この作品会社ニューロ バイオで賄われていた我々 感謝したい博士・ ジョヴァンニ ・ Ferrati、博士セルジオ ・ ロトンド (神経生物)、コメントや原稿にアドバイスを。

すべての動物の研究は、承認されたプロトコルの下で行われています。
注: このセクションで実験プロシージャの間に実行される一連の主なフェーズと推奨される時間間隔が表示される (図 1)。また、プロトコルの説明は脳除去から潜伏期間 (図 2) 後の組織の均質化に至るまで重要なアクションを見せて、説明パネルによって補われます。材料と装置と WB 解析のため後続のフェーズを構築する手順について詳細は、前述の22です。
1. 手術器具、Vibratome 設定と治療準備
注: は、実験の手順を開始する前に次の手順を実行します。
<ol><li>2 つ異なる人工髄液 (aCSFs)、脳をスライスするために使用される 1 つを準備し、29が前述に潜伏ステップのための他。簡単に言えば、2 つの aCSFs の (モル) で働く集中は、「スライス」アプライド: 120 塩化ナトリウム、5 KCl、20 NaHCO3、2.4 CaCl2、2 MgSO4、1.2 KH2PO4、および 10 のブドウ糖。6.7 HEPES 塩と 3.3 HEPES 酸;pH: 7.1。「記録」アプライドしばらく: 124 NaCl、3.7 KCl、26 NaHCO3、2 CaCl2、1.3 MgSO4、1.3 KH2PO4、および 10 のブドウ糖。pH: 7.1。</li>
	<li>ソリューションを混ぜると氷の上に置くし、脳とその後の断面を除去しながら寒さにスライスのアプライドに酸素を送り込みます。部屋の温度 (RT) と供給酸素記録アプライドに保ちます。</li>
	<li>斬首と脳の除去、すなわちギロチン、外科解剖キット (ハサミ、鉗子、羽根) とヘラの郭清のフードの下で楽器を配置します。</li>
	<li>Vibratome パラメーターを設定するには、スライス厚測定 (この準備で 300 μ m) と 7 の周波数と 6 で速度を選択します。その適切な場所に vibratome チャンバーを挿入し、氷とそれを囲みます。</li>
	<li>「スライス」(アプライド) 脳断面中に酸素を送り込むことカーボゲン ・ (95% O2 5% CO2) バルブを開きます。最小流量は 2 mL/分周辺を選択します。</li>
	<li>テストする条件を準備する 2 つの 15 mL チューブを取る。2 μ M (治療群) の濃度で T30 を豊かに「記録」アプライドの 10 mL 1 管 10 mL、「録音」アプライドだけで (コントロール群) のと、他を追加します。渦管し、セットアップ準備段階 (セクション 3) での使用まで氷のそれらを保ちます。</li>
</ol>2. 麻酔、脳領域抽出とスライス
注: この研究で 9 野生型 P14 雄 Wistar ラットが使用されました。脳の抽出とその後スライスを防止または組織の変性過程に少なくとも遅くために (10 分) 内ですぐに実行すべきのでプロトコルの重要な一部を構成します。
<ol><li>誘導室に綿の層にイソフルランの 100%/w の 1.5 mL を追加、中に動物を配置、ふたを閉じる。動物深く麻酔、ペダルの反射の有無を確認するまで待ち、その後、ギロチンを使用して動物の首をはねます。</li>
	<li>冷たい鍋含む内の動物の頭を保つ脳除去の段階アプライドをスライス (図 2 a -C)。</li>
	<li>頭蓋骨に手術の刃で皮膚を切開し、はさみ次の後部から、正中線を使って頭蓋骨をカット、前方 (図 2 a) OBs の上の骨に到達するまで続行します。</li>
	<li>脳 (図 2 b) へのアクセスを持つためにピンセットのペアを使用して頭蓋骨の横に 2 つの側面を引き出します。脳に腹へらを挿入し、優しくスクープ、それとそれは約 5 分 (図 2) のように冷たい「スライス」アプライドでうるおいをしっかりキープ。</li>
	<li>ろ紙上の脳を処分、ブレード (図 2 D) を用いた小脳を切り取る。Vibratome ディスクに垂直に脳を接着剤し断面のチャンバ内挿入、冷たい「スライス」のアプライドでそれ埋めるし、酸素 (図 2 e) を提供。</li>
	<li>切断間隔を調整し、脳断面 (図 2 f) に進みます。上記22内側中隔 (MS)、ブローカー (DBB) と黒 innominata (SI) の斜め帯など、関心の領域を含むセクションを収集します。上から下に、3 つのスライスには、吻側 (R)、(I)、中級、BF (図 2) の尾の (C) 部分が含まれています。</li>
	<li>小さなハサミで正中線に沿って各セクションを分割コントロールと同じ解剖学的平面で扱われる条件として個別に使用 2 つの鏡面 hemislices (図 2 H) を取得します。</li>
	<li>5-10 分の vibratome 室で、hemisections を維持し、ガラス ピペットで記録アプライドを含むバブル鍋にそれらを転送します。常温約 30 分を回復してください。</li>
	<li>記録アプライドだけ (以前に管理グループのための 1.6 の手順で準備) 3 mL で 3 つのガラス容器と扱われたグループの T30 (以前に 1.6 の手順で準備) を豊かにアプライドの 3 ml 3 バイアルを一方で、埋めます。</li>
</ol>3. セットアップ準備
<ol><li>制御グループ (緑枠、図 2 j) の 3 つの連続した hemislices (, 中間, 吻側と尾側の BF 地域を含む) を持つために彼らの対応するガラスの瓶 (図 2 i)、hemisections に転送、治療群 (赤枠、図 2 j) の対応を一致します。</li>
	<li>条件の間の任意の汚染を防ぐために制御 hemislices と処理対応の 2 つの異なるブラシを有する脳組織を転送します。</li>
	<li>対応するふたをオキシジェネイティング針 (21 G) (図 2 j) を提示、各バイアルをシールします。カーボゲン ・ ソース (図 2 K) に装置のメインのチューブを接続します。実験では脳組織と機械的損傷の任意の動きを避けるために 2 mL/min の最小流量では培養期間を通して hemislices に酸素を提供します。5 時間培養を開始します。5 分以内 3.1 3.3 の手順を実行します。 注: こまめにチェック (15-20 分毎) すべてのバイアルに酸素が供給されるかどうかと、その流れは底から組織を動いていません。</li>
</ol>4. サンプルの均質化
<ol><li>孵化後酸素の流れを停止し、バイアル瓶からすべての蓋を削除します。アイス (図 2 L) チューブを維持する換散バッファーの 250 μ L を含む 1.5 mL チューブに適切なブラシを使用して、hemislices に転送します。換散バッファー、PBS 1 をミックスするには、フォスファターゼ、プロテアーゼ阻害剤のカクテルと x で、1: 100 希釈し、サンプル間の汚染を防ぐために異なるすりこぎで組織をホモジナイズしてください。10-15 分内でこれらの操作を完了します。</li>
	<li>遠心分離機の 4 ° C で 5 分間 1,000 x g でライセートの脳上清を新しいチューブに転送し、-80 ° C のサンプル</li>
</ol>5. タンパク質濃度および西部のしみの分析を読む
<ol><li>各サンプルのタンパク質濃度を測定し、その後、上記22として西部のしみの分析試料を準備します。</li>
	<li>蛋白質の表現の治療の効果を評価するための統計分析を実行します。</li>
</ol>

<ol>
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X., Lidsky, T., Wegiel, J., Grundke-Iqbal, I., Iqbal, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolically+active+rat+brain+slices+as+a+model+to+study+the+regulation+of+protein+phosphorylation+in+mammalian+brain.">Metabolically active rat brain slices as a model to study the regulation of protein phosphorylation in mammalian brain.</a> Brain Research Protocols. 6 (3), 134-140 (2001).</li></ol>

著者は、競合する金銭的な利益を宣言します。スーザン ・ a. グリーン フィールドは、創設者兼社長神経バイオ限定、個人所有の会社と会社の株を保有しております。エマヌエーレ ブライとアントネラ Cogoni 神経バイオ株式会社の従業員であります。

特定のアプリケーションの後の彼らの応答を監視、同じ解剖学的面から取得した 2 つの鏡面 hemislices を同期的にテストすることができます確立前のヴィヴォ脳の技術に基づいて、このプロトコルの主要側面条件 (制御または扱われる);したがって、できるだけしっかりと制御実験パラダイムを提供します。神経変性イベント22日中に見られるように時間、線量 - と部位?...

広告は (OB)1,2,3、嗅 (EC)、BF、海馬 (HC)、嗅球などの異なった頭脳区域に影響を与える段階的な神経変性障害によって特徴づけられる慢性的な病理 4,5。広告開発の後期の段階は、この病気の約すべての場合6の 70% を占め、認知症の最も一般的なフォームを作り、進行性認知機能の低下に します。初期 AD の原因を理解する広範な試みにもかかわらずない現在、定義された実験的徴候がそれらを解明です。AD の病態も効果的な7,8 を証明している医薬品のターゲットを説明する完全なプロフィールを提供されていないまた、最も人気のある理論 -「アミロイド仮説」- は問わ ,9。
注目されている代替理論神経変性時に発生する初期のメカニズム広告3,10,11に主に影響を受けやすい神経クラスターに関連していることを示唆しています。,12,13,14. OB、HC、EC1615,などの複数の地域が BF、中脳、脳幹、プロジェクト内で包まれてこの異種細胞ハブ。神経細胞の形態、神経伝達物質合成の多様性、にもかかわらず細胞のこのコアは非酵素的関数17,18することも痛みを表現する共通の機能を共有します。シグナリング分子は、Ca2 +線量、可用性、および神経年齢17,18に関連して栄養や有害イベントを受けることができるニューロンにカルシウム (Ca2 +) の流れを仲介する小説としてこの非古典的な役割,19。
神経変性、中に観察された細胞損失かもしれないしたがってこの非酵素的関数17,18,20、関連付けられている痛みの C 末端から切断 30mer ペプチド (T30) に起因するであります。20.、T30 誘導 BF 構造を有するラット脳スライス前のヴィヴォに基づいて新規アプローチを通じて我々 は実証以前の結果は、細胞培養と光イメージング18,21の準備、実施に伴い広告のようなプロファイル22。この新しい手法は時間のタイム ・ ウィンドウのとはいえ、解剖学的から回路保全に至るまで、そのまま組織の特性の多くを維持するための細胞培養よりもより生理的シナリオ提供しています。T30 申請急性反応をモニタリング、神経変性疾患の初期の段階で行われるイベントを探索するこのプロトコルを適用されます。
脳の使い方の文献の大きいボディにもかかわらず神経損傷で暗黙の分子経路を調査するスライスまたはより即時初めて神経新生23,24, このプロトコルを提供し、読み出す敏感な比較脊髄スライスの共通使用。ただし、脳切片の場合、この急性スライス手順できますも採用される特定の工程で発生する主な分子の変化検出神経または神経毒性分子の評価など、いくつかの目的のため免疫組織化学的解析、および中枢神経系の薬理学的アッセイ関連病態です。

<table>
	<tbody>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>S7653</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>P9333</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (NaHCO3)</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>S5761</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Magnesium sulphate heptahydrate (MgSO4 (7H2O))</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>63138</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (KH2PO4)</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>P5655</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Hepes salt</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>H7006</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Hepes acid</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>H3375</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>G7528</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Calcium chloride dehydrate</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>223506</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>T30 peptide</td>
			<td>Genosphere Biotechnologies, France</td>
			<td></td>
			<td>AChE-derived peptide tested</td>
		</tr>
		<tr>
			<td>Surgical dissecting kit</td>
			<td>World Precision Instruments, USA</td>
			<td>Item #: MOUSEKIT</td>
			<td>Brain removal step</td>
		</tr>
		<tr>
			<td>Surgical blades</td>
			<td>Swann-Morton, UK</td>
			<td>BS 2982</td>
			<td>Brain removal step</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Fisher Scientific, USA</td>
			<td>11566873</td>
			<td>Brain preparation for slicing</td>
		</tr>
		<tr>
			<td>Glue</td>
			<td></td>
			<td></td>
			<td>Brain preparation for slicing</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica, Germany</td>
			<td>VT1000 S</td>
			<td>Slicing</td>
		</tr>
		<tr>
			<td>Brushes</td>
			<td></td>
			<td></td>
			<td>Tissue handling</td>
		</tr>
		<tr>
			<td>Oxygen canister</td>
			<td></td>
			<td></td>
			<td>Sectioning and incubation phase</td>
		</tr>
		<tr>
			<td>1x Phosphate buffer saline (PBS)</td>
			<td>Fisher Scientific, USA</td>
			<td>BP2438-4</td>
			<td>Homogenization step</td>
		</tr>
		<tr>
			<td>Phosphatase inhibitors</td>
			<td>Fisher Scientific, USA</td>
			<td>1284-1650</td>
			<td>Homogenization step</td>
		</tr>
		<tr>
			<td>Protease inhibitors</td>
			<td>Roche complete PIC, USA</td>
			<td>4693116001</td>
			<td>Homogenization step</td>
		</tr>
		<tr>
			<td>Pestles</td>
			<td>Starlab, UK</td>
			<td>I1415-5390</td>
			<td>Homogenization step</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce 660 nm Protein Assay</td>
			<td>Thermo Scientific, USA</td>
			<td>22660</td>
			<td>Protein concentration</td>
		</tr>
	</tbody>
</table>

an alternative approach to study primary events in neurodegeneration using ex vivo rat brain slices

基になる神経変性を処理するプライマリを反映して信頼性の高いモデル動物の開発を試みる多くの研究にもかかわらずいくつかの非常に幅広く受け入れられてまいりました。ここでは、新しいプロシージャをよく知られている前のヴィヴォ脳スライス テクニックから、近い体内適応を提案する-として細胞の変性をトリガーする初期のイベントを調査するための体外の準備よりシナリオのようなアルツハイマー病 (AD) で観測されました。このバリエーションは、シンプルで簡単に再現できる手順を実行し、選択した脳の領域と、ローカル機能を生理学的環境での解剖学的研究の保存を有効にするで構成されています。別の解剖学的領域は、サイト、線量、および時間依存的で問題の治療法と複数の実験を実行する機会を提供すること、同じ脳から入手できます。この方法論に関連する成果に影響を与えることができる潜在的な制限を組織、すなわち、スライスとインキュベーションのステップおよび切片厚中の解剖学的な整合性の維持保全に関連します。生化学的および免疫組織化学的解析に影響を与えることができます。このアプローチは、生理学的または病理学的条件、薬物スクリーニング、または用量反応アッセイに関与する分子機構の探索など、さまざまな目的に流用できます。最後に、このプロトコルはまた動物行動学で採用数を減らすことができます。ここで報告されたアプリケーションが最近説明し、含む、前脳基底部 (BF)、広告で主に影響を受ける脳の領域の一つであるラット脳スライス前のヴィヴォの最初の時間のためにテストします。具体的には、それはアセチルコリンエステラーゼ (AChE) の C 末端から派生した有毒ペプチド投与がトリガー、BF の前後軸に沿った発現の広告のようなプロファイルを求める可能性が実証されています。alpha7 ニコチン性受容体 (α 7-nAChR) など、広告の変更蛋白質は、タウ (p-タウ) とアミロイド β (a β) にリン酸化。

ここで提示されたプロトコルは、α 7-nAChR、p の式のサイト依存的に有毒ペプチド、T30 の管理が変調を示します-タウ ・ BF 含むセクション (図 3 a) で a β。ニコチン性受容体と比べてそのコントロール、吻側治療 hemislice の大幅な増加を示しています (スライス 1、 p = 0.0310) (図 3 b)、中間のスライスは 2 つの条件 (変更?...

Watch this Scientific Journal Video about An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices at JoVE.com

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

体内と体外の実験の利点を組み合わせてさらに神経変性疾患の基になる初期のイベントに対する洞察を提供することができますし、確立されたex vivo脳法に基づく手法を提案します。また、同じ解剖学的平面の処理と未処理のグループの直接の比較のためのユニークな機会を表します。

Ex Vivoラット脳切片を用いた神経変性疾患の主要なイベントを勉強する方法

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

an-alternative-approach-to-study-primary-events-neurodegeneration

Research

JoVE Journal

Neuroscience

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

6.8K ビュー.  Culham Science Centre. 体内と体外の実験の利点を組み合わせてさらに神経変性疾患の基になる初期のイベントに対する洞察を提供することができますし、確立されたex vivo脳法に基づく手法を提案します。また、同じ解剖学的平面の処理と未処理のグループの直接の比較のためのユニークな機会を表します。

ビデオ: An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

Cの生体レーザー軸索切断にエレガンス

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

神経科学

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

セミ厚い脳スライスにおける高解像度蛍光イメージングへの迅速なアプローチ

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

GABA-activated Single-channel and Tonic Currents in Rat Brain Slices

The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, respectively 4,5,6,7 The GABAA channel is a pentameric GABA-gated chloride channel. The channel subunits are grouped into 8 families (&alpha;1-6, &beta;1-3, &gamma;1-3, &delta;, &#949;, &theta;, &pi; and &rho;). Two alphas, two betas and one 3rd subunit form the functional channel 8. By combining studies of sub-type specific GABA-activated single-channel molecules with studies including all populations of GABAA channels in the neuron it becomes possible to understand the basic mechanism of neuronal inhibition and how it is modulated by pharmacological agents.
We use the patch-clamp technique 9,10 to study the functional properties of the GABAA channels in alive neurons in hippocampal brain slices and record the single-channel and whole-cell currents. We further examine how the channels are affected by different GABA concentrations, other drugs and intra and extracellular factors. For detailed theoretical and practical description of the patch-clamp method please see The Single-Channel Recordings edited by B Sakman and E Neher 10.

We use the patch-clamp technique to measure GABA-activated single-channel currents (GABAA channels, GABAA receptors) and the synaptic and tonic currents they generate in neurons. Activation of the channels decreases neuronal excitability in health and disease 1,2,3,4.

ラット脳スライスにおけるGABA活性化シングルチャネルとトニック電流

The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, ...

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

神経形態形成の研究のための方法：<em&gt;エキソビボ</em胎児マウス大脳皮質&gt; RNAiのエレクトロポレーション

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

超音波血液脳関門の破壊とマンガン増強MRIを用いた脳機能イメージングの試み

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

脳スライスビオチン：<em&gt;生体外</emアダルトニューロンにおいて地域固有の細胞膜のタンパク質輸送を測定するために&gt;の取り組み

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

Embryonic neurons are born in the ventricular zone of the brain, but subsequently migrate to new destinations to reach appropriate targets. Deciphering the molecular signals that cooperatively guide neuronal migration in the embryonic brain is therefore important to understand how the complex neural networks form which later support postnatal life. Facial branchiomotor (FBM) neurons in the mouse embryo hindbrain migrate from rhombomere (r) 4 caudally to form the paired facial nuclei in the r6-derived region of the hindbrain. Here we provide a detailed protocol for wholemount ex vivo culture of mouse embryo hindbrains suitable to investigate the signaling pathways that regulate FBM migration. In this method, hindbrains of E11.5 mouse embryos are dissected and cultured in an open book preparation on cell culture inserts for 24 hr. During this time, FBM neurons migrate caudally towards r6 and can be exposed to function-blocking antibodies and small molecules in the culture media or heparin beads loaded with recombinant proteins to examine roles for signaling pathways implicated in guiding neuronal migration.

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

Embryonic neurons are born in the ventricular zone of the neural tube, but migrate to reach appropriate targets. Facial branchiomotor (FBM) neurons are a useful model to study neuronal migration. This protocol describes the wholemount ex vivo culture of mouse embryo hindbrains to investigate mechanisms that regulate FBM migration.

マウス後脳<em&gt;生体外</em&gt;文化顔面Branchiomotorニューロンの移行を研究する

Embryonic neurons are born in the ventricular zone of the brain, but subsequently migrate to new destinations to reach appropriate targets. Deciphering ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

アン<em&gt;エクスビボ</em&gt;レーザー誘起脊髄損傷モデルがリアルタイムで軸索変性のメカニズムを評価するために

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

脳スライスにおける恐怖回路のex vivo光遺伝学解剖

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

生物発光カルシウムインジケーター、GFPエクオリンに基づいて生体内脳機能イメージングのアプローチで

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

Ex Vivoラット脳切片を用いた神経変性疾患の主要なイベントを勉強する方法

要約

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Cの生体レーザー軸索切断にエレガンス

セミ厚い脳スライスにおける高解像度蛍光イメージングへの迅速なアプローチ

ラット脳スライスにおけるGABA活性化シングルチャネルとトニック電流

神経形態形成の研究のための方法：

超音波血液脳関門の破壊とマンガン増強MRIを用いた脳機能イメージングの試み

脳スライスビオチン：

マウス後脳

アン

脳スライスにおける恐怖回路のex vivo光遺伝学解剖

生物発光カルシウムインジケーター、GFPエクオリンに基づいて生体内脳機能イメージングのアプローチで