すべての動物実験は、国立機関の健康動物使用のガイドラインに従って実施されています。この作品は、一部に C. 区国立衛生研究所 (R01NS093073 と R21AA024873) からの助成金によって支えられました。

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著者が明らかに何もありません。

この microbiomechanical のアッセイのプロシージャはまっすぐ進むです。そのすべてのステップは慎重に行われている場合信頼性の高い結果が生成されます。正常なデータ収集の妨げになる場合は、不適切な実行、いくつかの主要な手順があります。重要なステップが始まる上流たばこを吸う刺激の実際に適用しました。細かく、養殖と一次ニューロンの文化のケアが最も重要です。培養神経細胞が健全でない場合彼らが反応しない一貫して、以来、彼ら可能性がありますが既にされて準備万端ストレス。文化、初期セットアップと下肢が誘発される coverslip 表面から細胞の剥離は発生しませんが、根気よく行う必要があります、整合の圧力を提供するためにたばこを吸う装置の校正の下流。セッティングの正確な校正は、ピペットの目詰まり、チューブ、またはバルブの問題のための 1 時間をかかることがあります。皿にセルを配置し、ピペット チップを配置に進む前に最低限の抵抗とピペットから液が流れていることを確認する重要です。設定への変更が懸念している限りでは、血流、パッチク ランプ、その他顕微鏡ベースの計測器、顕微鏡の両側に無料の物理的なスペースに主に依存してたばこを吸う装置一組することができます。
このシステムには、注意が必要な 2 つの本質的な制限があります。設定は一貫してセルに噴き出し流体を介して培養海馬神経細胞の軸索で可逆的な下肢の形成を開始できます。しかし、同じたばこを吸うフィールド内のニューロンに正確な圧力値が同一でないことに注意する重要です。流体の圧力、細胞に影響を与える、微細加工シリコーン膜4,10から圧力負荷測定に基づいて計算、たばこを吸う、フィールドの中央で平均圧力を表します。したがって、正確なたばこを吸う圧力は、たばこを吸うフィールドとフィールドの端のまわりの最低重心で最高です。圧力強度相関発症、サイズ、および下肢の数4を誘発したことを示した.高い圧力はより大きくより速く発症およびより豊富な下肢4で起因しました。私たちの最近の研究では、確実にまだほとんどの軸索4で下肢を引き起こすことができる最小限の圧力 (たばこを吸うピペットの先端で 190 mmHg) を使用しました。この条件の下で若いニューロンの静脈瘤の発症は通常約 5 秒。神経細胞の種類、年齢、および細胞レベル下コンパートメントに依存だけでなく、たばこを吸うフィールド内のニューロンの位置にも左右されますが変化する正確なシステム、息を切らし同じ発症時間の値場合、注意してくださいすることが重要です。ニューロンによって受信されたたばこを吸う圧の変動、にもかかわらず、このアッセイ システムはまだ中枢ニューロンに与える力学的影響を研究するための信頼できる手段を提供します、高い一貫性と再現性の高い実験結果が得られます。
システムの第 2 の制限は、その目詰まりの問題です。生理学的に関連する圧力を達成するためにザ ・ ピペッツの開口部は小さい、45 μ m 周辺です。ただし、プラスチック製のチューブからの残骸を詰まらせる傾向にある小さな開口部、ピペット チップの残骸を顕微鏡下ではっきり見ることができます。これは、ピペットの目詰まりが発生した場合、セットアップを準備中の時間の量を増やします。したがって、すべてのソリューションは、注射器、チューブ、ピペットに置かれる前に慎重にフィルター処理されます。また、先頭とフィルター処理されたハンクのバッファーで洗浄後、一日の終わりに 70% エタノールでたばこを吸うシステムを洗浄することが重要です。潜在的な問題を解決するために複数のピペットをプルしようとしてお勧めします。その解決策は実際に煙を吐いて出て可視化実験を開始する前にピペットを確認が欠かせません。良いたばこを吸うシステムが確立されると、それは通常、その日の残りの部分を持続できます。
ニューロンの文化と組み合わせて、この生体力学的アッセイは、生理学的および病態生理学的条件を模倣した中枢神経形づくりを勉強するためのユニークな機会を提示します。このシステムは圧縮とニューロンのせん断の影響を調べることができます。対照的に、他のシステムの面内ストレッチ5,6,7を検討するため。たばこを吸う区域の中心のニューロンによる受信平均圧力は、私たちのシステム40.25 ± 0.06 nN/μ m2です。細胞のこの圧力は以前利用した走査型力顕微鏡とバルク レオロジー、光誘起変形13,14を識別するために測定される海馬ニューロンの弾性率に匹敵するだけでなく神経突起前縁in vitro でスキャンすることによって測定の成長を変えるため圧力力顕微鏡 (0.27 ± 0.04 μ m2)16。この圧力は、機械的に特定の蛍光細胞サイズリポソーム油微小油滴中 (1.6 ± 0.8 nN/μ m)2,14を用いて測定した E11 胚の間葉系細胞によって自然生成することができます圧力を超えない。
このシステムは既に正常にカルシウム イメージング、パッチク ランプ、電子顕微鏡、閉じた頭蓋骨 mTBI マウス モデル4の細胞解析との組み合わせで使用されています。アッセイは、下肢、内のタンパク質のターン オーバーを調べるだけでなく、勉強蛋白質蛋白質の相互作用を変更する心配する (FRAP) 退色後蛍光回復などの顕微鏡試験と組み合わせても使用ことができます。静脈瘤形成。ことができる任意のメソッド本質的に、このたばこを吸うアッセイ顕微鏡とライブセル イメージング セットアップを使用してペアリングが可能します。このアッセイの多様性あるマイクロメカニックスによるストレスによって神経細胞の形態と機能に関する様々 な効果の基礎となる分子メカニズムの勉強に非常に貴重です。

静脈瘤の形成、または腫れ/ビーズ, 軸索に沿っては多くの障害やパーキンソン病、アルツハイマー病、多発性硬化症を含む中枢神経系の損傷で観察した神経変性疾患の顕著な特徴と外傷性脳損傷1,2。活動電位伝搬とシナプス伝達3軸索下肢の生理学的な影響にもかかわらず、下肢の生成方法は不明のまま。最近、齧歯動物から培養海馬神経細胞の新設 microbiomechanical 試金を使用して、機械的刺激が下肢機能を非常に魅力的なこれらのニューロンを引き起こすことができることがわかった。最初に、静脈瘤の誘導は急速な (< 10 s)、このプロセスは予期せず元に戻せる。第二に、静脈瘤開始が圧力を吹きかける強さ依存: より高い圧力、高速の開始。第三に、静脈瘤の発生は神経年齢に依存します。若いニューロンの軸索はより古いニューロンのそれらと比較して、機械的ストレスに対する応答表示されます。第四に、樹枝状結晶およびこれらのニューロンの軸索の初期セグメント中の海馬ニューロンの軸索に沿って下肢のフォームには、同じたばこを吸う条件変更は表示されません。したがって、我々 の研究は、神経細胞の極性の新規機能を明らかにしました。体外システムこれらの発見は、生理学的に関連しています。軽度外傷性脳損傷 (mTBI)生体内でモデルを使用して、軸索の下肢が閉じる頭蓋骨への影響、私たち体外で一貫した後すぐにマウスの体性感覚野における多焦点方式で開発を示した結果4。私たちの染色・ イメージング mTBI マウスの神経の形態学的変化のスナップ ショットを提供するだけで、体内の実行以来機械的衝撃に神経細胞形態のタイムラプス イメージングはまだ不可能することが重要です。
この液体に吹きかけるシステム機械的応力誘起静脈瘤形成に関連するユニークな機能をキャプチャするのみならず、基になるメカニズムを決定することができました。細胞外のさまざまなソリューション、ブロッカーと機械刺激感受性イオン チャネル、細胞電気生理学の別候補のオープナをテストすることにより一時的な受容器の潜在的な陽イオン チャネル亜科 V メンバー 4 (TRPV4) ことがわかりました吹き込むことによってアクティブ化される Ca2 + +の Na に透過性チャネルは軸索静脈瘤形成4の間に初期の機械的応力を検出を主に担当。これはさらに、siRNA ノックアウト アプローチで確認されました。まとめると、海馬ニューロンの文化を開発したこの新しいアッセイ システム、他の技術との組み合わせでは特に、脳のニューロンのマイクロメカニックスによるプロパティの勉強にとって非常に貴重です。
設けてこのマイクロ システムはユニークないくつかの主要な側面で既存システムとは異なります。まず、このシステムは、ニューロンは圧縮とせん断の形態で面外応力を経験します。機械的衝撃に神経突起 coverslip 表面に接続したまま、移動しないでください。その他実験的曲げ、平面でストレッチを主に含んだシステム (または張力) からこれとは異なり、文字列5,6を移動またはマイクロパターニングゲル上に成長した軸索を伸ばす例えば、バンドルされた軸索の偏向のようなチャンネルと伸縮性膜7,8。また、軸索の下肢に誘起することが流体に吹きかけるシステムでこれらの設定のプロセスのようなこれらの試金が表示されます (数時間6,7,810 分) からのより多くの時間がかかると不可逆的です。最後に、ローカル流体息を切らしを用いてできます静脈瘤形成の空間的特徴の検討 (e.g。、樹状突起スパイン、相馬、軸索の初期セグメント、軸索端末)、時空間特徴のほか。このシステムを使用すると、予期しない、ユニークな機能がいくつか軸索静脈瘤形成、特に急激な発症、低速の可逆性と軸索・樹状突起の極性を発見しました。
本稿で述べるシステムは分子の多くの技術との互換性と、細胞生物学。例えば、機械的ストレスが神経細胞の形態と機能に及ぼす影響を研究するそれと共に使用できますミエリン共、タイムラプス蛍光共鳴エネルギー移動 (FRET) の全内部反射蛍光 (TIRF) イメージングカルシウム イメージング、およびパッチ ・ クランプ記録。本稿で我々 はシステムのコア ・ コンポーネントに焦点を当てます。海馬ニューロンの文化、流体パフィング セットアップ、軸索輸送のための高分解能のタイムラプス イメージングおよびカルシウム イメージングを以下順を追って示します。

<table border="0" cellpadding="0" cellspacing="0" style="width:765px;" width="765">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">12 mm coverslips&#160;</td>
			<td style="width:139px;">Warner Instruments&#160;</td>
			<td style="width:119px;">64-0702</td>
			<td style="width:167px;">for 24-well plate&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">25 mm coverslips&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>12-545-102&#160;</td>
			<td>for 6-well plate&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Acetic acid</td>
			<td>Fisher Scientific&#160;</td>
			<td>A38-212</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Poly-D-lysine</td>
			<td>Sigma&#160;</td>
			<td>P6407</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Rat tail collagen&#160;</td>
			<td>Roche&#160;</td>
			<td>11 179 179 001&#160;</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">10X PBS&#160;</td>
			<td>National Diagnostics&#160;</td>
			<td>CL-253</td>
			<td></td>
		</tr>
		<tr height="40">
			<td align="left" height="40" style="height:40px;">Na2SO4</td>
			<td>Fisher Scientific&#160;</td>
			<td>S373-500</td>
			<td></td>
		</tr>
		<tr height="40">
			<td align="left" height="40" style="height:40px;">K2SO4</td>
			<td>Fisher Scientific&#160;</td>
			<td>P304-500</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HEPES&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>BP410-500</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">D-glucose&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>D16-500</td>
			<td></td>
		</tr>
		<tr height="40">
			<td align="left" height="40" style="height:40px;">MgCl2</td>
			<td>Fisher Scientific&#160;</td>
			<td>BP214-500</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">NaOH</td>
			<td>Fisher Scientific&#160;</td>
			<td>SS255-1</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Protease enzyme&#160;</td>
			<td>Sigma&#160;</td>
			<td>P4032</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">FBS</td>
			<td>Gibco&#160;</td>
			<td>26140</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodium pyruvate&#160;</td>
			<td>Gibco&#160;</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-glutamine&#160;</td>
			<td>Gibco&#160;</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Penicillin/Streptomycin 100x (P/S)</td>
			<td>Gibco&#160;</td>
			<td style="width:119px;">15140122</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MEM Earle&#39;s Salts&#160;</td>
			<td>Gibco&#160;</td>
			<td>11090</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">B27 supplement&#160;</td>
			<td>Gibco&#160;</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Neurobasal&#160;</td>
			<td>Gibco&#160;</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Arabinosylcytosine (Ara-C)</td>
			<td>Sigma&#160;</td>
			<td>147-94-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Opti-MEM media&#160;</td>
			<td>Gibco&#160;</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamine 2000</td>
			<td>Invitrogen&#160;</td>
			<td>1854313</td>
			<td>transfection reagent&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate rods&#160;</td>
			<td>World Precision Instruments Inc.&#160;</td>
			<td>PG52151-4</td>
			<td>for puffing pipette&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">rubber tubing&#160;</td>
			<td style="width:139px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">14-169-1A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10cc plastic syringe and plunger</td>
			<td>Becton Dickinson&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">micromanipulator&#160;</td>
			<td>Sutter Instruments&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>S640-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>BP366-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">CaCl2</td>
			<td>Fisher Scientific&#160;</td>
			<td>C70-500</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:341px;">Cell culture dish (35 mm x 10 mm)</td>
			<td style="width:139px;">Corning&#160;</td>
			<td style="width:119px;">3294</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Fluo-4 AM</td>
			<td style="width:139px;">Molecular Probes</td>
			<td style="width:119px;">F14201</td>
			<td style="width:167px;">for calcium imaging&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Mito-YFP construct&#160;</td>
			<td style="width:139px;">Takara Bio Inc.&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">for cell transfection&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">YFP-N1 construct&#160;</td>
			<td style="width:139px;">Takara Bio Inc.&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">for cell transfection&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Model P-1000 Flaming/Brown Micropipette puller&#160;</td>
			<td style="width:139px;">Sutter Instruments&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Eclipse TE2000-U Mcroscope&#160;</td>
			<td style="width:139px;">Nikon</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Plan Fluor ELWD 20x lens&#160;&#160;</td>
			<td style="width:139px;">Nikon</td>
			<td style="width:119px;">062933</td>
			<td style="width:167px;">objective&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Apo TIRF 100x/1.49 oil lens&#160;</td>
			<td style="width:139px;">Nikon</td>
			<td style="width:119px;">MRD01991</td>
			<td style="width:167px;">objective&#160;</td>
		</tr>
	</tbody>
</table>

a microbiomechanical system for studying varicosity formation and recovery in central neuron axons

軸索の下肢は、不均一性の高度で軸索の軸に沿って拡大構造です。彼らが神経変性疾患や外傷、脳は通常の脳だけではなく存在です。ここでは、我々 静脈瘤形成と異種タンパク質組成を支配するメカニズムを理解すること急速に、確実に、かつ可逆的に軸索の下肢を誘導するために新設された微小力学系をについて説明します。このシステムは、ニューロン、他の培養システム主に及ぼす伸張の影響に焦点を当てる別の細胞内コンパートメントに及ぼす圧縮と剪断応力を評価するための新しい手段を表します。重要なは、我々 のシステムの固有の機能のおかげで我々 は最近、加圧流体のアプリケーション迅速かつ可逆的に誘発することが一時的な受容器の潜在的なチャネルの活性化を介して軸索の下肢を示す新規発見をしました。私たちの生体力学的システムは、薬剤灌流、ライブセル イメージング、カルシウム イメージング、およびパッチ ・ クランプ記録との組み合わせで便利に利用できます。したがって、このメソッドは、機械刺激感受性イオン チャネル、軸索輸送規制、軸索細胞骨格のダイナミクス、カルシウム シグナル伝達、および形態変化関連外傷性脳損傷を研究するため採用することができます。

息を切らし前、軸索は通常小さな静脈瘤形成を示します。次は私たちの標準圧力 (190 mmH2O 高さ)、多くのビーズのような下肢を開発する軸索開始と息を切らし。下肢の形成は、次の 10 分回復期 (図 2A B) 事前息切れの状態を返す軸索の領域で示すように、部分的に可逆です。回復の長い期間後 (> 20 分)、いくつかの軸索は完全に回復します。急速に、190 mm ステージ上で注射器の正確な位置決めと同様にたばこを吸うピペットの最適位置決めも下肢は生成可能性があります。最適な条件は、若いニューロン (約 7 DIV) 約 5 秒4の軸索静脈瘤形成になります。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57202/57202fig1.jpg"> 図 1: 回路図と写真の装置に息を切らしします。(A) 写真顕微鏡ネタケース。(B) が保有するマニピュレーター ガラス ピペットを示す装置を吹きかけるとゴム管に接続されています。(C) 位相コントラスト フォーカスが右下でピペットの影を示す一次海馬ニューロンの平面画像。ピペット チップを膨化に焦点を当てた細胞平面の (D) のうち。(E) 距離と下肢を誘導するための計算された圧力の模式図。スケールバー = 50 μ m.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57202/57202fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57202/57202fig2.jpg"> 図 2: 次の息を切らし静脈瘤形成の代表的なイメージです。(A) 7DIV、トランスフェクション GFP マウス海馬ニューロン軸索を事前に表示、後 10 分回復期間に続いて、息を切らしてのイメージング。(B) (A) のニューロンのタイムラプス イメージングの Kymograph。赤い矢印は次の 150 を形成している下肢を示す 190 mmH2O を吹かしの s (30 から始まる s)。スケール バー = 15 μ m.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57202/57202fig2large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td colspan="2">Coverslip コーティング ソリューション</td>
		</tr>
<tr>
<td>780 Μ L</td>
			<td>酢酸 acid17 mM 在庫: 50 μ L 酢酸 50 mL の H2O の</td>
		</tr>
<tr>
<td>200 Μ L</td>
			<td>ポリ-D-リジン (0.5 mg/mL 在庫)</td>
		</tr>
<tr>
<td>20 Μ L</td>
			<td>ラット尾コラーゲン (3 mg/mL 在庫)</td>
		</tr>
<tr>
<td colspan="2">総容積はコーティングする coverslips の数に基づいて決定されます。</td>
		</tr>
</tbody></table>表 1: Coverslip コーティング ソリューションです。Coverslips に対する培養細胞の付着する使用される coverslip コーティング ソリューションを準備するためのレシピを提供します。
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td colspan="2">1 x スライス解離ソリューション (SLD) をフィルター処理、pH 7.4、4 ° C でストアを =</td>
		</tr>
<tr>
<td>1 L</td>
			<td>ddH2O</td>
		</tr>
<tr>
<td>82 mM</td>
			<td>ナ24
</td>
		</tr>
<tr>
<td>30 mM</td>
			<td>K2その4
</td>
		</tr>
<tr>
<td>10 mM</td>
			<td>HEPES (遊離酸)</td>
		</tr>
<tr>
<td>10 mM</td>
			<td>D-グルコース</td>
		</tr>
<tr>
<td>5 mM</td>
			<td>MgCl2
</td>
		</tr>
<tr>
<td>0.00%</td>
			<td>フェノール レッド (オプション)</td>
		</tr>
<tr>
<td colspan="2">メディアをめっき (午後、フィルター、4 ° C で保存)</td>
		</tr>
<tr>
<td>439 mL</td>
			<td>MEM アールの塩</td>
		</tr>
<tr>
<td>50 mL</td>
			<td>政府短期証券</td>
		</tr>
<tr>
<td>11.25 mL</td>
			<td>20% グルコース</td>
		</tr>
<tr>
<td>5 mL</td>
			<td>ピルビン酸ナトリウム (100 mM)</td>
		</tr>
<tr>
<td>62.5 Μ L</td>
			<td>L-グルタミン (200 mM)</td>
		</tr>
<tr>
<td>5 mL</td>
			<td>ペニシリン/ストレプトマイシン (P/S, 100 x)</td>
		</tr>
<tr>
<td colspan="2">保守メディア (MM、フィルター、4 ° C で保存)</td>
		</tr>
<tr>
<td>484 mL</td>
			<td>Neurobasal</td>
		</tr>
<tr>
<td>10 mL</td>
			<td>B27 50 x サプリメント</td>
		</tr>
<tr>
<td>1.25 mL</td>
			<td>L-グルタミン (200 mM)</td>
		</tr>
<tr>
<td>5 mL</td>
			<td>100 P/S ×</td>
		</tr>
<tr>
<td colspan="2">すべてのソリューションは、0.2 mm 孔径フィルターを使用して滅菌されています。</td>
		</tr>
</tbody></table>表 2: 細胞培養媒体レシピ。プライマリ海馬神経細胞の培養に利用されているメディアを準備するためのレシピを提供します。
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td colspan="2">ハンクのバッファー (フィルター、pH 7.4 4 で店を =° C</td>
		</tr>
<tr>
<td>150 mM</td>
			<td>塩化ナトリウム</td>
		</tr>
<tr>
<td>4 mM</td>
			<td>KCl</td>
		</tr>
<tr>
<td>1.2 mM</td>
			<td>MgCl2
</td>
		</tr>
<tr>
<td>1 mM</td>
			<td>CaCl2
</td>
		</tr>
<tr>
<td>10 mg/mL</td>
			<td>D-グルコース</td>
		</tr>
<tr>
<td>20 mM</td>
			<td>HEPES</td>
		</tr>
</tbody></table>テーブル 3: ハンクのバッファー調理法。たばこを吸うプロトコルで、住セルイメージ投射の間に利用するバッファーを準備するためのレシピを提供します。
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td colspan="6">ピペットのパラメーターを引っ張って</td>
		</tr>
<tr>
<td>熱</td>
			<td>プル</td>
			<td>速度</td>
			<td>遅延</td>
			<td>圧力</td>
			<td>ランプ</td>
		</tr>
<tr>
<td>501</td>
			<td>0</td>
			<td>15</td>
			<td>1</td>
			<td>500</td>
			<td>530</td>
		</tr>
</tbody></table>表 4: パラメーターを引っ張ってピペットします。直径約 45 μ m にある開口部を取得するピペットの引き手に設定するパラメーターを示します。

Watch this Scientific Journal Video about A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons at JoVE.com

A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons

このプロトコルでは、ニューロンの下肢の急速かつ可逆的誘導の生理学的に関連する、加圧液体のアプローチについて説明します。

静脈瘤形成と中枢神経の軸索の回復を研究するための Microbiomechanical システム

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with ...

a-microbiomechanical-system-for-studying-varicosity-formation

Nanyang Technological University

Research

JoVE Journal

Neuroscience

6.2K ビュー.  The Ohio State University. このプロトコルでは、ニューロンの下肢の急速かつ可逆的誘導の生理学的に関連する、加圧液体のアプローチについて説明します。

ビデオ: A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution in assessing therapeutic efficacy. Between the two major classifications of administration, local vs. systemic, systemic delivery approaches are often preferred due to ease of administration. However, systemic delivery may result in suboptimal drug concentration being achieved in the CNS, and lead to erroneous conclusions regarding agent efficacy. Local drug delivery methods are more invasive, but may be necessary to achieve therapeutic CNS drug levels. Here, we demonstrate proper technique for three routes of systemic drug delivery: intravenous injection, intraperitoneal injection, and oral gavage. In addition, we show a method for local delivery to the brain: convection-enhanced delivery (CED). The use of fluorescently-labeled compounds is included for in vivo imaging and verification of proper drug administration. The methods are presented using murine models, but can easily be adapted for use in rats.

Thorough preclinical testing of drugs that act in the central nervous system often involves assessing and comparing drug biodistribution in association with specific routes of administration. Here, three commonly used methods of systemic delivery (intravenous, intraperitoneal, and oral) as well as a method for local delivery (convection-enhanced delivery) are demonstrated in mice.

齧歯類モデルで中枢神経系の疾患を治療するための全身および局所薬物送達

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution ...

神経科学

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

視神経切断：中枢神経系における成人ニューロンのアポトーシスのモデル

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

の形態素解析ショウジョウバエ</em遺伝モザイクを使用>幼虫末梢感覚ニューロンの樹状突起と軸索

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

ドーパミン作動性ニューロンの開発における遺伝子機能を研究するためのニワトリ中脳でのOVOエレクトロポレーションに

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

中枢神経系から細胞を変換するためのレンチウイルスベクターの生産

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

A Lightweight, Headphones-based System for Manipulating Auditory Feedback in Songbirds

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and sensorimotor plasticity1. Consistent sensory perturbations result in compensatory changes in motor output, reflecting changes in feedforward motor control that reduce the experienced feedback error. By quantifying how different sensory feedback errors affect human behavior, prior studies have explored how visual signals are used to recalibrate arm movements2,3 and auditory feedback is used to modify speech production4-7. The strength of this approach rests on the ability to mimic naturalistic errors in behavior, allowing the experimenter to observe how experienced errors in production are used to recalibrate motor output.
Songbirds provide an excellent animal model for investigating the neural basis of sensorimotor control and plasticity8,9. The songbird brain provides a well-defined circuit in which the areas necessary for song learning are spatially separated from those required for song production, and neural recording and lesion studies have made significant advances in understanding how different brain areas contribute to vocal behavior9-12. However, the lack of a naturalistic error-correction paradigm - in which a known acoustic parameter is perturbed by the experimenter and then corrected by the songbird - has made it difficult to understand the computations underlying vocal learning or how different elements of the neural circuit contribute to the correction of vocal errors13.
The technique described here gives the experimenter precise control over auditory feedback errors in singing birds, allowing the introduction of arbitrary sensory errors that can be used to drive vocal learning. Online sound-processing equipment is used to introduce a known perturbation to the acoustics of song, and a miniaturized headphones apparatus is used to replace a songbird's natural auditory feedback with the perturbed signal in real time. We have used this paradigm to perturb the fundamental frequency (pitch) of auditory feedback in adult songbirds, providing the first demonstration that adult birds maintain vocal performance using error correction14. The present protocol can be used to implement a wide range of sensory feedback perturbations (including but not limited to pitch shifts) to investigate the computational and neurophysiological basis of vocal learning.

We describe the design and assembly of miniaturized headphones suitable for replacing a songbird&#x2019;s natural auditory feedback with a manipulated acoustic signal. Online sound processing hardware is used to manipulate song output, introduce real-time errors in auditory feedback via the headphones, and drive vocal motor learning.

鳴禽類には聴覚フィードバックを操作するための軽量、ヘッドフォンベースのシステム

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

齧歯中枢神経系への薬物の直接脳室内デリバリー

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

ラット中枢神経系および末梢リンパ節の組織切片での免疫組織化学的解析

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

小脳軸索のレーザーNanosurgery<em&gt;インビボ</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

齧歯類の中枢神経系の生体光遺伝学的刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...