資金調達：この作品は、財団Bertarelli補助金（DJL）、MED-ELの助成金（DJL）、国立衛生研究所補助金のDC01089（MCB）によってサポートされていました。

注：すべての実験手順は、実験動物の愛護管理と使用に関する公衆衛生サービスポリシーを含む全国の動物保護ガイドラインに従うマサチューセッツアイ、耳診療所とハーバード大学医学部の動物実験委員会、に従って行われる、 ILARガイド、および動物福祉法。左DCNの詳細暴露下に表示実験手順。生存手術を行いながら、無菌の楽器を使用してください。 1.プライマリ開頭と背側蝸牛神経核露出<ol><li>麻酔<ol><li>キシラジンの腹腔内投与による20mg / kgのケタミンを100mg / kgの18〜24グラム体重、高齢者8-12週間、マウスを麻酔。心拍数、呼吸数、ならびに足指ピンチ引っ込め反射を監視することによって、適切な麻酔を確認する。麻酔下ながら乾燥を防ぐために、目に獣医軟膏を置きます。一度適切に麻酔下で、髪の上に重なるTを剃る彼は、手術部位への遮るもののないアクセスを提供するために頭皮。 </li></ol></li><li>外科ポジショニング<ol><li>鼻クランプにより所定位置に保持小動物定位ホルダー、に安全にマウスを置きます。 </li><li>鼻クランプが十分な呼吸を可能にするのに十分な緩いが、完全にマウスの頭部を固定するのに十分タイトであることを確認してください。マウスヘッドが緩んでいる場合、動物は処置の開頭部分の間にヘッドホルダから脱落する可能性がある。 </li><li>心拍数のモニタリングを可能にし、標準的な方法、で聴性脳幹インプラント電極を配置します。呼吸数は、視覚化することによって監視されるべきである。通常の心拍数や呼吸数は、マウスの年齢に応じて変化するであろうことに注意してください。 </li><li>恒温加熱ブランケット上に配置介して温度計を配置し、euthermiaが確保される。 </li></ol></li><li>皮膚切開と頭蓋骨のinterparietalと後頭部の骨の露出<ol><li> microscの下でOPE、直接耳介の間で、正中線から始まる、と後頭部の尾部に至る皮膚を介して垂直切開する。正中皮膚切開の後、横方向に皮膚を移動させる。頭蓋骨の左側の後部側面をカ​​バーする筋肉を視覚化する。 </li><li>メスまたはアイリスはさみと、それぞれの骨に筋肉の添付ファイルの離断によって筋肉左頭頂、interparietalの上にある、と頭蓋骨の後頭部の骨を削除します。カット筋縁に沿って、出血の小さな度合いを観察します。 10〜15秒間綿棒で穏やかな圧力によって、これを最小化します。 </li></ol></li><li>開頭その上の蝸牛神経核<ol><li>矢状及びラムダ縫合ライン（ 図1、左パネル）を含む関連縫合線を、特定します。 </li><li>骨鉗子を使用して、interparietal骨の上に開頭手術をし、正中線のラムダ縫合線へ〜2mmの尾を残した。この領域は、DCNの上に重なる。 </li><li> FollowinGの開頭、小脳の上に重なる硬膜の薄い層を観察する。手術用メスの刃を使用して、硬膜を削除します。 （ 図1、中央のパネル）。 注：硬膜の除去は、出血の小さな度合いを引き起こす可能性があります。硬膜を除去するために外科用メスの刃を使用したこのプロセスは、脳幹の表面とDCNの曖昧な同定にプールう小脳吸引の間の血液損失の量を低減することを目的とする。マウスの全血液量を15％以上&gt;〜1.5ミリリットルを超えてはならないです。出血がこの量を超えた場合、マウスは、流体補充を用いて提供されるべきである。 </li><li>綿棒アプリケーターで小脳を重ね凝固した血液を削除します。必要に応じて、優しく血液を血液を離れてクリアする小脳の上に歯科ポイントを使用して生理食塩水を滴下する。 </li></ol></li><li>小脳吸引<ol><li> DCNのVIになるまで5フランス語吸引を使用して、DCNの上に重なる左小脳の横ほとんどの部分を吸引UAL（ 図1、右パネル）。左小脳の約1/4 1/3を削除します。 DCNに隣接した主なランドマークは、優れた半規管の膨大部である。 注：小脳の吸引がプロトコルの重要なステップである。それが出血原因になります。このような吸引の複数のパスを持つ単一の試行で発生していない場合は、小脳の監督吸引が最も成功している。吸引を補助するために、小脳の表面にわずかに遠位されるであろう、CNの予想される深さに顕微鏡の焦点面を設定する。 CNに焦点面を設定すると、可視化を改善し、鮮明な画像が保証されます。 </li><li> DCNの以下の吸引、さらに出血、脳脊髄液（CSF）、ビルドアップ、及び小脳変位が期待されている。血液凝固を防ぐためにすぐに開頭術に滅菌生理食塩水0.5ccのを植え付ける。歯科ポイントと吸引優しい軽くたたくの組み合わせは、その後かもしれDCNの直接可視化するためのパスを離れてクリアするために使用。直接DCNの表面に接触しないでください。 </li></ol></li></ol>ウイルス媒介遺伝子導入および外科回復2.圧力マイクロインジェクション<ol><li> DCNは、その上の血液およびCSFの自由であり、はっきりと見えるした後、2分間にわたって10μlのハミルトンシリンジを使用して、DCNに圧力マイクロインジェクションを作る。 </li><li>先端がDCNの表面の下に見えなくなるまで、マイクロマニピュレーターで針を導入しない。 </li><li>最適なパフォーマンスを得るには、33または34ゲージの針を使用する薄いである、鈍的外傷を最小限にし、DCN内の注入量をローカライズするために、そのような45°と、浅いベベルと（34ゲージ針で気密注射器を使用します）と、浅い脳幹構造体（&lt;厚さ300μm）。 注：他のマイクロインジェクション器具ベースを利用することができる。理想的には、注射器は若干ベンを可能にするために柔軟であるべきであるD、直接DCNをターゲットにする必要がある場合。マウスは呼吸による手続きを通じて少し移動するとさらに、楽器、運動の少量に耐えるために十分に堅牢でなければなりません。 </li></ol> 3.外科回復<ol><li>すぐに次の注射は、皮膚を再近似するとマウスが標準の回復手順ごとに回復することができます。残りの小脳および瘢痕組織が小脳の吸引に起因する空洞内に記入します。十分な意識が胸骨横臥位を維持するために回復するまで、常に動物を監視します。心拍数、呼吸数、ならびに供給する能力をモニターする。 注：完全に回復するまでは、動物の手術を受けた他の動物とのケージに戻している。 </li><li>マウスは、円術後（症例の&lt;5％）の中を歩くが開始された場合、それはおそらく困難な時期送りを持っていると同じように、通常は2〜4時間皮膚切開の閉鎖後、すぐに、マウスを生け贄に捧げる。 T彼の副作用に起因小脳の吸引に起因する可能性が高い。動物は、術後の痛みのために監視し、適切な麻薬は、制度の基準に基づいて、動物の快適性を確保するために与えられるべきである。感染の証拠があれば抗生物質は、必要であり得る。 </li></ol> 4.セカンダリ開頭と蝸牛神経核露出<ol><li>癒しとウイルス媒介遺伝子導入のインキュベーションの2-4週間後、再麻酔前に注射したマウスを繰り返しステップ1.1〜1.5の最適なインキュベーション時間は、導入遺伝子の種類によって異なります。 </li><li>ピンセット、外科用メス、および骨鉗子の組み合わせによって開頭サイト上の瘢痕組織を削除します。 </li><li>開頭術を可視化した後、残りの小脳及びDCNを覆う瘢痕組織の組み合わせを特定する。その上小脳/（前の小脳吸引と同様）瘢痕組織を吸引する5フランスの吸引を使用してください。 </li><li>吸引後、さらに出血、CSF bを期待するuildアップ、および小脳の残りのビット。すぐに血液凝固を防ぐために開頭術に生理食塩水を導入。 DCNの直接可視化するためのパスをクリアするために穏やかな歯科ポイント軽くたたくと吸引を組み合わせて使用​​してください。 </li><li> DCNの表面を観察します。 DCNがアクセスできるようになりましたように、光遺伝学ベースの生理学実験を行う。光遺伝学ベースの実験を駆動するために、光ファイバ（ 図2）と、例えば、光刺激を導入する。 </li></ol> 5.組織学<ol><li>実験の終了後、ケタミンの過剰摂取でマウスを安楽死させる。 4％パラホルムアルデヒド、続いて生理食塩水でマウスを灌流する。 </li><li> 2時間頭蓋骨とポストフィックスから脳幹を抽出します。 24〜48時間、30％スクロース中で脳幹をCryoprotect。第60ミクロン切片を用いて、標準的なクライオスタットを用いて、脳幹。 </li></ol>

<ol>
	<li>Lalwani, A., Mhatre, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlear+gene+therapy.">Cochlear gene therapy.</a> Ear Hear. 24 (4), 342-348 (2003).</li><li>Lalwani, A., Jero, J., Mhatre, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+issues+in+cochlear+gene+transfer.">Current issues in cochlear gene transfer.</a> Audiol Neurootol. 7 (3), 146-151 (2002).</li><li>Jero, J., 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=Cochlear+gene+delivery+through+an+intact+round+window+membrane+in+mouse.">Cochlear gene delivery through an intact round window membrane in mouse.</a> Hum Gene Ther. 12 (5), 539-548 (2001).</li><li>Koh, S., Pettis, R., Mhatre, A., Lalwani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlear+microinjection+and+its+effects+upon+auditory+function+in+the+guinea+pig.">Cochlear microinjection and its effects upon auditory function in the guinea pig.</a> Eur Arch Otorhinolaryngol. 257 (9), 469-472 (2000).</li><li>Lalwani, A., Walsh, B., Reilly, P., Muzyczka, N., Mhatre, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+in+vivo+gene+therapy+for+hearing+disorders:+introduction+of+adeno-associated+virus+into+the+cochlea+of+the+guinea+pig.">Development of in vivo gene therapy for hearing disorders: introduction of adeno-associated virus into the cochlea of the guinea pig.</a> Gene Ther. 3 (7), 588-592 (1996).</li><li>Wareing, M., Lalwani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlear+gene+therapy:+current+perspectives.">Cochlear gene therapy: current perspectives.</a> Int J Pediatr Otorhinolaryngol. 5 (49), 27-30 (1999).</li><li>Han, J., 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=Transgene+expression+in+the+guinea+pig+cochlea+mediated+by+a+lentivirus-derived+gene+transfer+vector.">Transgene expression in the guinea pig cochlea mediated by a lentivirus-derived gene transfer vector.</a> Hum Gene Ther. 10 (11), 1867-1873 (1999).</li><li>Akil, O., 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=Restoration+of+hearing+in+the+VGLUT3+knockout+mouse+using+virally+mediated+gene+therapy.">Restoration of hearing in the VGLUT3 knockout mouse using virally mediated gene therapy.</a> Neuron. 75 (2), 283-293 (2012).</li><li>Hernandez, V. H., 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=Optogenetic+stimulation+of+the+auditory+pathway.">Optogenetic stimulation of the auditory pathway.</a> J Clin Invest. 124 (3), 1114-1129 (2014).</li><li>Adamantidis, 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=Optogenetic+interrogation+of+dopaminergic+modulation+of+the+multiple+phases+of+reward-seeking+behavior.">Optogenetic interrogation of dopaminergic modulation of the multiple phases of reward-seeking behavior.</a> J Neurosci. 30 (31), 10829-10835 (2011).</li><li>Kim, 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=Optogenetic+mimicry+of+the+transient+activation+of+dopamine+neurons+by+natural+reward+is+sufficient+for+operant+reinforcement.">Optogenetic mimicry of the transient activation of dopamine neurons by natural reward is sufficient for operant reinforcement.</a> PloS One. 7 (4), e33612 (2012).</li><li>Britt, J., Bonci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+interrogations+of+the+neural+circuits+underlying+addiction.">Optogenetic interrogations of the neural circuits underlying addiction.</a> Curr Opin Neurobiol. 23 (4), 539-545 (2013).</li><li>Abbott, S., Coates, M., Stornetta, R., Guyenet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+stimulation+of+c1+and+retrotrapezoid+nucleus+neurons+causes+sleep+state-dependent+cardiorespiratory+stimulation+and+arousal+in+rats.">Optogenetic stimulation of c1 and retrotrapezoid nucleus neurons causes sleep state-dependent cardiorespiratory stimulation and arousal in rats.</a> Hypertension. 61 (4), 835-841 (2013).</li><li>Carter, M., 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=Tuning+arousal+with+optogenetic+modulation+of+locus+coeruleus+neurons.">Tuning arousal with optogenetic modulation of locus coeruleus neurons.</a> Nat Neurosci. 13 (12), 1526-1533 (2010).</li><li>Darrow, 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=Optogenetic+control+of+central+auditory+neurons.">Optogenetic control of central auditory neurons.</a> Assoc. Res. Otolaryngol. Abstr. (695), (2012).</li><li>Shimano, T., 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=Assessment+of+the+AAV-mediated+expression+of+channelrhodopsin-2+and+halorhodopsin+in+brainstem+neurons+mediating+auditory+signaling.">Assessment of the AAV-mediated expression of channelrhodopsin-2 and halorhodopsin in brainstem neurons mediating auditory signaling.</a> Brain Res. 1511, 138-152 (2013).</li><li>Doucet, J., Ryugo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projections+from+the+ventral+cochlear+nucleus+to+the+dorsal+cochlear+nucleus+in+rats.">Projections from the ventral cochlear nucleus to the dorsal cochlear nucleus in rats.</a> J Comp Neurol. 385, 245-264 (1997).</li><li>Brown, M., Drottar, M., Benson, T., Darrow, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commissural+axons+of+the+mouse+cochlear+nucleus.">Commissural axons of the mouse cochlear nucleus.</a> J Comp Neurol. 521, 1683-1696 (2013).</li><li>Verma, R., 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=Auditory+responses+to+electric+and+infrared+neural+stimulation+of+the+rat+cochlear+nucleus.">Auditory responses to electric and infrared neural stimulation of the rat cochlear nucleus.</a> Hear Res. 310, 69-75 (2014).</li><li>Taberner, A. M., Liberman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+properties+of+single+auditory+nerve+fibers+in+the+mouse.">Response properties of single auditory nerve fibers in the mouse.</a> J Neurophysiol. 93 (1), 557-569 (2005).</li><li>Rolls, 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=Optogenetic+disruption+of+sleep+continuity+impairs+memory+consolidation.">Optogenetic disruption of sleep continuity impairs memory consolidation.</a> Proc Natl Acad Sci. 108 (32), 13305-13310 (2011).</li><li>Huff, M., Miller, R., Deisseroth, K., Moorman, D., LaLumiere, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posttraining+optogenetic+manipulations+of+basolateral+amygdala+activity+modulate+consolidation+of+inhibitory+avoidance+memory+in+rats.">Posttraining optogenetic manipulations of basolateral amygdala activity modulate consolidation of inhibitory avoidance memory in rats.</a> Proc Natl Acad Sci. 110 (9), 3597-3602 (2013).</li><li>Stortkuhl, K., Fiala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Smell+of+Blue+Light:+A+New+Approach+toward+Understanding+an+Olfactory+Neuronal+Network.">The Smell of Blue Light: A New Approach toward Understanding an Olfactory Neuronal Network.</a> Front Neurosci. 5 (72), (2011).</li><li>Hira, R., 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=Transcranial+optogenetic+stimulation+for+functional+mapping+of+the+motor+cortex.">Transcranial optogenetic stimulation for functional mapping of the motor cortex.</a> J Neurosci Methods. 179 (2), 258-263 (2009).</li><li>Ayling, O., Harrison, T., Boyd, J., Foroshkov, A., Murphy, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+light-based+mapping+of+motor+cortex+by+photoactivation+of+channelrhodopsin-2+transgenic+mice.">Automated light-based mapping of motor cortex by photoactivation of channelrhodopsin-2 transgenic mice.</a> Nat Methods. 6 (3), 219-224 (2009).</li><li>Boyden, E., Zhang, F., Bamberg, E., Nagel, G., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Millisecond-timescale,+genetically+targeted+optical+control+of+neural+activity.">Millisecond-timescale, genetically targeted optical control of neural activity.</a> Nat. Neurosci. 8, 1263-1268 (2005).</li></ol>

著者らは、開示することは何もない。

本稿では、中央聴覚系を操作するためのマウスモデルにおけるDCNの直接可視化の技術が記載されている。直接可視化の概略アプローチは、定位アプローチである主要な代替、上の大きな利点を提供する。定位のアプローチが直接可視化を与えないのに対し、主に、DCNの直接可視化は、脳幹の部位の即時確認することができます。注入は、ターゲットの場所を「ミス」した場合場合、ウイルス媒介遺伝子移入にあるように、延長インキュベーション期間を必要とする実験では、低い感染効率の可能性がある。さらに、直接可視化のアプローチは、広い範囲の動物の年齢に注入することができます。類似の外科的アプローチは、ラットおよびモルモットのような他の動物モデルと生存実験で使用することができる。 このプロトコルでいくつかの重要なステップがあります。まず、マウスは定位ホルダーに適切に配​​置する必要があります。頭の任意の動きが困難開頭術を作ることになります。第二に、開頭の場所は非常に重要です。開頭術が適切な位置で行われていない場合は、小脳のD​​CN以下吸引の可視化は、困難、不可能ではないであろう。最後に、プロトコルの最も重要なステップは、小脳の部分的な指示吸引です。小脳の吸引には2つの技術的アプローチがあります。最初の吸引のアプローチでは、吸引は常に脳幹から上方に持ち上げ、小脳上に保持されている。このアプローチでは、DCNは、部分的にCNが完全に可視化し、小脳が除去されるまで小脳および吸引保持されているの下に可視化することができる。 DCNが可視化されるまで、第2のアプローチでは、小脳は、部分的に、いくつかの層状のパスで吸引される。歯科のポイントは、血液または残りの小脳のfragmeをクリアするために穏やか軽くたたくで使用することができるそれは完全に可視化することができない場合、CNからのnts。 出血は直接可視化アプローチの主要な欠点​​であると、主に初期の開頭術中および小脳吸引後に発生します。最初の開頭術の際、多くの場合、出血は2-5分以内の時間で停止します。出血部位でのティッシュペーパーまたは歯科ポイントを配置し、止血を可能にする。小脳吸引後の出血の面では、血液を希釈し、CNのレベルで血餅形成を防止するために開頭術部位における生理食塩水（0.5 ccで）を注入することが重要である。吸引と穏やかな歯科ポイント軽くたたくの組み合わせは、血液や生理食塩水を離れてクリアするために使用することができる。実際、このアプローチの主要な制限は、手順の侵襲性である。 プロトコルは、聴覚システムの操作を超えた意味を有する。中枢神経系へのインビボ遺伝子導入のdivの操作を可能に神経経路をERSEとメモリ、モータ制御、嗅覚、およびオーディションを含む複数の神経機能への洞察を提供している。16,21-26外科的アプローチ、遺伝子導入及び刺激のために直接的な可視化を可能にすると、潜在的に他の領域にも適用することができる脳幹の、上に列挙されたものと他のものの両方。 DCNの直接可視化の習得後、実験のホストは、CNへのオプシンの注入及び青色レーザーを用いたその後の刺激を含む、可能である。そのような電気刺激と神経インジェクタトレーシングのような他の神経生理学実験は、18も可能である。可視化のレベルと、このアプローチの刺激の持続時間は、実験の広い範囲のための複数のアプリケーションを可能にする。

Virus-mediated gene transfer to reverse hearing loss has largely been focused on the peripheral auditory system.1 Targeting the cochlea, investigators have examined a host of delivery routes, including osmotic minipump infusion2, vector-transgene complex-soaked Gelfoam&#174;2 or gelatin sponge3, direct microinjection4; numerous gene transfer vectors, including adeno-associated viral vectors5,6, lentiviral vectors7, and cationic liposomes2; and the dissemination of gene transfer vectors beyond the target tissue2. Most recently, adeno-associated virus (AAV)-1 has been introduced in the cochlea in order to treat deafness in mice due to loss of vesicular glutamate transporter-3.8 Further, the application of optogenetics in peripheral auditory system has recently been described.9
Few studies, however, have examined gene transfer to the central auditory system. The dorsal cochlear nucleus (DCN) of the brainstem contain second order neurons of the auditory pathway. While gene transfer techniques in the cochlear nucleus (CN) may be utilized for a host of investigations, gene transfer of opsins, light-sensitive proteins, to the DCN may also be utilized to enable optogenetics-based experimental techniques. Following virus-mediated gene transfer delivery of an opsin, such as channelrhodopsin-2 (ChR2), the neurons of the DCN becomes sensitive to light stimuli. Optogenetic gene transfer has been previously attempted in several brainstem regions, including the rat retrotrapezoid nucleus, mouse locus coeruleus, monkey superior colliculus, and mouse ventral tegmental area.10-14
Recently, investigators have examined the use of optogenetics in the DCN.15,16 The DCN is the location of placement of auditory brainstem implants in humans, making it an attractive part of the auditory system to study for translational studies on auditory neuroprostheses. However, given the location of the DCN, surgical exposure is challenging. The technique described herein provides a protocol for maximal exposure of the DCN via posterior fossa approach to enable viral vector gene transfer and optogenetics-based experiments in a murine model. Previous studies used stereotactic microinjection into the DCN with channelrhodopsin-2.16 Stereotaxic injections, however, are potentially less accurate than injections made by direct visualization, especially in a nucleus as small and deep along the brainstem as the DCN. Transgenic mice expressing tissue specific proteins in the CN are also an attractive option and would obviate the need for gene transfer. Our protocol for exposure of the DCN would also work in transgenic mice as the DCN would need to be directly exposed for optical stimulation. This technique for surgical exposure of the DCN is adapted from previous protocols involving recordings from the auditory nerve and cochlear nucleus in mice and rat models.15,17-20
The overall goal of the protocol is to provide direct exposure to the CN to allow for gene transfer techniques. More specifically, the approach is compatible with both acute and survival surgery and the preparation can be repeated in the same animal for subsequent neurophysiological testing. The direct exposure of the DCN protocol has implications for optogenetics- and virus-mediated gene transfer-based experimentation in other nuclei of the brainstem.

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direct visualization of the murine dorsal cochlear nucleus for optogenetic stimulation of the auditory pathway

逮捕または難聴逆転するウイルス媒介遺伝子導入の使用の調査は、主に末梢聴覚系に追いやられています。いくつかの研究は、中枢聴覚系への遺伝子導入を検討した。聴覚路の二次ニューロンが含まれている脳幹の背側蝸牛神経核（DCN）は、遺伝子導入のための潜在的部位である。このプロトコルでは、後頭蓋窩アプローチを経由してネズミDCNの直接的かつ最大の露出のための技術が実証されている。このアプローチは、急性または生存のどちらかの手術が可能になります。 DCNの直接可視化に続いて、実験のホストは、青色光レーザに結合された光ファイバによる蝸牛神経核およびその後の刺激へのオプシンの注射を含む、可能である。そのような電気刺激と神経インジェクタトレーシングのような他の神経生理学実験は、また可能である。visualizaのレベルると、刺激の持続時間は、達成可能な実験の広範囲にこのアプローチを適用する。

部分的な小脳吸引は蝸牛神経核へのアクセスをデモ 頭蓋骨を覆う皮膚と筋肉がそのような冠状及びラムダ縫合線として、頭蓋骨の表面ランドマークを、除去された後、開頭のおおよその局在を実証する。鉗子との開頭術の後、小脳が可視化される。小脳の小部分を注意深く吸引し、次に注入することができ、CN、（ 図1）の可視化を実証する。 ブルーライトレーザーを刺激するための二次蝸牛神経核露出 オプシンとDCN、遺伝子導入、及びインキュベーション期間の最初の曝露に続いて、DCNは、青色光レーザーによって刺激され得る。光刺激のためのDCNの表面を露出させる二次開頭術は、プライマリ開頭術（SUPP。動画1）に技術的アプローチに似ています。さDCN、厚さが300μm未満である表面構造。実験的なネズミの準備には、最大6時間のための実行可能なままです。 <img alt="図1" fo:content-width="7in" src="/files/ftp_upload/52426/52426fig1highres.jpg" width="700" /> 図1：部分的な小脳吸引は背側蝸牛神経核へのアクセスを示しています。左パネル ：マウスは、ホルダ内に配置された後、切開を首の軟組織への頭蓋骨の後面に沿って皮膚および軟組織を通って垂直に形成されている。皮膚は左右で非対称で、冠状縫合線の上にある筋肉が削除され、中央のパネル ：。開頭術は、その後、骨の骨鉗子と小脳とその下の背側蝸牛神経核の領域にわたって完了した右パネル ：背側蝸牛神経核の暴露について、5フレンチの吸引は、次に、小脳を吸引するために使用され背側蝸牛神経核を可視化されるまで、蝸牛神経核の上に重なる。 <img alt="図2" fo:content-width="5in" src="/files/ftp_upload/52426/52426fig2highres.jpg" width="500" /> 図2：青色レーザーを用いた刺激のための蝸牛神経核の表面の曝露は、光学的に誘発聴性脳幹反応を生成する背側蝸牛神経核の表面上に青色光レーザに結合された光ファイバの配置は、光刺激を可能にする。オプシン感染背側蝸牛核。ブルーバー=400μmの。

Watch this Scientific Journal Video about Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway at JoVE.com

Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model.

聴覚路の光遺伝学的刺激のためのマウス背側蝸牛神経核の直接可視化

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. ...

direct-visualization-murine-dorsal-cochlear-nucleus-for-optogenetic

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

9.2K ビュー.  Harvard Medical School. The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model. 

ビデオ: Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

慢性のためのアダルト生まれの嗅覚ニューロンの選択的なウイルス形質導入 in vivoで Optogenetic刺激

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

神経科学

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

でProprioceptorsの可視化<em&gt;ショウジョウバエ</em&gt;幼虫および蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard for analyzing patterns of synaptic connectivity, but paired electrophysiological recordings can be both cumbersome and experimentally limiting. The development of optogenetics has introduced an elegant method to stimulate neurons and circuits, both in vitro1 and in vivo2,3. By exploiting cell-type specific promoter activity to drive opsin expression in discrete neuronal populations, one can precisely stimulate genetically defined neuronal subtypes in distinct circuits4-6. Well described methods to stimulate neurons, including electrical stimulation and/or pharmacological manipulations, are often cell-type indiscriminate, invasive, and can damage surrounding tissues. These limitations could alter normal synaptic function and/or circuit behavior. In addition, due to the nature of the manipulation, the current methods are often acute and terminal. Optogenetics affords the ability to stimulate neurons in a relatively innocuous manner, and in genetically targeted neurons. The majority of studies involving in vivo optogenetics currently use a optical fiber guided through an implanted cannula6,7; however, limitations of this method include damaged brain tissue with repeated insertion of an optical fiber, and potential breakage of the fiber inside the cannula. Given the burgeoning field of optogenetics, a more reliable method of chronic stimulation is necessary to facilitate long-term studies with minimal collateral tissue damage. Here we provide our modified protocol as a video article to complement the method effectively and elegantly described in Sparta et al.8 for the fabrication of a fiber optic implant and its permanent fixation onto the cranium of anesthetized mice, as well as the assembly of the fiber optic coupler connecting the implant to a light source. The implant, connected with optical fibers to a solid-state laser, allows for an efficient method to chronically photostimulate functional neuronal circuitry with less tissue damage9 using small, detachable, tethers. Permanent fixation of the fiber optic implants provides consistent, long-term in vivo optogenetic studies of neuronal circuits in awake, behaving mice10 with minimal tissue damage.

The development of optogenetics now provides the means to precisely stimulate genetically defined neurons and circuits, both in vitro and in vivo. Here we describe the assembly and implantation of a fiber optic for chronic photostimulation of brain tissue.

脳組織の慢性Optogenetic刺激のための光ファイバ注入

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

で逃避行動のOptogenetic刺激<em&gt;キイロショウジョウバエ</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

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 ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

聴覚神経の光遺伝学的刺激

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

視床前核の脳深部刺激後のラット海馬における遺伝子発現の変化の解析

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

後根神経節モデル：癌性神経浸潤のin vitroモデルでは

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, dystonia or tremor. DBS is based on the delivery of electrical stimuli to specific deep anatomic structures of the central nervous system. However, the mechanisms underlying the effect of DBS remain enigmatic. This has led to an interest in investigating the impact of DBS in animal models, especially in rats. As DBS is a long-term therapy, research should be focused on molecular-genetic changes of neural circuits that occur several weeks after DBS. Long-term DBS in rats is challenging because the rats move around in their cage, which causes problems in keeping in place the wire leading from the head of the animal to the stimulator. Furthermore, target structures for stimulation in the rat brain are small and therefore electrodes cannot easily be placed at the required position. Thus, a set-up for long-lasting stimulation of rats using platinum/iridium electrodes with an impedance of about 1 M&#937; was developed for this study. An electrode with these specifications allows for not only adequate stimulation but also recording of deep brain structures to identify the target area for DBS. In our set-up, an electrode with a plug for the wire was embedded in dental cement with four anchoring screws secured onto the skull. The wire from the plug to the stimulator was protected by a stainless-steel spring. A swivel was connected to the circuit to prevent the wire from becoming tangled. Overall, this stimulation set-up offers a high degree of free mobility for the rat and enables the head plug, as well as the wire connection between the plug and the stimulator, to retain long-lasting strength.

A method for implanting electrodes into the subthalamic nucleus (STN) of rats is described. Better localization of the STN was achieved by using a microrecording system. Furthermore, a stimulation set-up is presented that is characterized by long-lasting connections between the head of the animal and the stimulator.

微小電極は、長期的な脳深部刺激のためのラットの視床下核に電極の移植ガイド付き

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

並列の単核 RNA 配列のため大人の脊髄核の分離

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...