この作品は、革新的な領域研究費補助金によってサポートされていました &quot;メゾスコピックNeurocircuitry&quot;（番号22115002）と文部科学省の &quot;総合的な脳科学のネットワーク&quot;（第221S0003）技術、日本と補助金日本学術振興会の若手研究（B）（番号21700344）（日本学術振興会）にHK

1。幼虫の準備<ol><li>標準のフライ食品を含むプラスチックバイアル中UAS-NpHR2、UAS-ChR2をまたはOK6-Gal4の、OK6-Gal4ののフライラインを維持する。 </li><li>適切な濃度で含まれている全ト​​ランスレチナールスプレッド酵母ペースト（ATR）（ChR2を、1 mMの、リンゴジュース寒天プレート上NpHR2ための10ミリメートル（略して &quot;NpHR&quot;）。 </li><li>バイアルからの第 2または第 3齢幼虫をピックアップし、ATR含有プレート上に置く。 </li><li> 25でそれらをリア適切な期間のために暗闇の中で℃（NpHRためにChR2を、2日間、1日。） </li></ol> 2。顕微鏡のセットアップ<ol><li> Cマウントアタッチメント（倍率0.35x）と、従来の共焦点顕微鏡（我々の場合、FV1000、オリンパス）とCCDカメラ（XCD-V60、ソニー）を取り付けます。 </li><li>対物レンズからのレーザスキャン中に閉じCCDカメラへの光路に沿ってシャッターがある場合は、慎重にそれを削除します。このステップではミクロスに依存する必要に応じてセットアップ対応、技術サポートのために顕微鏡の製造元にお問い合わせください。 </li></ol> 3。解剖<ol><li>身体から残留食物を除去するために水でATR-飼育幼虫をすすぐ。 </li><li> SYLGARDコーティングディッシュ、背側を上（背側には、背側正中線の両側に1を縦方向に実行している2つの気管チューブを持っています）に幼虫を置く。 SYLGARDの厚さが5mm程度である。 </li><li>鉗子（＃5イノックス、デュモンによってFST、スイス）と気管チューブの間に尻尾に虫ピン（アウステルリッツの昆虫ピン、Φ0.10mm、ステンレス製）を挿入します。ピンは、10mmの長さ約、曲がったり、対物レンズの表面を打撃し、損傷を避けるために（〜2ミリメートルの長さ）十分に短くなるようにカットする必要があります。その後、口フック、前端に黒い爪のような構造の近くに幼虫の頭の中に第​​二虫ピンを置く。 </li><li>のCa 2 +を含まない生理食塩水を（NaClを140mMの、塩化カリウム2mMの、MgCl 2を 6 mMの、HEPES-NaOHを5mMの追加、幼虫は湿った保つためにショ糖36 MM（pH7.1））。 </li><li>マイクロはさみ（MB-50-7、Napox、日本）と尾の近くに小さな切開を行います。 </li><li>切開から、頭に向かって背側正中線に沿って縦方向のカットをする。腹側神経索（VNC）と軸索を損傷しないように注意してください。 </li><li>横方向に頭に小さな切開を加えます。 </li><li>解剖bodywallの各コーナーに4つのピンを配置します。体壁は、体壁の分節パターンを視覚化するのに十分な伸びが、蠕動運動を妨げないようにあまりにも多くする必要がある。 </li><li>脳やVNCを除き内臓を外し、+フリー生理食塩水のCa 2でサンプルをすすいでください。 </li><li>体壁に虫ピンの位置および向きを変化させることによって左右対称に腹神経索の向きを調整する。腹側神経索の位置を修正するには、SYLGARDまでフック脳と口の間の組織を通してピンを挿入します。 </li> &lt;李は&gt; 2 mMのCa 2 +のリンゲル液（130mMのNaClを、KClを5mmのMgCl 2を 2mmのCaCl 2を 2 mMの、HEPES-NaOHを5mMの、ショ糖36 MM（pH7.3））でバッファを交換してください。 </li></ol> 4。レーザー照明とイメージング<ol><li>共焦点顕微鏡の4倍乾燥系対物レンズ（UPlanSApo 4倍、NA 0.16、オリンパス、日本）を取り付け、ステージ上の幼虫の準備を設定します。 </li><li>共焦点顕微鏡によって腹側神経索を見つけるために赤色レーザー（633nmの）で解剖準備の透過画像を得る。このスキャンでは、それぞれ、ChR2をまたはNpHRの不要な刺激を誘発するか、または488nmの559nmレーザーを使用しないようにしてください。 </li><li>透過像に関心領域（ROI）を定義します。ズームモードでは、ROIの詳細を決定するために有用である可能性があります。 </li><li> 488nmのまたは559nmの光を照射するために顕微鏡のフィルタセットを変更します。 </li><li>体壁を可視化するためにハロゲンランプと準備を照らす。木曜日電子製剤は、共焦点顕微鏡に取り付けられたCCDカメラで監視することができる。 </li><li>体壁の動きを記録し、動きを監視しながらオン·オフレーザビームを切り替える。 </li></ol>

<ol>
	<li>Cattaert, D., Birman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blockade+of+the+central+generator+of+locomotor+rhythm+by+noncompetitive+NMDA+receptor+antagonists+in+Drosophila+larvae.">Blockade of the central generator of locomotor rhythm by noncompetitive NMDA receptor antagonists in Drosophila larvae.</a> J. Neurobiol. 48, 58-73 (2001).</li><li>Fox, L. E., Soll, D. R., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordination+and+modulation+of+locomotion+pattern+generators+in+Drosophila+larvae:+effects+of+altered+biogenic+amine+levels+by+the+tyramine+beta+hydroxlyase+mutation.">Coordination and modulation of locomotion pattern generators in Drosophila larvae: effects of altered biogenic amine levels by the tyramine beta hydroxlyase mutation.</a> J. Neurosci. 26, 1486-1498 (2006).</li><li>Cheng, L. E., Song, W., Looger, L. L., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+TRP+channel+NompC+in+Drosophila+larval+and+adult+locomotion.">The role of the TRP channel NompC in Drosophila larval and adult locomotion.</a> Neuron. 67, 373-380 (2010).</li><li>Gomez-Marin, A., Louis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+sensation+during+orientation+behavior+in+the+Drosophila+larva:+more+sense+than.">Active sensation during orientation behavior in the Drosophila larva: more sense than.</a> Curr. Opin. Neurobiol. , (2011).</li><li>Gershow, 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=Controlling+airborne+cues+to+study+small+animal+navigation.">Controlling airborne cues to study small animal navigation.</a> Nat. Methods. , (2012).</li><li>Suster, M. L., Bate, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+assembly+of+a+central+pattern+generator+without+sensory+input.">Embryonic assembly of a central pattern generator without sensory input.</a> Nature. 416, 174-178 (2002).</li><li>Yizhar, O., Fenno, L. E., Davidson, T. J., Mogri, M., 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=Optogenetics+in+neural+systems.">Optogenetics in neural systems.</a> Neuron. 71, 9-34 (2011).</li><li>Miesenbock, G., Kevrekidis, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+and+control+of+genetically+designated+neurons+in+functioning+circuits.">Optical imaging and control of genetically designated neurons in functioning circuits.</a> Annu. Rev. Neurosci. 28, 533-563 (2005).</li><li>Schroll, C., 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=Light-induced+activation+of+distinct+modulatory+neurons+triggers+appetitive+or+aversive+learning+in+Drosophila+larvae.">Light-induced activation of distinct modulatory neurons triggers appetitive or aversive learning in Drosophila larvae.</a> Curr. Biol. 16, 1741-1747 (2006).</li><li>Hwang, R. Y., 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=Nociceptive+neurons+protect+Drosophila+larvae+from+parasitoid+wasps.">Nociceptive neurons protect Drosophila larvae from parasitoid wasps.</a> Curr. Biol. 17, 2105-2116 (2007).</li><li>Pulver, S. R., Pashkovski, S. L., Hornstein, N. J., Garrity, P. A., Griffith, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+dynamics+of+neuronal+activation+by+Channelrhodopsin-2+and+TRPA1+determine+behavioral+output+in+Drosophila+larvae.">Temporal dynamics of neuronal activation by Channelrhodopsin-2 and TRPA1 determine behavioral output in Drosophila larvae.</a> J. Neurophysiol. 101, 3075-3088 (2009).</li><li>Pulver, S. R., Hornstein, N. J., Land, B. L., Johnson, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetics+in+the+teaching+laboratory:+using+channelrhodopsin-2+to+study+the+neural+basis+of+behavior+and+synaptic+physiology+in+Drosophila.">Optogenetics in the teaching laboratory: using channelrhodopsin-2 to study the neural basis of behavior and synaptic physiology in Drosophila.</a> Adv. Physiol. Educ. 35, 82-91 (2011).</li><li>Inada, K., Kohsaka, H., Takasu, E., Matsunaga, T., Nose, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+dissection+of+neural+circuits+responsible+for+Drosophila+larval+locomotion+with+halorhodopsin.">Optical dissection of neural circuits responsible for Drosophila larval locomotion with halorhodopsin.</a> PLoS One. 6, e29019 (2011).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>Venken, K. J., Simpson, J. H., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulation+of+genes+and+cells+in+the+nervous+system+of+the+fruit+fly.">Genetic manipulation of genes and cells in the nervous system of the fruit fly.</a> Neuron. 72, 202-230 (2011).</li><li>Boyden, E. S., 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><li>Nagel, 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=Channelrhodopsin-2,+a+directly+light-gated+cation-selective+membrane+channel.">Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 13940-13945 (1073).</li><li>Hildebrand, E., Dencher, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+photosystems+controlling+behavioural+responses+of+Halobacterium+halobium.">Two photosystems controlling behavioural responses of Halobacterium halobium.</a> Nature. 257, 46-48 (1975).</li><li>Takahashi, T., Mochizuki, Y., Kamo, N., Kobatake, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+the+long-lifetime+photointermediate+of+s-rhodopsin+is+a+receptor+for+negative+phototaxis+in+Halobacterium+halobium.">Evidence that the long-lifetime photointermediate of s-rhodopsin is a receptor for negative phototaxis in Halobacterium halobium.</a> Biochem. Biophys. Res. Commun. 127, 99-105 (1985).</li><li>Zhang, F., 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=Multimodal+fast+optical+interrogation+of+neural+circuitry.">Multimodal fast optical interrogation of neural circuitry.</a> Nature. 446, 633-639 (2007).</li><li>Brent, J. R., Werner, K. M., McCabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+larval+NMJ+dissection.">Drosophila larval NMJ dissection.</a> J. Vis. Exp. (24), e1107 (2009).</li><li>Fenno, L., Yizhar, O., 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=The+development+and+application+of+optogenetics.">The development and application of optogenetics.</a> Annu. Rev. Neurosci. 34, 389-412 (2011).</li></ol>

利害の衝突は宣言されていない。

神経活動の時間的局所摂動は、神経回路網のダイナミクスを分析するための貴重なテクニックです。このプロトコルでは、レーザーを用いて光遺伝学によって神経活動を操作する手法を提案する。レーザーの高い​​方向性は水銀やキセノンランプを用いた広視野刺激よりもローカライズoptogenetic刺激を可能にします。レーザ照明が既に以前の研究で光遺伝学に適用されてきたが、ガラス繊維、マイクロマニピュレーター、レーザ光源などの特殊な設定は、ほとんどの先行研究で必要とされる。このプロトコルでは、我々は地元の照明のために、従来の共焦点顕微鏡を使用しています。共焦点顕微鏡システムが広く使用されているので、この方法は、多くのラボで高解像度の光遺伝学を適用する機会を開く。 幼虫の解剖とレーザーパワー：プロトコルは、2つの重要なポイントがあります。まず、脳、腹側神経索や運動神経はドゥリが破損している場合ngの解剖、幼虫は以下またはnone自発蠕動運動を示すであろう。精密スプリングはさみで背側に切り込みを作り、鉗子で内臓を取り外す場合は特に、不可欠です。解剖の詳細は以前に報告されている21。第二に、もし十分な刺激は、約0.1のレーザパワー〜ChR2をNpHR又は1〜10mWのための/ mm 2でが要求されるための1 mWの/ mm 2で達成されるべきである。我々は、照明のおおよその面積で割った対物レンズ下の総光パワーのレーザパワーを評価した。我々は、パワー·メータ（mobiken、三和MIテクノス、日本）を用いてトータル光パワーを測定した。私たちは、円形の定数倍とほぼ回折限界の照明領域を与えるレーザーの波長の2乗を照明の面積を推定した。試料上の有効な照明の電力は、走査速度およびしびれを変化させることにより、レーザの出力パワーによるものだけを調整することができるが、繰り返しのER。我々は63回20-100μ秒/ピクセルでレーザーをスキャンしました。また、光遺伝学蛋白質及びATRの量の発現レベルはまた、刺激効率のために重要である。従って、幼虫はないoptogenetic応答を示さなかった場合は、以下の点をチェックし、修正すべき：1）レーザパワー（顕微鏡システムを最適化することにより）、2）光遺伝学タンパク質の発現レベル（遺伝子型および飼育温度をチェックすることにより）、及び3）量ATR（ATR、供給期間の濃度を調整することにより）。 NpHR 13未知の理由により、ChR2を行いよりATRの高い濃度を必要とします。 ChR2をここで説明するよりも低いATR（ 例えば 0.1 mm）を（1 mM）を含む食品で飼育幼虫でうまく動作します。 1ミリメートルATRに幼生を供給すると、ChR2を光反応を行うために十分であるとして、我々はChR2を実験で最初の裁判のため、この濃度をお勧めします。 ATRの濃度は、その後、1 mMのから降りて滴定することができます。 ATRの露光期間と同様に、私たちはお勧め期間は堅牢optogenetic制御のため、ここで説明。私たちはしばしば、短い期間ATRに幼生を供給する際にoptogenetic応答を観察することができませんでした。 このプロトコルでは、レーザ照明および画像取得が別のコンピュータによって操作される。したがって、CCDカメラによるレーザ照射の時空間パターンを明確に検出、データ解析のために重要である。レーザスポット又はラインはCCDイメージに暗い場合には、ハロゲンランプと体壁を可視保ちながらレーザー照射を視覚化するCCDカメラのゲイン値のパワーを最適化する必要があります。 このプロトコルで示さ時空照明が幼虫のモーター回路に関する情報を提供しますが、方法はいくつかの制限があります。 &quot;空間&quot;の態様としては、照明の位置は、体壁の動きをビデオテープに録画に使用される低倍率のCCDイメージによって記録される。これにより、照射領域は、セグメント·レベルではなく、細胞レベルで割り当てることができる。 &quot;tまでのようにemporal &quot;の態様では、レーザーによる共焦点顕微鏡照明によりレーザ走査スイッチオン時間差は、共焦点顕微鏡システムとスキャン条件に依存するので、試行錯誤によっていくつかのテストは、照明タイミングを調整する必要がある。照射のタイミングにできるので、 ImageJのような適切なソフトウェアを使用してCCDイメージムービーで決定することが、時間分解能の重要な要因は、CCDカメラ画像のフレームレートです。我々は通常、毎秒7.5から15フレームのフレームレートを設定します。 optogeneticツールの進歩は、私たちは、このプロトコルを修正する機会を提供します。我々は第 3齢幼虫OK6-Gal4のを有するとUAS-ChR2を[H134R]またはUAS-NpHR2を使用していました。その他optogeneticツールの代わりにChR2を[H134R]またはNpHR2などChR2を[T159C/E123T]、NpHR3またはアーチがアクティビティコントロール22の効率を高めることができますように。加えて、他のGAL4線を使用したり、他の発達段階に分析することで、モータcircuiに関する詳細情報を提供することができるTS。 このプロトコルでは、運動活性をCCDカメラでbodywall収縮の透過画像でモニターした。 ChR2をまたはNpHRで神経活動を操作しながら、カルシウム感受性蛍光分子（ 例えば GCaMP）と運動ニューロンの活性を直接監視することができます。この場合には、広域かつ高感度CCDカメラ（ 例えば、カメラEMCCD）で青色光照明はハロゲンランプの代わりに用い、通常のCCDカメラが以前に本明細書に記載されている。低倍率の対物レンズによる試料と監視bodywall上方から高倍率の対物レンズ（40倍など ）による腹神経索の照明：追加の対物レンズを用いて、bodywallの動きを監視しながら、刺激の空間分解能を向上させるためには、より高度な方法とすることができる（ 例えば 4倍）サンプルの下。この二重レンズ系は、私たちは、より高い空間分解能で神経系を刺激することを可能にする。

ショウジョウバエの幼虫でフォワード蠕動運動は、前のセグメントの後方から筋収縮の伝播によって生じる。この運動は、長手方向の体軸に沿って腹神経索内で運動ニューロンの逐次的活性化によって実現される。このパターン化された伝播の活動の背後にある回路を調べるには、神経活動の局所摂動は有益なアプローチである可能性があります。薬理アッセイは神経組織でこのような神経伝達物質などの特定物質を、制御することができますが、幼虫の腹側神経索が縦軸に沿って似neuromeres成る反復構造であるため、薬理薬の効果は、ほぼすべてのセグメントの中で同じようなことができます。あるいは、一つのセグメントの少数のoptogeneticまたは温度感受性プローブ​​分子を発現することができる。しかしながら、このような特定のGAL4線を生成することは非常に困難である。ここで我々はいくつかのセグメントにニューロンを操作するための方法を提示レーザ照明を用いメンツ。共焦点顕微鏡における空間的に閉じ込められた照明の利点生かし、1は、ローカルエリアのニューロンの活動を混乱させることができます。ニューロン特異GAL4行のサブセットによってプローブの発現パターンと組み合わせることで、このレーザー照射法が大幅に摂動のために空間分解能を向上させます。この方法は唯一、従来の共焦点顕微鏡を必要とするためと、追加の実験装置の購入は通常必要ありません。 この手法を用いて、モータ出力13の伝搬時のモータ神経活動の役割を調べた。蠕動運動中にいくつかのセグメント内の運動ニューロンの活動を遮​​断することによって、我々は、運動ニューロン自体の活性はモータ出力信号の伝播のために必要とされるかどうかをテストすることができた。運動ニューロンのローカルおよび過渡封鎖は蠕動運動の伝播を逮捕した。封鎖は、プロを除去した後pagationは波が逮捕されたセグメントに現れた。この現象は、モータニューロンの活性化せず、伝播波が腹神経索に沿ってそれ以上進行しないことを示唆し、従って、運動ニューロンの活性化は、蠕動運動が必要である。我々は以前稲田ら （2011）13に詳細なデータと、この現象についての議論を報告している。ここでは、解剖幼虫の動きを監視しながら、インタラクティブに神経活動を混乱させる方法について説明します。さまざまなGAL4ラインと活動操作のための時空間パターンのシリーズを使用して、1つはモータ回路の摂動応答特性を通して回路ロジックを調べることができます。

<table border="1">
<tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td >Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>BX61WI Microscope</td>
 <td>Olympus Corporation</td>
 <td>BX61WI</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluoview FV1000 confocal system</td>
 <td>Olympus Corporation</td>
 <td>FV1000</td>
 <td></td>
 </tr>
 <tr>
 <td>CCD camera</td>
 <td>Sony</td>
 <td>XCD-V60</td>
 <td></td>
 </tr>
 <tr>
 <td>all-trans retinal</td>
 <td>Sigma</td>
 <td>R2500-500MG</td>
 <td>200 mM stock in 95% EtOH</td>
 </tr>
 <tr>
 <td>Silpot184</td>
 <td>Dow Corning Toray</td>
 <td>3255981</td>
 <td></td>
 </tr>
 </tbody>
</table>

optogenetic perturbation of neural activity with laser illumination in semi-intact drosophila larvae in motion

ショウジョウバエ幼虫の運動は、回路1-6基礎となる神経細胞の成分の遺伝アクセシビリティーのおかげで、発達と生理的神経科学の素晴らしいモデルシステムです。幼虫の神経回路における光遺伝学7,8の適用は、私たちが空間的かつ時間的にパターン化された方法は9-13に神経活動を操作することができます。典型的には、試験片を広く水銀ランプで照明またはLEDので、ターゲットニューロンの特異性は、例えばたGal4-UAS装置14,15などのバイナリ遺伝子発現系によって制御されている。本研究では、 &quot;サブ遺伝解像度&quot;に空間分解能を向上させるために、我々は、ローカルで、従来の共焦点顕微鏡で実装レーザーを使用して腹側神経索のニューロンのサブセットを点灯。半無傷の幼虫の体壁の動きを監視している間、私たちは、対話的にアクティブ化またはチャネルロドプシン16,17 Oと神経活動を抑制したそれぞれRハロロドプシン18-20、。神経組織の空間的かつ時間的に制限された照明により、我々は行動の特定の段階で回路内の特定の神経細胞の活動を操作することができます。この方法では、腹側神経索とモータ出力の時空間パターンにおける局所神経アセンブリの活動との関係を研究するために有用です。

<html> ChR2をと運動ニューロンのローカルおよび過渡活性化。 私たちは、運動ニューロンにChR2をを表明した。各VNCセグメントプロジェクトから体壁の対応するセグメントに新たな運動ニューロンの軸索。我々は、青色レーザVNCの1つまたは2つのセグメントを刺 ​​激すると、対応するセグメント内の筋肉が収縮（ 図1）を示した。 NpHRと運動ニューロンのローカルおよび過渡抑制。 私たちは、運動ニューロンにおけるNpHRを表明した。我々は蠕動筋肉の収縮時に黄色のレーザーでVNCの少数（1〜2）セグメントを刺 ​​激した場合には、伝搬波は、体壁の対応するセグメント（ 図2）で逮捕された。我々は、レーザー照射13を切ったときに、その後波が逮捕されたセグメントから再開。 50513fig1.jpg &quot;ALT =&quot;図1 &quot;のfo：コンテンツ幅=&quot; 5.5インチ &quot;のfo：SRC =&quot; / files/ftp_upload/50513/50513fig1highres.jpg &quot;/&gt; 図1。フィレット準備の地元活性化のChR2を。A.スキームと運動ニューロンの局所活性化 。運動ニューロンにChR2をを表現する腹側神経索のいくつかのセグメントの青色光照射により、対応するセグメントの筋肉が（矢頭）と契約されています。解剖幼虫のB.伝送イメージは。腹神経索のローカル調光（矢印の頭）は、体壁の筋肉（矢印）の分節収縮を誘発する。 <img src="/files/ftp_upload/50513/50513fig2.jpg" alt="図1" fo:content-width="5.5in" fo:src="/files/ftp_upload/50513/50513fig2highres.jpg"/> 図2。フィレット準備のローカル阻害NpHR。A.スキームと運動ニューロンの局所阻害 。黄色Lで運動ニューロン、自発的に発生して蠕動運動（上の矢印頭）の間の筋肉の収縮緩和さでNpHRを表現腹神経索のいくつかのセグメントのightの照明解剖幼虫の（下の矢印頭。）B.透過画像 。腹側神経索の局所照明（矢頭）自発的に契約体壁セグメント（矢印）をリラックス。

Watch this Scientific Journal Video about Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion at JoVE.com

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

ここ半無傷でモータ出力の変化（筋収縮）を監視しながら、我々は運動神経活動のoptogenetic操作のためのプロトコルを記述<em&gt;ショウジョウバエ</em従来の共焦点顕微鏡内にレーザーを使用して&gt;幼虫。この技術は、モータ回路のダイナミクスを解明するために、いくつかのneuromeresにおける神経活動の局所摂動を達成するために研究者を可能にします。

半無傷でレーザー照射による神経活動のOptogeneticの摂動<em&gt;ショウジョウバエ</emイン·モーション&gt;幼虫

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the ...

optogenetic-perturbation-neural-activity-with-laser-illumination-semi

Research

JoVE Journal

Neuroscience

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

11.5K ビュー.  The University of Tokyo. ここ半無傷でモータ出力の変化（筋収縮）を監視しながら、我々は運動神経活動のoptogenetic操作のためのプロトコルを記述ショウジョウバエ幼虫。この技術は、モータ回路のダイナミクスを解明するために、いくつかのneuromeresにおける神経活動の局所摂動を達成するために研究者を可能にします。

ビデオ: Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

胚の脊髄から交連ニューロンの解剖と文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

神経科学

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

行動内分泌学と実験経済学を組み合わせること：テストステロンと社会的意思決定

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

In vivo Laser Axotomy in C. elegans

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

In vivo Laser Axotomy in C. elegans

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

Cの生体レーザー軸索切断にエレガンス

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

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 ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

で食欲連想嗅覚学習<em&gt;ショウジョウバエ</em&gt;幼虫

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

ローカスの神経活動を特徴づけるためにマルチユニット記録方法（<em&gt; Schistocercaアメリカーナ</em&gt;）嗅覚回路

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

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 ...

Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in the hippocampus. The transverse hippocampal slice preparation is the most common tissue preparation to study hippocampus electrophysiology. A longitudinal hippocampal slice was also developed in order to investigate longitudinal connections in the hippocampus. The intact mouse hippocampus can also be maintained in vitro because its thickness allows adequate oxygen diffusion. However, these three preparations do not provide direct access to neural propagation since some of the tissue is either missing or folded. The unfolded intact hippocampus provides both transverse and longitudinal connections in a flat configuration for direct access to the tissue to analyze the full extent of signal propagation in the hippocampus in vitro. In order to effectively monitor the neural activity from the cell layer, a custom made penetrating micro-electrode array (PMEA) was fabricated and applied to the unfolded hippocampus. The PMEA with 64 electrodes of 200 &#181;m in height could record neural activity deep inside the mouse hippocampus. The unique combination of an unfolded hippocampal preparation and the PMEA provides a new in-vitro tool to study the speed and direction of propagation of neural activity in the two-dimensional CA1-CA3 regions of the hippocampus with a high signal to noise ratio.

We have developed an in vitro unfolded hippocampus which preserves CA1-CA3 array of neurons. Combined with the penetrating micro-electrode array, neural activity can be monitored in both the longitudinal and transverse orientations. This method provides advantages over hippocampal slice preparations as the propagation in the entire hippocampus can be recorded simultaneously.

地中マイクロ電極アレイと非折り畳み海馬準備神経活動の伝播

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

覚醒マウスにおける神経活性物質のMicroiontophoreticアプリケーションと組み合わせることで神経活動の細胞外記録

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

冷痛覚のための新規アッセイ<em&gt;ショウジョウバエ</em&gt;幼虫

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...