyclerのための特定の</td></tr><tr><td> RT 2 ProfilerのPCRアレイ</td><td> SABiosciences </td><td> PARN - 011A </td><td></td></tr></tbody></table>プロテオミクスアッセイ<table border="1"><tbody><tr><td> 試薬/装置の名前 </td><td> 会社 </td><td> カタログ/モデル番号</td><td> コメント </td></tr><tr><td>細胞溶解キット</td><td> Bio - Rad社</td><td> 171-304011 </td><td></td></tr><tr><td>マイクロ遠心機</td><td>フィッシャーサイエンティフィック</td><td>マイクロ7 </td><td></td></tr><tr><td> BSAタンパク質の株式</td><td> Bio - Rad社</td><td> 500-0007 </td><td></td></tr><tr><td> BCAタンパク質アッセイキット</td><td>サーモサイエンティフィック</td><td> 23225 </td><td></td></tr><tr><td>免疫96マイクロウェルプレート</td><td>ヌンク</td><td> 449824 </td><td></td></tr><tr><td>ガムテープ</td><td> Bio - Rad社</td><td> 2239444 </td><td></td></tr><tr><td> 96ウェルプレートリーダー</td><td>パーキンエルマー</td><td> 1420 - mulilabelカウンタ</td><td></td></tr><tr><td> BioplexラットTNF -αのサイトカインアッセイ</td><td> Bio - Rad社</td><td> X0000001T </td><td></td></tr></tbody></table>免疫染色<table border="1"><tbody><tr><td> 試薬/装置の名前 </td><td> 会社 </td><td> カタログ/モデル番号 </td><td> コメント </td></tr><tr><td> 16％パラホルムアルデヒド</td><td>アルファAesar </td><td> 43368 </td><td></td></tr><tr><td>リジン</td><td>シグマ</td><td> L - 6001 </td><td></td></tr><tr><td>リン酸ナトリウム</td><td>フィッシャーサイエンティフィック</td><td> S374 - 1 </td><td></td></tr><tr><td>ヨウ素酸ナトリウム</td><td>シグマ</td><td> S - 1878 </td><td></td></tr><tr><td>アジ化ナトリウム</td><td>シグマ</td><td> S - 2002 </td><td></td></tr><tr><td>過酸化水素（30％） </td><td>シグマ</td><td> H1009 </td><td></td></tr><tr><td>ヤギ血清</td><td>コロラド血清会社</td><td> CS 0920 </td><td></td></tr><tr><td>トリトンX - 100 </td><td>シグマ</td><td> T - 8787 </td><td></td></tr><tr><td>染色皿</td><td>テッドペラ、（株） </td><td> 14511 </td><td></td></tr><tr><td>プレートシェーカー</td><td>ラボラインインスツルメンツ株式会社</td><td> 3520 </td><td></td></tr><tr><td> ＃1フィルタペーパー（185ミリメートル） </td><td>ワットマン紙</td><td> 1001-185 </td><td></td></tr><tr><td> 3,3 - ジアミノベンジジン</td><td>シグマ</td><td> D8001 </td><td></td></tr><tr><td>ジメチルスルホキシド</td><td>シグマ</td><td> D8418 </td><td></td></tr><tr><td>エタノール</td><td> Pharmco - AAPER </td><td> 111000200 </td><td></td></tr><tr><td>キシレン</td><td>フィッシャーサイエンティフィック</td><td> X3S - 4 </td><td></td></tr><tr><td> Permountマウントメディア</td><td>フィッシャーサイエンティフィック</td><td> SP15 - 100 </td><td></td></tr><tr><td>クライオスタット</td><td>ライカ</td><td> cm 3の 050 S </td><td></td></tr><tr><td>組織 - Tek社</td><td>テッドペラ、（株） </td><td> 2705​​0 </td><td></td></tr><tr><td>精密チップペイントブラシ</td><td>テッドペラ</td><td> 11806 </td><td></td></tr><tr><td> PAPブロッキングペン</td><td>テッドペラ、（株） </td><td> 22309 </td><td></td></tr><tr><td> SFXシグナルエンハンサー</td><td>インビトロジェン</td><td> A31631 </td><td></td></tr><tr><td>退色を延長</td><td>インビトロジェン</td><td> P7481 </td><td></td></tr></tbody></table>

strategies for study of neuroprotection from cold-preconditioning

神経損傷は、全身麻酔と、効果的な管理が容易で安全なプレコンディショニング治療法の開発により軽減することができる関連する外科的処置の罹患率と死亡率の原因となることが多い。我々は、傷害の発症前に脳を保護するための治療薬の開発のための新たなターゲットを特定する手段として、コールドプレコンディショニングを担当し神経免疫シグナルを定義しようとしています。時間の経過に伴う変化をシグナリング低レベルのプロ炎症性メディエーターは、冷間プレコンディショニング神経保護のために不可欠です。このシグナリングは、刺激性の刺激が明らかになることを保護するための刺激に適応するための十分な時間と閾値の大きさに達することを必要と生理的なコンディショニングのホルミシスの基本的な教義と一致しています。 従って、冷間プレコンディショニング神経保護に関与する免疫シグナルの線引きは、生物学的システムと実験操作に加え、技術的能力が高い再現性と小文字を区別することが必要です。我々のアプローチは密接に成熟しており、静止大膠細胞/ミクログリアの影響を受けた多シナプスニューラルネットワークによる生体のカウンターでを反映するin vitroモデルとしての海馬のスライス培養を使用することです。彼らはフェムトモル範囲の手術であるサイトカイン、の主要な供給源であるため、このグリア状態では、ミクログリアのために特に重要です。また、スライス培養、活性化刺激を呼び起こすと適応応答を評価するために十分な時間である、数週間のためにin vitroで維持することができます。最後に、環境条件を正確にそのサイトカインがコールドプレコンディショニングのシグナリングようにスライス培養を用いて制御することができるが、測定模倣、および重要なノードの側面を分析するように変調することができます。サイトカインシグナル伝達系の解析では、高感度で再現性の多重化技術を使用する必要があります。我々は、組織全体のサイトカインの変化を評価する定量的リアルタイム定量PCRアレイのスクリーニングに続いてミクログリアの活性化のために画面にTNF -αのために定量PCRを使用してください。後者は、同時に変更を通知する複数のサイトカインのシステムを測定する最も高感度で再現性の手段です。重要な変更は、対象となる定量PCRしてから、蛋白質の検出により確認されています。我々はLuminexの技術を使用して多重化ミクロスフェアのフローサイトメトリーアッセイを用いて組織ベースのサイトカインタンパク質の変化を探る。細胞特異的サイトカイン産生は、二重ラベルの免疫組織化学で決定されます。一緒になって、提案された調査の戦略に結合この脳組織の調製および使用のスタイルは、、コールドプレコンディショニングの利点を模倣することができる新たな治療法の開発のための潜在的なターゲットを特定するための最適なアプローチかもしれない。

Watch this Scientific Journal Video about Strategies for Study of Neuroprotection from Cold-preconditioning at JoVE.com

Strategies for Study of Neuroprotection from Cold-preconditioning

我々は、傷害の発症前に脳を保護するための治療薬の開発のための新たなターゲットを特定する手段として、コールドプレコンディショニングを担当し神経免疫シグナルを定義しようとしています。我々は生物学的システム、実験操作に加え、再現性の高いと小文字が区別される技術的能力を必要とするような作業のための戦略を提示する。

コー​​ルド - プレコンディショニングによる神経保護の研究のための戦略

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by ...

strategies-for-study-of-neuroprotection-from-cold-preconditioning

Research

JoVE Journal

Neuroscience

14.8K ビュー.  The University of Chicago Medical Center. 我々は、傷害の発症前に脳を保護するための治療薬の開発のための新たなターゲットを特定する手段として、コールドプレコンディショニングを担当し神経免疫シグナルを定義しようとしています。我々は生物学的システム、実験操作に加え、再現性の高いと小文字が区別される技術的能力を必要とするような作業のための戦略を提示する。

ビデオ: Strategies for Study of Neuroprotection from Cold-preconditioning

Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the 
neuro-metabolic-vascular relationship. Development of new pO2 measurement modalities with simultaneous monitoring of pO2 in larger fields of view with higher 
spatial and/or temporal resolution will enable greater insight into the functioning of the normal brain and will also have significant impact on diagnosis 
and treatment of neurovascular diseases such as stroke, Alzheimer's disease, and head injury.
Optical imaging modalities have shown a great potential to provide high spatiotemporal resolution and quantitative imaging of pO2 based on hemoglobin absorption in visible and near infrared range of optical spectrum. However, multispectral measurement of cerebral blood oxygenation relies on photon migration through the highly scattering brain tissue. Estimation and modeling of tissue optical parameters, which may undergo dynamic changes during the experiment, is typically required for accurate estimation of blood oxygenation. On the other hand, estimation of the partial pressure of oxygen (pO2) based on oxygen-dependent quenching of phosphorescence should not be significantly affected by the changes in the optical parameters of the tissue and provides an absolute measure of pO2. Experimental systems that utilize oxygen-sensitive dyes have been demonstrated in in vivo studies of the perfused tissue as well as for monitoring the oxygen content in tissue cultures, showing that phosphorescence quenching is a potent technology capable of accurate oxygen imaging in the physiological pO2 range. 
Here we demonstrate with two different imaging modalities how to perform measurement of pO2 in cortical vasculature based on phosphorescence lifetime imaging. In first demonstration we present wide field of view imaging of pO2 at the cortical surface of a rat. This imaging modality has relatively simple experimental setup based on a CCD camera and a pulsed green laser. An example of monitoring the cortical spreading depression based on phosphorescence lifetime of Oxyphor R3 dye was presented. In second demonstration we present a high resolution two-photon pO2 imaging in cortical micro vasculature of a mouse. The experimental setup includes a custom built 2-photon microscope with femtosecond laser, electro-optic modulator, and photon-counting photo multiplier tube. We present an example of imaging the pO2 heterogeneity in the cortical microvasculature including capillaries, using a novel PtP-C343 dye with enhanced 2-photon excitation cross section.
Click here to view the related article <a href="http://www.jove.com/Details.php?ID=1731">Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems.</a>

We present an experimental procedure for measuring the partial pressure of oxygen (pO2) in cerebral vasculature based on oxygen-dependent quenching of phosphorescence. Animal preparation and imaging procedures were outlined for both large field of view CCD-based imaging of pO2 in rats and 2-photon excitation based imaging of pO2 in mice.

燐光の酸素依存性の焼入に基づいて脳の血液の酸素化の測定

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the 
...

神経科学

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

のレーザーキャプチャーマイクロダイセクションショウジョウバエ末梢ニューロン

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

老化とアミロイド病理の間にシナプス変化の研究のためのラットおよびトランスジェニックマウスからの急性海馬スライスの準備

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

シナプス伝達とイメージシナプス前終末を研究するために齧歯類の脳スライスからの神経細胞のVibrodissociation

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

シーケンシャル神経筋接合部におけるシングルシュワン細胞の輪郭を描くのにフォトブリーチング

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

グルコース感知を勉強する成体マウス由来の膜視床下部腹内側部のニューロンの電位色素イメージング

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) induced in acute hippocampal slices are widely accepted as cellular models of learning and memory. There are numerous studies that use live cell imaging or immunohistochemistry approaches to visualize activity dependent protein dynamics. However these methods rely on the suitability of antibodies for immunocytochemistry or overexpression of fluorescence-tagged proteins in single neurons. Immunoblotting of proteins is an alternative method providing independent confirmation of the findings.&#160;The first limiting factor in preparation of subcellular fractions from individual tetanized hippocampal slices is the low amount of material. Second, the handling procedure is crucial because even very short and minor manipulations of living slices might induce activation of certain signaling cascades. Here we describe an optimized workflow in order to obtain sufficient quantity of nuclear enriched fraction of sufficient purity from the CA1 region of acute hippocampal slices from rat brain. As a representative example we show that the ERK1/2 phosphorylated form of the synapto-nuclear protein messenger Jacob actively translocates to the nucleus upon induction of LTP and can be detected in a nuclear enriched fraction from CA1 neurons.

We provide a detailed protocol for induction of long-term potentiation in the CA1 region of the hippocampus and the subsequent isolation of nuclear enriched fractions from the tetanized area of the slice. This approach can be used to determine activity dependent nuclear protein import in cellular models of learning and memory.

Synapto核メッセンジャータンパク質の活性に依存する核内移行を研究するために海馬スライスからCA1原子力エンリッチ画分の単離

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular ...

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations &#8211; most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

リアルタイム神経細胞株に神経毒性および神経保護に関与する分子経路を描写するツールとしてのインピーダンスベースの細胞分析装置

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

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. ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

マイクロ電極アレイにおけるニューロン細胞培養の刺激に対するネットワーク応答の時間依存的増加

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

コー​​ルド - プレコンディショニングによる神経保護の研究のための戦略

コールド - プレコンディショニングによる神経保護の研究のための戦略