この作品は、PTR に健康の国民の協会 (R01 NS084941)、(ADHS16-162406) アリゾナ州医学研究委員会からの補助金によって支えられました。ワシントン州は、過去の国家機関の健康研修助成金 (T32 HL007249) 博士前期予定からサポート受けています。

下記すべての手続機関動物ケアおよび使用委員会 (IACUC) によって承認されているし、国立衛生研究所 (NIH) と動物研究に準拠: In Vivo実験 (到着) ガイドラインを報告します。プロトコルの手順のフローは、図 1に描かれています。
1. セットアップ手順について
<ol><li>脳微小血管バッファー (BMB) を準備します。54.66 g D-マンニトール、1.90 g グリコールエーテルジアミン四酢酸、および 1.46 g 2-amino-2-(hydroxymethyl)-1,3-propanediol (すなわち、トリス) ベースをきれいなビーカーに計量して起動します。1.0 L の脱イオン水を追加します。磁性攪拌器を使用して BMB バッファーのコンポーネントをミックスします。ソリューションが完全に混合されて、一度 7.4 1.0 M HCl を使用してに pH を調整します。</li>
	<li>動物の麻酔前に 26% デキストラン (75,000 MW) ソリューション (w/v) を準備します。次の手順で説明するように、デキストラン ソリューションを準備します。 注: それがデキストランは緩衝水溶液で分解する約 1.5-2 時間を取ることができるので、早めにこのソリューションを準備する重要です。<ol>
<li>きれいなビーカーに粉末デキストラン (100 mL バッファーあたり 26 g) を重量を量る。</li>
		<li>BMB の適切な量を測定し、独立した、きれいなビーカーに分注します。</li>
		<li>BMB を粉末デキストランを含むビーカーにゆっくりと注ぐ。手動で BMB ガラス攪拌棒を使用しての注入中に粉末のデキストランをミックスします。</li>
		<li>使用してすぐに前 1.0 M HCl で 7.4 に pH を調整します。 注: 12 個別 Sprague-dawley ラット脳微小血管の典型的な分離 (男性または女性; 生後 3 か月; 200-250 g 各) デキストラン溶液 (すなわち、BMB の 200 mL で 52 g デキストラン) 200 mL が必要です。</li>
	</ol>
</li>
</ol>2. ラット脳組織の抽出
<ol><li>(50 mg/kg; 2.5 mL/kg i. p.) ケタミン ・ キシラジンを使用してラットの麻酔希望の実験的治療 (10 mg/mL; 2.5 mL/kg I、p.)。それぞれケタミン ・ キシラジンの 20 mg/mL と 4.0 mg/mL の最終濃度 0.9% 生理食塩水で希釈します。斬罪に IACUC ガイドラインに従って削ったギロチンを使用して動物を安楽死します。男性と女性の両方の Sprague-dawley ラットの同じ手順を使用します。</li>
	<li>外科はさみを使用して単一横カットすることによってラット頭蓋骨から皮膚を切除します。</li>
	<li>Rongeurs を使用して、慎重に頭蓋骨プレートを削除し、脳を公開します。</li>
	<li>ヘラを使って脳を削除します。大脳を外し、BMB の 5 mL を含む 50 mL の円錐管に隔離した脳細胞の場所します。プロテアーゼ阻害剤を使用する前にすぐに BMB のバッファーの 1.0 mL あたりカクテルの 1.0 μ L を追加します。</li>
</ol>3. 脳処理
<ol><li>きれいなペトリ皿に 50 mL の円錐管から脳組織を転送します。</li>
	<li>12.5 cm 径フィルター紙上大脳皮質に接着された疎外髄膜を削除する脳をロールバック鉗子を使用して、優しく。優しくフィルター ペーパーに対して脳組織を押しながら再び組織のロールします。この手順中に頻繁にろ紙をオンにします。</li>
	<li>鉗子による大脳半球から脈絡叢を区切ります。 注: 脈絡叢は、大脳の脳室の表面にローカライズされた明確な膜質の組織として表示されます。</li>
	<li>優しく脳組織を平らにし、残りの髄膜と鉗子による嗅球を削除します。</li>
	<li>冷えたグラスのモルタルに皮質の脳組織を配置します。モルタルに BMB 含むプロテアーゼ阻害剤カクテルの 5.0 mL を追加します。</li>
	<li>頭上式の電力ホモジナイザーを使用して、3,700 rpm ストローク上下 15 を使用して脳組織をホモジナイズしてください。10 mL の乳鉢と乳棒組織グラインダーを使用して均質化を実行します。それぞれの個々 のサンプルの均質化、間 70% のエタノールを使用して杵をクリーンアップします。 注: 均質化ペースの面で一貫しているべきストロークと大きさ。</li>
	<li>ラベル付き遠沈管に磨砕液を注ぐ。</li>
</ol>4. 遠心分離の手順
<ol><li>脳のホモジネートを含む各ラベル付き遠心管に 26% デキストラン溶液 8.0 mL を追加します。</li>
	<li>2 回チューブを反転し、徹底的に渦サンプル。脳ホモジネート液の 26% デキストランの徹底的な混合できるように複数のアングルを使用して各サンプルのボルテックスを実施します。</li>
	<li>4 ° C で 15 分間 5,000 × gで遠心分離機サンプル 注: いくつかの分析に便利です脳実質、脳微小循環を比較します。脳実質におけるタンパク質発現の評価すべき必要、この遠心分離のステップ (すなわち、脳実質分数) から清収集でき将来の使用に備えて-80 ° C で保存します。</li>
	<li>真空吸引装置を使用して上清を除去し、パスツール ピペットをガラスです。 注: ペレットを邪魔しないように注意する必要があります。そうでなければ、その大幅このプロシージャから蛋白質収量を低下させるが、収集した脳微小循環の量が削減されます。</li>
	<li>BMB 含むプロテアーゼ阻害剤のカクテル (すなわち、1.0 μ L プロテアーゼ抑制剤のカクテル BMB バッファーの 1.0 mL あたり) 5.0 mL にペレットを再懸濁します。渦ように徹底した混合ペレット。</li>
	<li>各遠心管と渦このプロトコルの手順 4.1 4.2 で説明されているように 26% デキストランの 8.0 mL を追加します。</li>
	<li>4 ° C で 15 分間 5,000 × gで遠心分離機サンプル</li>
	<li>真空フラスコ、ガラス ピペットを使用して、上清を吸引、脳微小血管を含むそのペレットが中断されないことを確認します。 注: 管壁に付着した余分な材料微小血管が含まれていないとする必要がありますが丁寧に掃除して削除します。</li>
	<li>手順 4.5 4.8 追加を 2 回。</li>
	<li>一度 4 デキストラン遠心分離手順が完了、各ペレットとサンプルを再懸濁しますに渦に BMB の 5.0 mL を追加します。 注: 4.10 のステップが完了した後、タンパク質局在の解析の全体の微小血管を収集できます。これは、再浮遊微小ペレットの分注 50 μ L を取り、ガラス顕微鏡スライドの上に塗りつけによって実現できます。微小血管が冷たいエタノールで固定後に追加 10 分スライドは 4 ° c までのイメージングに必要な格納できる暖房のブロック上で 10 分間 95 ° C で熱固定になります。</li>
</ol>5. 遠心合計脳微小血管膜のサンプルを準備するには
<ol><li>冷えたグラス ホモジナイザーにサンプルを転送します。</li>
	<li>頭上式の電力ホモジナイザーを使用して、3,000 rpm で 8-10 上下のストロークを使用して脳組織をホモジナイズしてください。均質化は、10 mL の乳鉢と乳棒組織グラインダーを使用して行われています。次のそれぞれの個別のサンプルの均質化 70% エタノールを使用して杵をクリーンアップします。</li>
	<li>超遠心機チューブをきれいにするサンプルを転送します。数し、それぞれの個々 のチューブの重量を量る。超遠心機のローターに読み込む前にチューブのペアし、のバランスします。 注: すべての重さと遠心チューブの組み合わせ実施しなければならないキャップと上管重量の正確な測定を確保します。</li>
	<li>4 ° C で 1 時間 150,000 × gで遠心分離機サンプル</li>
	<li>上清を吸引真空フラスコとガラス パスツール ピペットを使用して中空糸膜ペレットを妨害しないように気を使っています。</li>
	<li>ペレットのサイズに基づいては脱イオン水と BMB の 1:1 (v/v) 比で構成されるストレージ バッファーの適切なボリュームを追加します。通常、ペレットはストレージ バッファーの 400-500 μ L で再停止されます。プロテアーゼ阻害剤カクテルをストレージ バッファーの 1.0 mL あたり 1.0 μ L の割合でストレージ バッファーに追加します。</li>
	<li>渦サンプル ストレージ バッファーでペレットを再懸濁します。</li>
	<li>ガラス パスツール ピペットを使用すると、ラベル付きの 1.5 mL 遠心チューブに中空糸膜サンプルを転送します。</li>
	<li>針を通して渡すサンプルは、サンプルが完全に混合し、細胞凝集体が存在しないことを確保するため 5 x を注射器します。</li>
	<li>-80 ° c まで解析に必要なサンプルを格納します。 注: 各微小血管膜サンプルのタンパク質含量は、ブラッドフォード法を使って測定します。</li>
</ol>

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T., Bendayan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+viral+envelope+glycoprotein+gp120+produces+oxidative+stress+and+regulates+the+functional+expression+of+multidrug+resistance+protein-1+(Mrp1)+in+glial+cells.">HIV-1 viral envelope glycoprotein gp120 produces oxidative stress and regulates the functional expression of multidrug resistance protein-1 (Mrp1) in glial cells.</a> J Neurochem. 106 (3), 1298-1313 (2008).</li><li>Pustylnikov, S., Sagar, D., Jain, P., Khan, Z. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+C-type+lectins-mediated+host-pathogen+interactions+with+dextran.">Targeting the C-type lectins-mediated host-pathogen interactions with dextran.</a> J Pharm Pharm Sci. 17 (3), 371-392 (2014).</li><li>Obermeier, B., Daneman, R., Ransohoff, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development,+maintenance+and+disruption+of+the+blood-brain+barrier.">Development, maintenance and disruption of the blood-brain barrier.</a> Nat Med. 19 (12), 1584-1596 (2013).</li><li>Abdullahi, W., Davis, T. P., Ronaldson, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Expression+of+P-glycoprotein+and+Organic+Anion+Transporting+Polypeptides+at+the+Blood-Brain+Barrier:+Understanding+Transport+Mechanisms+for+Improved+CNS+Drug+Delivery?.">Functional Expression of P-glycoprotein and Organic Anion Transporting Polypeptides at the Blood-Brain Barrier: Understanding Transport Mechanisms for Improved CNS Drug Delivery?.</a> AAPS J. 19 (4), 931-939 (2017).</li></ol>

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この記事では新鮮なラット脳組織から分離された微小血管からの膜蛋白質のサンプルの準備のシンプルで効果的な方法を説明します。文献13,20,21,22で孤立した血管からラット脳微小血管の分離および/または膜の準備世代のためのいくつかの方法が報告されています。,24. ...

血液脳関門 (BBB) は、中枢神経系 (CNS) と全身循環間のインターフェイスに存在して、脳のホメオスタシスの維持に重要な役割を果たしています。具体的には、正確にコントロール溶質濃度1中枢神経系のかなり代謝要求を満たすために脳組織に必要なこれらの栄養素を効率よく供給して脳細胞外液中に BBB の機能。これらの役割は、微小血管内皮細胞のレベルに主に存在する、BBB は脳実質に潜在的に有害物質ができませんを確保しながらアクセスするいくつかの物質を有効に離散のメカニズムを持つ必要がありますを意味します。蓄積されます。確かに、脳微小血管内皮細胞は穴あきではなく、非選択的透過性2の欠如を保証する限られた pinocytosis を展示します。脳微小血管内皮細胞が物理的な隣接する血管内皮細胞間の「封印」と大きく脳に血液媒介物質の傍細胞拡散を制限するフォームに動作するタイトのジャンクションと adherens ジャンクション蛋白質を表現するさらに、実質。確かに、内因性および外因性物質の選択的な透磁率は、吸収と排出トランスポーター3の機能発現を必要とします。全体的に、タイトな接合、アドヘレンスジャンクション、トランスポーターは、BBB のユニークなバリア性を維持するためにコンサートで動作します。
BBB は生理学的、病態生理学的、および薬理学的刺激に応答する動的な障壁であります。たとえば、低酸素/再酸素化ストレスの増加傍細胞透過性このような血管マーカーに関連付けられている (すなわち、オクルディン、ギャップ occluden 1 (ZO-1))、重要なタイト結合タンパク質の発現を調節するのに示されています。ショ糖4,5,6。外傷性脳損傷7末梢炎症性疼痛8,9設定で bbb と同様の観測が行われました。これらの同じ病気の BBB10、11,12,13,14トランスポート メカニズムを調節することがまた。確かに、低酸素/再酸素化障害を高める有機アニオン輸送ポリペプチド 1a4 の機能発現 (Oatp1a4)、BBB でつながることができる特定ファミリートランスポーターの輸送基質の血液-脳輸送に大幅に増加するなどタウロコール酸とアトルバスタチンとの13。薬物療法自体は、脳の薬の効果の両方の深遠な変更および薬物-薬物相互作用の基礎を形成することができますメカニズムによって BBB プロパティを変更することも。たとえば、脳微小血管内皮細胞におけるアセトアミノフェン ターゲット核内受容体シグナル機構重要な排出トランスポーター P 糖タンパク質 (P ・ gp) の発現を増加し、時間依存性鎮痛を変更オピオイド鎮痛薬と確立された P gp は輸送基板15モルヒネによって与えられました。BBB の変更、病気や薬によって誘発されることができますの徹底的な理解は、これらの変更を制御する特定の規制メカニズムの同定と解析にも必要です。確かに、離散のシグナル伝達経路はタイト結合蛋白質16,17 , トランスポーター15,の分子の発現を制御する脳微小血管内皮細胞で確認されている18,19。一緒に取られて、これらの観察は、複雑な分子経路が健康および病気のトランスポーターの BBB のタイトな接合の規制に関与していることを示します。
BBB の研究で重要な課題は、微小血管に由来する実験動物及びその後膜サンプルの脳組織からの分離のためのシンプルで効果的なメソッドの絶対要件です。これらのサンプルは、脳微小血管内皮細胞の濃縮し、他の細胞型の存在の限定、両方準備されなければなりません。過去数年間、齧歯動物の脳から血管の隔離のための複数の方法論は、科学文献13,20,21,22で報告されています。ここでは、シンプルで堅牢な再現可能な方法ラット脳から微小血管の分離および蛋白質の表現の分析に使用することができます内皮膜濃縮試料の調製をについて説明します。この微小血管の隔離のプロトコルの利点は、個々 の実験動物から十分な蛋白質収量と高品質のサンプル準備を取得する機能です。これにより、間動物可変性蛋白質の表現の検討。このプロトコルのような進歩は、BBB で蛋白質の変更の本当の大きさの推定過剰 (または過少評価) を回避できますので BBB 研究の堅牢性を大幅に改善が。また、デキストランの遠心分離手順で複数の包含は、ニューロンなど不要な細胞成分の除去を促進しながら実験サンプルの微小血管についての改良濃縮できます。

<table>
	<tbody>
		<tr>
			<td>Protease Inhibitor Cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>#P8340</td>
			<td>Component of brain microvessel buffer</td>
		</tr>
		<tr>
			<td>D-mannitol</td>
			<td>Sigma-Aldrich</td>
			<td>#M4125</td>
			<td>Component of brain microvessel buffer</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>#E3889</td>
			<td>Component of brain microvessel buffer</td>
		</tr>
		<tr>
			<td>Trizma Base</td>
			<td>Sigma-Aldrich</td>
			<td>#T1503</td>
			<td>Component of brain microvessel buffer</td>
		</tr>
		<tr>
			<td>Dextran (MW 75,000)</td>
			<td>Spectrum Chemical Mftg Corp</td>
			<td>#DE125</td>
			<td>Dextran used in centrifugation steps to separate microvessels from brain parenchyma</td>
		</tr>
		<tr>
			<td>Zetamine</td>
			<td>MWI Animal Health</td>
			<td>#501072</td>
			<td>General anesthetic</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Western Medical Supply</td>
			<td>#5530</td>
			<td>General anesthetic</td>
		</tr>
		<tr>
			<td>0.9% saline solution</td>
			<td>Western Medical Supply</td>
			<td>N/A</td>
			<td>General anesthetic diluent</td>
		</tr>
		<tr>
			<td>Filter Paper (12.5 cm diameter)</td>
			<td>VWR</td>
			<td>#28320-100</td>
			<td>Used for removal of meninges from brain tissue</td>
		</tr>
		<tr>
			<td>Centrifuge Tubes</td>
			<td>Sarstedt</td>
			<td>#60.540.386</td>
			<td>Disposable tubes used for dextran centrifugation steps</td>
		</tr>
		<tr>
			<td>Pierce&#8482; Coomassie Plus (Bradford) Assay</td>
			<td>ThermoFisher Scientific</td>
			<td>#23236</td>
			<td>Measurement of protein concentration in membrane preparations</td>
		</tr>
		<tr>
			<td>Wheaton Overhead Power Homogenizer</td>
			<td>DWK Life Sciences</td>
			<td>#903475</td>
			<td>Required for homogenization of samples</td>
		</tr>
		<tr>
			<td>10.0ml glass mortar and pestle tissue grinder</td>
			<td>DWK Life Sciences</td>
			<td>#358039</td>
			<td>Required for homogenization of samples</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Sigma-Aldrich</td>
			<td>#H1758</td>
			<td>Required for pH adjustment of buffers</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>ThermoFisher Scientific</td>
			<td>#23210</td>
			<td>Protein standard for Bradford Assay</td>
		</tr>
		<tr>
			<td>Standard Forceps</td>
			<td>Fine Science Tools</td>
			<td>#91100-12</td>
			<td>Used for dissection of brain tissue</td>
		</tr>
		<tr>
			<td>Friedman-Pearson Rongeurs</td>
			<td>Fine Science Tools</td>
			<td>#16020-14</td>
			<td>Used for opening skull to isolate brain</td>
		</tr>
		<tr>
			<td>50 ml conical centrifuge tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>#352070</td>
			<td>Used for collection of brain tissue following isolation</td>
		</tr>
		<tr>
			<td>Glass Pasteur Pipets</td>
			<td>ThermoFisher Scientific</td>
			<td>#13-678-20C</td>
			<td>Used for aspiration of cellular debris following dextran spins</td>
		</tr>
		<tr>
			<td>Ethanol, anhydrous</td>
			<td>Sigma-Aldrich</td>
			<td>#459836</td>
			<td>Used for cleaning tissue grinder; diluted to 70% with distilled water</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes</td>
			<td>Beckman-Coulter</td>
			<td>#41121703</td>
			<td>Used for ultracentrifugation of samples</td>
		</tr>
	</tbody>
</table>

a simple and reproducible method to prepare membrane samples from freshly isolated rat brain microvessels

血液脳関門 (BBB) は、様々 な病態生理学的および薬理学的刺激に応答する動的なバリア組織です。これらの刺激の結果としてそのような変更大幅脳への薬物送達を調節でき、延長によって、病気の原因のかなりの挑戦中枢神経系の治療に (CNS)。薬物治療学に影響を与える多くの BBB 変更は、ローカライズは、内皮細胞のレベルで表現するタンパク質を含みます。確かに、健康および病気の BBB の生理学的な知識は、これらの膜タンパク質の研究に大きな関心を呼んでいます。基本的な科学研究の観点から、これは単純なが堅牢で再現性のある実験動物から採取した脳組織から血管の分離法のための要件を意味します。新鮮単離血管から膜サンプルを準備するためにサンプル準備が血管内皮細胞に富むが、単位 (すなわち、アストロ サイト、ミクログリア、ニューロンの他の細胞型の存在下で限定が肝要です。ペリサイト)。付加的な利点は、個々 の動物から実験群におけるタンパク質発現の真の変動を捕捉するためにサンプルを準備する機能です。本稿では、ラット脳微小血管の分離と膜試料の調製に利用されるメソッドに関する詳細情報が提供されます。派生した、サンプルの微小血管濃縮サンプル バッファーのデキストランをふくむ 4 遠心分離手順を使用して実現されます。このプロトコルは、特定のアプリケーションの他の所で簡単に対応できます。生理学的、病態生理学的、および薬理学的刺激への BBB 対応の理解を助けることができる大きくタンパク質解析実験から堅牢な実験データを生成するため、このプロトコルから生成されるサンプルを示されています。

ラット脳微小血管の分離と微小血管膜試料の準備のための実験の流れは図 1に示します。ここに示す手順を使用して、ラットの脳からそのまま微小血管の正常な分離を示す (図 2 a)。これらの血管は、デキストランと膜サンプル (すなわち、完了ステップ 4.10) を準備する遠心を開始する前にすぐに遠心分離が完了できた。こ?...

Watch this Scientific Journal Video about A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels at JoVE.com

A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels

ここでは、ラット脳微小血管の分離膜試料の調製の方法を説明します。このプロトコルには、個々 の動物から得られる許容可能なタンパク質試料濃縮微小血管を生産の明確な利点があります。サンプルは、脳微小血管内皮細胞で堅牢なタンパク質解析に使用できます。

新鮮単離ラット脳微小循環から膜のサンプルを準備するシンプルで再現性のある方法

The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting ...

a-simple-reproducible-method-to-prepare-membrane-samples-from-freshly

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

10.0K ビュー.  University of Arizona, Tucson. ここでは、ラット脳微小血管の分離膜試料の調製の方法を説明します。このプロトコルには、個々 の動物から得られる許容可能なタンパク質試料濃縮微小血管を生産の明確な利点があります。サンプルは、脳微小血管内皮細胞で堅牢なタンパク質解析に使用できます。

ビデオ: A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

春雨とカッペリーニの処理試験：ラットとマウスの器用な前足の機能の簡単な定量的指標

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

神経科学

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

非心室プラスミド注入し、エレクトロポレーションにより産後のラット脳への遺伝子デリバリー

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

FM染料を用いて培養小脳顆粒神経細胞のシナプス小胞プールの補充の定量分析

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

A Visual Description of the Dissection of the Cerebral Surface Vasculature and Associated Meninges and the Choroid Plexus from Rat Brain

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain proper, that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis. The tissue harvested is suitable for biochemical and physiological analysis, and the MAV has been shown to be sensitive to damage produced by amphetamine and hyperthermia 1,2. As well, the major and minor cerebral vasculatures harvested in MAV are of potentially high interest when investigating concussive types of head trauma. The MAV dissected in this presentation consists of the pial and some of the arachnoid membrane (less dura) of the meninges and the major and minor cerebral surface vasculature. The choroid plexus dissected is the structure that resides in the lateral ventricles as described by Oldfield and McKinley3,4,5,6. The methods used for harvesting these two tissues also facilitate the harvesting of regional cortical tissue devoid of meninges and larger cerebral surface vasculature, and is compatible with harvesting other brain tissues such as striatum, hypothalamus, hippocampus, etc. The dissection of the two tissues takes from 5 to 10 min total. The gene expression levels for the dissected MAV and choroid plexus, as shown and described in this presentation can be found at GSE23093 (MAV) and GSE29733 (choroid plexus) at the NCBI GEO repository. This data has been, and is being, used to help further understand the functioning of the MAV and choroid plexus and how neurotoxic events such as severe hyperthermia and AMPH adversely affect their function.

This video presentation shows a method of harvesting the two most important highly vascular structures that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis.

ラットの脳から脳表面血管系および関連髄膜の解剖と脈絡叢のビジュアル解説

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

マウスにおける低温感受性と適応を決定するためのシンプルで安価な方法

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies using Drosophila have contributed important breakthroughs in understanding neurological processes. Thus, with the goal of understanding the cellular and molecular basis of TBI pathologies in humans, we developed the High Impact Trauma (HIT) device to inflict closed head TBI in flies. Flies subjected to the HIT device display phenotypes consistent with human TBI such as temporary incapacitation and progressive neurodegeneration. The HIT device uses a spring-based mechanism to propel flies against the wall of a vial, causing mechanical damage to the fly brain. The device is inexpensive and easy to construct, its operation is simple and rapid, and it produces reproducible results. Consequently, the HIT device can be combined with existing experimental tools and techniques for flies to address fundamental questions about TBI that can lead to the development of diagnostics and treatments for TBI. In particular, the HIT device can be used to perform large-scale genetic screens to understand the genetic basis of TBI pathologies.

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Here we describe a method to inflict closed head traumatic brain injury (TBI) in Drosophila. This method provides a gateway to investigate the cellular and molecular mechanisms that underlie TBI pathologies using the vast array of experimental tools and techniques available for flies.

で閉鎖性頭部外傷性脳損傷を負わせる方法<em&gt;ショウジョウバエ</em

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.

MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

miRNAの脳特異的ノックダウンのための定位注射への単純な代替

MicroRNAs (miRNAs) are key regulators of gene expression.  In the brain, vital processes like neurodevelopment and neuronal functions depend on the ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

使用して報酬を求める行動の変化を測定するための簡単な方法<em&gt;キイロショウジョウバエ</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

ひと成人皮膚線維芽細胞から誘導される神経細胞の高収率文化の生成の単純です

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

新鮮単離ラット脳微小循環から膜のサンプルを準備するシンプルで再現性のある方法

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