This work is supported by an R21 grant from the National Institutes of Health and by a grant from Hearing Research, Incorporated.

注：すべての手順や動物の取り扱いは、NIHの倫理ガイドラインを遵守し、カリフォルニア大学の制度的動物実験委員会、サンフランシスコのプロトコル要件を承認した。 1.手術のための動物の準備<ol><li>きれいな、専用スペースでの外科的処置を行ってください。手術前にガラスビーズ滅菌器で滅菌する、すべての手術器具をオートクレーブ。 注：このプロトコルでは、生後10〜12（P10-12）FVBマウスを使用しています。異なる年齢のマウスの株は、特定のプロジェクトのニーズを満たすために使用することができる。水疱の骨が難しくなりますので、P40より古いマウスは、挑戦することができます。 </li><li>塩酸ケタミン（100mg / kg）の混合物の腹腔内注射、塩酸キシラジン（10mg / kgの）、およびアセプロマジン（2mg / kgの）でマウスを麻酔。動物は、もはやそのようなつま先のピンチのような痛みを伴う刺激に応答した後にのみ、外科準備を開始します。もしnecessaryは、オリジナルの麻酔薬平面を復元するために麻酔薬カクテルのブースター用量（五分の一のオリジナル用量）を管理する。 注：この年齢で観測された死亡率のほとんどは麻酔薬の過剰投与によって引き起こされるので、この段階では注意してください。 </li><li>動物のまばたき反射を抑制し、麻酔中湿った目を保つために、保護眼軟膏で動物の目をカバーしています。 </li><li>マウスが完全に目を覚ましになるまで、麻酔後の低体温を防ぐため、作業中は加熱パッド上に拡張され、首にマウスを置きます。 </li><li>バリカンで左ポスト耳介地域を剃ると外科的操作の前に70％エタノール及びポビドンヨードで消毒する。 </li></ol> 2.手術手順とベクター注入<ol><li>鼓室胞を露出させるためにポスト耳アプローチを使用してください。ポスト耳介筋肉を露出させるために、小さなハサミで皮下組織を切開。目に脂肪組織を後退後切開部の電子後方側は、側頭骨を露出させるために切開に垂直な左右の筋肉を分離する。この切開は十分な可視化と快適に作業するのに十分な長さであることを確認してください。 </li><li> 25 G針で鼓膜胞を穿孔し、蝸牛の基底回転へのアクセスを許可するように戻って骨を剥離することにより、鉗子で、必要に応じて穴を展開し、[あぶみ骨動脈と丸い窓膜を可視化するために十分に広げる（RWM） 。 </li><li>ホウケイ酸キャピラリーピペットで中央に優しくRWMをパンク。この時点ではRWMを通して流体流出を観察する。これは正常です。流出が安定するまで待ちます（蝸牛から流出した流体は、滅菌濾紙で乾燥させる）。 注：保持とRWMの穿孔ができるだけ小さくなるように、ガラス針を進める際には注意してください。使用される顕微鏡に応じて、ピペットがピペットを用いてマイクロマニピュレーターを用いて、または手で保持するホルダー。 </li><li>パッチクランプピペットが用意されているのと同じ方法プラーピペットを用いて注入するためのピペットを準備します。内と外のウイルスをピペットが簡単に行われますように（直径約15μm​​）先端径が大きいことを確認してください。 </li><li>インジェクションピペットにAAV1-VGLUT3の1から2μL、AAV1-GFPまたはAAV2-GFPを策定する。流出（5〜10分）、安定化、microinject同じ穴を通して鼓室階にこの固定ボリュームは、以前RWMに行われた後。 </li><li>筋肉の小さなプラグをピペットを引き出した後、すぐにRWのニッチを密封し、ラウンド窓からの漏れを回避するために、筋肉に置かれた組織接着剤の小滴で固定します。 注：非常によく、針が蝸牛-このステップからの外リンパの漏出を防止するために、筋肉の小さなプラグを除去した後、この穴をシール聴力を維持することが重要です。完全にRWMをシールに失敗すると、時間をかけて難聴になります。 </li><li>注射後、カバー脂肪組織と聴覚の水疱の穴、彼らの通常の位置にポスト耳介筋や脂肪組織を返し、6-0または小さい吸収性クロム縫合糸で層に傷を縫合。 </li><li>ポビドンヨードと傷口を消毒する。 </li></ol> 3.術後ケア<ol><li>暖かいケージにマウスを置き、彼らが完全に戻っ母親とそれらを移動回収されるまで、それらを無人のままにしないでください。それは、母親と一緒に戻って子犬を置く前に、ケージから男性を削除することを推奨します。 </li><li>炎症および疼痛を管理するために、3日間その後術後鎮痛と、24時間の皮下カルプロフェン（2ミリグラム/ kg）を投与する。苦痛、異常な体重減少、痛み、または感染の徴候について毎日動物を監視します。一般的に、すべてのマウスが正常に機能するべきである手術後3 日目による。苦痛や病気の徴候は3日後にマウスに表示された場合は、Tを安楽死を検討施設のガイドラインに従って、彼はマウス。 </li></ol>聴覚脳幹反応（ABR）録画を用いてウイルス送達後の蝸牛機能4.評価 NOTE：聴性脳幹反応（ABR）は、聴覚が潜在脳における進行中の電気的活動から抽出され、頭皮の下に配置された電極を介して記録された誘発である。動物は、音で刺激される。結果の記録は、聴覚刺激の発症後最初の10ミリ秒で聴覚路に連続する点の電気的活動を反映して5波で構成されています。 <ol><li>このプロトコルのセクション1.2で説明したようにアセプロマジンなし除き、塩酸ケタミンとキシラジン塩酸塩の混合物の腹腔内注射によりマウスを麻酔。 </li><li>マウスが完全に目を覚ましになるまで、麻酔後の低体温を防ぐために、聴力検査を通じて加熱パッド上にマウスを置きます。 注：使用この実験では、次の音の刺激：（5ミリ秒の期間; 31 Hzのプレゼンテーション率）をクリックします。 </li><li>頂点で、左耳（参照）の耳介の下、および対耳（グランド）の下に皮下針電極を配置し、以前に防音室27,28で説明したようにのABRの記録を開始。ダウン最大振幅から5デシベルの音圧レベル毎のレコードABR波形。 </li><li>早けれウイルス送達後4日間などの術後のABR閾値を決定 注：閾値は波の応答のピークI-Vは、明らかに繰り返し目視上に存在していた最低の刺激レベルとして定義される。 </li><li>私は蝸牛神経からのアクティビティを分析するために波を測定します。検出可能なABR波形が得られる最低刺激レベルを閾値として定義される。 </li></ol>免疫蛍光を用いて5蝸牛導入遺伝子タンパク質発現<ol><li> intrapの過剰摂取によりマウスを麻酔塩酸ケタミンとキシラジン塩酸塩の混合物のeritoneal注入アセプロマジンなしを除いて、このプロトコルのセクション1.2で説明したように。 </li><li>動物は、もはやそのようなつま先のピンチのような痛みを伴う刺激に応答した後にのみ頭を首を切る。 </li><li> 26を説明するように全体のマウント免疫蛍光のためにアウトとプロセス蝸牛を解剖。抗VGLUT3および抗ミオシン7aの抗体を用いて蝸牛全体マウント免疫蛍光法の詳細なプロトコルはアキールら 、2013 29に記載されている。 </li><li> 250希釈：GFP標識のために、1でウサギ抗GFP抗体を用いて4℃で一晩蝸牛全体のマウントをインキュベートする。 </li><li> PBSで10分間、二回蝸牛をリンスした後、Cy2とコンジュゲートしたヤギ抗ウサギIgGで2時間インキュベート1に希釈した：PBSで4000。 </li><li> 10分間、2回PBSで蝸牛をすすぎ、15分間DAPIでそれらを培養する。 </li><li>スライドガラス上に蝸牛をマウントし、ウントを観察共焦点免疫蛍光顕微鏡えー。 </li></ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus-mediated+gene+delivery+into+the+scala+media+of+the+normal+and+deafened+adult+mouse+ear.">Adeno-associated virus-mediated gene delivery into the scala media of the normal and deafened adult mouse ear.</a> Gene Ther. 18 (6), 569-578 (2011).</li><li>Yamasoba, T., Yagi, M., Roessler, B. J., Miller, J. M., Raphael, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inner+Ear+Transgene+Expressionafter+Adenoviral+Vector+Inoculation+in+the+Endolymphatic+Sac+Hum.">Inner Ear Transgene Expressionafter Adenoviral Vector Inoculation in the Endolymphatic Sac Hum.</a> Gene Ther. 10 (5), 769-774 (1999).</li><li>Praetorius, M., Baker, K., Weich, C. M., Plinkert, P. K., Staecker, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hearing+preservation+after+inner+ear+gene+therapy:+the+effect+of+vector+and+surgical+approach.">Hearing preservation after inner ear gene therapy: the effect of vector and surgical approach.</a> ORL J. Otorhinolaryngol. Relat. Spec. 65 (4), 211-214 (2003).</li><li>Carvalho, G. J., Lalwani, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cochleaostomy+and+intracochlear+infusion+on+auditory+brain+stem+response+threshold+in+the+guinea+pig.">The effect of cochleaostomy and intracochlear infusion on auditory brain stem response threshold in the guinea pig.</a> Am. J. Otol. 20 (1), 87-90 (1999).</li><li>Kawamoto, K., Oh, S. H., Kanzaki, S., Brown, N., Raphael, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Functional+and+Structural+Outcome+of+Inner+Ear+Gene+Transfer+via+the+Vestibular+and+Cochlear+Fluids+in+Mice.">The Functional and Structural Outcome of Inner Ear Gene Transfer via the Vestibular and Cochlear Fluids in Mice.</a> Mol. Ther. 4 (6), 575-585 (2001).</li><li>Lalwani, A. K., Han, J. J., Walsh, B. J., Zolotukhin, S., Muzyczka, N., Mhatre, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+fluorescent+protein+as+a+reporter+for+gene+transfer+studies+in+the+cochlea.">Green fluorescent protein as a reporter for gene transfer studies in the cochlea.</a> Hear Res. 114 (1-2), 139-147 (1997).</li><li>Lalwani, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+in+vivo+cochlear+transgene+expression+mediated+by+recombinant+adeno-associated+virus.">Long-term in vivo cochlear transgene expression mediated by recombinant adeno-associated virus.</a> Gene Ther. 5 (2), 277-281 (1998).</li><li>Raphael, Y., Frisancho, J. C., Roessler, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral-mediated+gene+transfer+into+guinea+pig+cochlear+cells+in+vivo.">Adenoviral-mediated gene transfer into guinea pig cochlear cells in vivo.</a> Neurosci. Lett. 207 (2), 137-141 (1996).</li><li>Weiss, M. A., Frisancho, J. C., Roessler, B. J., Raphael, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+mediated+gene+transfer+in+the+cochlea.">Viral mediated gene transfer in the cochlea.</a> Int. J. Dev. Neurosci. 15 (4=5), 577-583 (1997).</li><li>Pettis, R. M., Han, J. J., Mhatre, A. N., Lalwani, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracochlear+infusion+of+recombinant+adeno+associated+virus:+Analysis+of+its+dissemination+to+near+and+distant+tissues.">Intracochlear infusion of recombinant adeno associated virus: Analysis of its dissemination to near and distant tissues.</a> Assoc. Res. Otolaryngol. Abstr. 21, 673 (1998).</li><li>Konish, i. M., Kawamoto, K., Izumikawa, M., Kuriyama, H., Yamashita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+into+guinea+pig+cochlea+using+adeno-associated+virus+vectors.">Gene transfer into guinea pig cochlea using adeno-associated virus vectors.</a> J. Gene Med. 10 (6), 610-618 (2008).</li><li>Kaplitt, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+gene+expression+and+phenotypic+correction+using+adeno-associated+virus+vectors+in+the+mammalian+brain.">Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain.</a> Nature Genetics. 8 (2), 148-154 (1994).</li></ol>

No conflicts of interest declared.

本研究では、遺伝的欠陥によって損なわれている通常の聴覚機能を回復または救助を目的とし、詳細に蝸牛遺伝子治療に使用することができる技術が記載されている。それは典型的に非外傷性であるように、このアプローチは、人工遺伝子導入または他の潜在的な分子の治療法30に安全である。蝸牛療法のための他のアプローチは、腹側のアプローチ24、マウスおよびモルモ?...

遺伝子治療は、長い遺伝性難聴のための潜在的治療法として提案されているが、この分野での成功は、1とらえどころのないままである。現在までに、ウイルス媒介性の方法論は、比較的アクセス蝸牛内の特定の細胞型を標的とする理論的能力により優勢いる。アデノウイルス（AV）、アデノ随伴ウイルス（AAV）の両方が蝸牛遺伝子送達のために使用されている。のAAVは、多くの理由のために蝸牛で有利である。彼らは、複製欠損ウイルスであると効率的にニューロン、難聴の原因の数の重要な標的を含む、異なる種類の細胞に遺伝子導入する分子を転送することができます。細胞へのAAVのエントリは、特定の受容体2によって媒介される。このように、特定の血清型の選択は、形質導入される細胞型に適合しなければならない。のAAVは、効率的に有毛細胞3をトランスフェクトし、トラヒックの安定した長期発現をもたらす、宿主ゲノムに組み込むことができnsgenicタンパク質や細胞4における表現型の変化。毛髪細胞再生短期用途には必ずしも有利ではないが、長期発現は、遺伝的欠陥を安定救助のために非常に重要である。 AAVは、任意のヒト疾患または感染に関連しない耳毒性5,6,7を示さないれていないので、彼らは損失8聴力の遺伝型の遺伝子治療に使用するための理想的な候補である。 ウイルスベクターを用いて哺乳動物の内耳への外因性遺伝物質の移動は、過去10年間研究されており、損失9聴力の遺伝的および後天両方の形態を治療するための有望な技術として浮上している。蝸牛は、潜在的にいくつかの理由のために、遺伝子治療のための理想的な標的である：1）その小さな容積は、必要なウイルスの限定された量を必要とする。他の器官系の制限副作用2）の相対的な単離; 3）その流体充填チャンバは、ウイルスを容易にする迷路10、11,12,13,14、15を通じて配信。 先天性難聴のマウスモデルは、系統的、複製可能な方法で、内耳の開発を監視するための研究の多くの方法の使用を可能にする。マウス蝸牛の小さいサイズは、いくつかの外科的困難を提示しませんが、マウスが他の種16に比べていくつかの実験的な利点で、遺伝性難聴の研究に極めて重要なモデルとなる。マウスモデルは、遺伝的連鎖解析、詳細な形態学的観察の収集、および病原性のシナリオをシミュレートを通して特性の範囲の評価を可能にする。のような、それらがウイルス媒介遺伝子治療のための良好な候補である。技術の進歩と組み合わせたマウスでの広範な遺伝学的研究は研究室17,18、19、20,21全体で再現可能な方法で、遺伝子改変されたマウスを作製することが可能になりました。 Furthermor両方を取得し、この動物モデル22、23,24で厳密なテストを可能にする、マウスにおける難聴表現型を継承するための電子、多数のモデルが存在する。従って、マウスモデルにおいてウイルス媒介遺伝子治療を用いて聴覚を補正するヒト疾患の治療法の探索において適切な最初のステップである。 我々は以前胞性グルタミン酸輸送体3（VGLUT3）を欠くトランスジェニックマウスによるIHCリボンシナプス25におけるグルタミン酸放出の欠如のために聴覚障害者が生まれていることが示されている。この変異は感覚毛細胞の一次変性につながらないので、これらの変異マウスは、潜在的に先天性難聴のための蝸牛遺伝子治療をテストするに優れたモデルである。 現在までに、蝸牛遺伝子治療のためのウイルス送達技術の数が正円窓膜拡散、正円窓膜注射、および蝸牛を介した送達を含む、記載されている。強力ありますIAL利点及びこれらのアプローチ9のそれぞれの短所。 ここでは、正円窓膜（RWM）を介してVGLUT3 KOマウス内耳へのウイルス媒介遺伝子送達のための外科的方法を報告している。耳介RWM注入法は、優れた聴覚保護と低侵襲性であり、かつ比較的高速である。我々は以前に公開しているように、このマウスモデルで聴力回復するための努力で、VGLUT3遺伝子（AAV1-VGLUT3）を運ぶAAV1ベクターは、生後12日目にこれらの聴覚障害のマウスの蝸牛に導入した（P！@）、その結果26聴力の回復。導入遺伝子のタンパク質発現を免疫蛍光（IF）を使用して確認しながらVGLUT3 KOマウスにおいて聴力は、聴性脳幹反応（ABR）によって確認した。この方法論は、このように、ウイルス媒介遺伝子治療は、そうでなければ難聴になるだろう遺伝的欠陥を修正することができることを実証している。

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			<td height="40" style="height:40px;width:355px;">Borosilicate capillary pipette</td>
			<td style="width:177px;">World precision instruments, inc.</td>
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			<td height="24" style="height:24px;width:355px;">Tissue glue (Vetcode)</td>
			<td style="width:177px;">Butler Schein</td>
			<td align="right" style="width:161px;">31477</td>
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			<td height="24" style="height:24px;width:355px;">Rabbit Anti-GFP antibody</td>
			<td style="width:177px;">Invitrogen</td>
			<td style="width:161px;">A11122</td>
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			<td height="20" style="height:20px;">Dissecting microscope&#160;&#160;&#160;&#160;&#160;</td>
			<td>Leica</td>
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			<td height="20" style="height:20px;">Flaming/ Brown Micropipette&#160;&#160;&#160;&#160;&#160;</td>
			<td>Sutter Instrument Co</td>
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			<td height="20" style="height:20px;">Puller Model P-97&#160;&#160;</td>
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			<td height="20" style="height:20px;">TDT BioSig III System&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>&#160;Tucker-Davis Technologies</td>
			<td></td>
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surgical method for virally mediated gene delivery to the mouse inner ear through the round window membrane

感音難聴からの機能的回復を達成するために使用される遺伝子治療は、難聴に寄与する根本的な分子的および遺伝的メカニズムのより良い理解を与えることを約束する。内耳へのベクターの導入は、既存の構造への損傷を最小限に抑えながら、広く蝸牛全体にエージェントを配布する方法で行われなければならない。この原稿は、分子、薬理学的、およびマウス生後10日目と丸い窓膜（RWM）を介して、古いのウイルス送達を用いてマウス蝸牛治療に用いることができる後の耳介の外科的アプローチについて説明します。血液の損失を最小化し、動物の死亡を回避しながら、この外科的アプローチは、鼓室階への迅速かつ直接の受け渡しが可能になります。完全に聴力を維持しながら、この手法は、無視できるか、インナーと中耳の本質的な構造への損傷だけでなく、首の筋肉を伴う。この手術法、水疱glutamの有効性を実証するために、食べトランスポーター3ノックアウト（VGLUT3 KO）マウスは、アデノ随伴ウイルス（AAV-1）を使用して内耳にVGLUT3の送達後に聴力回復先天性難聴のマウスモデルの例として使用される。

蝸牛分子治療のための技術的特徴とポスト耳アプローチの有用性を確認するには、AAV1-VGLUT3、AAV1-GFPおよびAAV2-GFPはRWM経由P10-12マウス内耳に送達された。このアプローチは、内有毛細胞内で正常に導入遺伝子発現（IHC）（VGLUT3 図1及びGFP 図2及びGFP 図3A）、外有毛細胞（OHC）（GFP 図2）および支持細胞（GFP、図2および図3A?...

Watch this Scientific Journal Video about Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane at JoVE.com

Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane.

円窓膜を介してマウスインナーイヤーにウイルスにより媒介遺伝子送達のための手術方法

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and ...

surgical-method-for-virally-mediated-gene-delivery-to-mouse-inner-ear

Research

JoVE Journal

Neuroscience

15.2K ビュー.  University of California, San Francisco. The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane. 

ビデオ: Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

クローズ頭蓋窓を使用してマウス脳の微小循環の生体顕微鏡

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

神経科学

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

脊髄電気生理学II：細胞外吸引電極の作製

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

フローサイトメトリーを用いてマウスの内耳の組織からのLacZの発現細胞を単離する

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

開発マウス内耳への遺伝子導入による<emインビボで&gt;</em&gt;エレクトロポレーション

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

マウス脳の長期イメージングのためのポリッシュと強化間引き頭蓋骨ウィンドウ

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

Extinction Training During the Reconsolidation Window Prevents Recovery of Fear

Fear is maladaptive when it persists long after circumstances have become safe. It is therefore crucial to develop an approach that persistently prevents the return of fear. Pavlovian fear-conditioning paradigms are commonly employed to create a controlled, novel fear association in the laboratory. After pairing an innocuous stimulus (conditioned stimulus, CS) with an aversive outcome (unconditioned stimulus, US) we can elicit a fear response (conditioned response, or CR) by presenting just the stimulus alone1,2 . Once fear is acquired, it can be diminished using extinction training, whereby the conditioned stimulus is repeatedly presented without the aversive outcome until fear is no longer expressed3. This inhibitory learning creates a new, safe representation for the CS, which competes for expression with the original fear memory4. Although extinction is effective at inhibiting fear, it is not permanent. Fear can spontaneously recover with the passage of time. Exposure to stress or returning to the context of initial learning can also cause fear to resurface3,4.
Our protocol addresses the transient nature of extinction by targeting the reconsolidation window to modify emotional memory in a more permanent manner. Ample evidence suggests that reactivating a consolidated memory returns it to a labile state, during which the memory is again susceptible to interference5-9. This window of opportunity appears to open shortly after reactivation and close approximately 6hrs later5,11,16, although this may vary depending on the strength and age of the memory15. By allowing new information to incorporate into the original memory trace, this memory may be updated as it reconsolidates10,11. Studies involving non-human animals have successfully blocked the expression of fear memory by introducing pharmacological manipulations within the reconsolidation window, however, most agents used are either toxic to humans or show equivocal effects when used in human studies12-14. Our protocol addresses these challenges by offering an effective, yet non-invasive, behavioral manipulation that is safe for humans.
By prompting fear memory retrieval prior to extinction, we essentially trigger the reconsolidation process, allowing new safety information (i.e., extinction) to be incorporated while the fear memory is still susceptible to interference. A recent study employing this behavioral manipulation in rats has successfully blocked fear memory using these temporal parameters11. Additional studies in humans have demonstrated that introducing new information after the retrieval of previously consolidated motor16, episodic17, or declarative18 memories leads to interference with the original memory trace14. We outline below a novel protocol used to block fear recovery in humans.

Conditioned fear can be diminished through an inhibitory process called extinction, but can resurface under conditions such as the passage of time or exposure to stress. Our protocol presents a novel way of preventing fear recovery by introducing extinction during the reconsolidation window (the re-storage phase of a reactivated memory).

再固定ウィンドウの間に絶滅の訓練は恐怖の回復を防ぎ

Fear is maladaptive when it persists long after circumstances have become safe. It is therefore crucial to develop an approach that persistently prevents ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

内耳有毛細胞におけるパッチクランプ記録は、単離されたゼブラフィッシュから

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

イメージング初代培養マウス神経細胞における形質膜に受容体の挿入をpHluorinタグ付き

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

軸索分岐と神経形態形成中に形質膜配信の役割を定義するために組み合わせる方法論を活用

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

エレクトロポレーションによる齧歯類坐骨神経内のシュワン細胞へのインビボ遺伝子導入で

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...