プロジェクトは、もっぱら著者の責任であり、必ずしも母子保健と人間開発や国立研究所の公式見解を表していないコンテンツをタグ付けするために母子保健と人間開発の国立研究所からの助成金番号R01HD050725によってサポートされていました説明国立衛生研究所。私たちは、原稿とビデオへの入力のためGodenschwegeラボだけでなく、バ​​ーバラSchreaderのメンバーに感謝。

1。腹神経索の解剖 GF充填解剖と染料は、室温で行うことができます。すべての解剖が冷たいな手順については（4℃）生理食塩水は細胞の新陳代謝を遅くして、より長い神経細胞が生き続けるために使用する必要があります。 <ol><li> CO 2の成人のハエ（4日以上）を麻酔またはそれらが固定化されるまで、氷上で冷やします。氷で麻酔すると、より時間がかかります（最大30分）が長いために固定化したハエ続けます。 </li><li> Sylgardコーティングしたペトリ皿内の1つフライを置きます。この解剖は、高倍率（80 - 100X）解剖の実体顕微鏡を使用する必要があります。 </li><li> Vannasのはさみで、足、テングザル、そして翼を取り外します。 </li><li> Sylgard皿の中で配向するフライ背側を粗鉗子と細かい虫ピンを（minutienピン）を使用してください。第一腹部を固定してから（図2を参照）首がわずかに伸張しており、アクセス可能であるように、頭の後ろ、首の両側にある2つのピンを配置することによって、わずかに動物を細長い。 </li><li>新鮮な、冷たいショウジョウバエの生理食塩水のフライ水没。最も一般的に使用されるショウジョウバエのSalinesのは解剖のために十分な、しかしここで我々は、区とオダウド、2006年4で説明した生理食塩水を使用していました。気泡は、フライで囲まれます。シリンジで空気を取り外します。 </li><li>腹部の背側端に短く、横方向の切開を加えます。切開から始まり、胸部に向かって腹部の背側正中線に沿って浅い、ストレートカットを行います。その後、結合に向かって正中線に沿って可能な限り正確な胸部の浅い、長手方向のカットを継続する。それは、VNC（図1）を損傷しないようにカットが浅いために重要です。 </li><li>慎重に胸部を開くには2微細な昆虫ピンを使用してください。体がピンを使用して離れて半分に広がり、飛翔筋（図2）を介してピンを配置。キューティクルのリッピングを避けるために、図2に示すように胸部がさらに開かれるべきではない。 VNCの直線方向を確保するためのピンは両側で同じ前方後方位置に配置する必要があります。 </li><li>色素注入と、適切なイメージングのためのVNCへのアクセスは、内部器官によって妨害されることがあります。したがって、胃、腸、心臓、生殖器官を削除するには鉗子やハサミを使う。 VNCの両側に横たわっている唾液腺を引き出します。フローティング組織と脂肪を除去するために新鮮な生理食塩水で慎重に解剖をすすぐ。 </li><li>子宮頸結合（CVC）の下から体脂肪を取り除きます。脂肪体は、高倍率での最善表示されます。慎重にサイドから組織を把握し、それを引き出すために微細な鉗子を使用してください。 </li></ol>解剖は、5〜10分以内に行うことができます。新鮮な、冷たい生理食塩水を使用して解剖し、無傷の神経系は、少なくとも1時間のために生きているままになります。 2。細胞内GFフィルそれは解剖後できるだけ早く充填色素を実行することをお勧めします。 <ol><li>固定ステージ（図3）と10倍の空気を目的とした正立顕微鏡下で解剖フライでSylgard皿を置きます。センター染料フィル電極ホルダーに向けて配置さハエの後端とサンプル。 CVCの微分干渉コントラスト（DIC）モードとフォーカスを使用してください。 GFSの大きな軸索は、CVCの背側表面上にはっきりと見えるはずです。 </li><li> 1％の濃度まで脱イオン水でルシファーイエローCHジリチウム塩を希釈する。約の抵抗にフィラメントとホウケイ酸ガラス電極を引き出します。 MΩ60から80。ガラス電極の先端が長く、シャープにする必要があります。約のためのルシファーイエローソリューションへの電極の先端の反対側の端に電極を配置することにより1％ルシファーイエロー溶液を電極の先端を埋める。 30〜60秒。その後、染料とLiClの間に気泡を残してハミルトンシリンジを使用して3M LiClを持つ電極をバックフィル。ヘッドステージに染料充填電極を接続してください。 </li><li>生理食塩水に塩化コーティングされた銀線の接地電極を挿入し、3次元油圧マイクロマニピュレーターを使用してVNCのフライ平行上記の色素の塗り電極を配置する。色素充填電極はそれぞれ、ハイパーまたは現在の脱分極を渡すことによって悪影響を解放したり、電極からの正に帯電した染料にするために電流を流すことができるアンプに接続する必要があります。 </li><li> 40倍水浸レンズに切り替えて、GFSの一つに色素充填電極の先端を下げる。 GFの軸索は、CVCの背側表面に近い置く。 GFに電極を挿入するために前方に（頭に向かって）動きを使用してください。 </li><li>落射蛍光光とルシファーイエローフィルターのスイッチに光源を変更します。 GFにルシファーイエローを注入するために、アンプと染料フィル電極に分極電流を適用する。色素注入の量は目で制御することができます現在の電子の量は、渡されただけでなく、持続時間の電流が渡されます。注射部位または損傷GF外にこぼれることから色素を防ぐために、それを1〜5分間にわたり低電流（約3〜5 NA）を用いて注入することをお勧めします。 </li><li> VNCでのGFの端末がはっきりと見ることができるまで、現在適用されます（図4A）。シナプス後ニューロンのシナプス経由の染色に、染料もシナプス後ニューロンに見られるようになるまでに電流を流すために引き続き。現在のスイッチをオフにし、色素充填電極を取り外します。 </li></ol> 3。代表的な結果： 代表的な色素ルシファーイエローを使用すると、ワンGFの塗りつぶしは、図4Aに示されています。画像は、顕微鏡に搭載されたスポットのカメラを使用してニコンの要素のソフトウェアで画像をスタックとして買収された。このサンプルでは、​​現在は約2分間電極を通過させた。 GFは、徹底的に満ちていると胸郭の大きなGF端子がよく（図4A、楕円）というラベルが付いています。ルシファーイエローは、ギャップジャンクショ​​ンを通過するのに十分小さいですが、何シナプス後ニューロンは、この場合は表示されません。信号が検出されるには余りにも弱いため染料がGFに十分な長さに注入されていない場合は、トランスシナプスシナプス後ニューロンの塗りつぶしが見られていない可能性があります。 GFSへのシナプス後ニューロンが（図4B、矢印の頭）などNeurobiotinのようなさらに小さな神経トレーサーを使用して、より確実に可視化することができる。 2分間脱分極電流と2 M酢酸カリウム溶液で7％Neurobiotinを注入することを強くシナプス後ニューロンを標識するために十分です。 Neurobiotinは、異なる波長の色素に結合したストレプトアビジンを用いて共焦点顕微鏡のための視覚化できます。 Neurobiotinは蛍光色素、同じソリューション内にローダミン-デキストランまたはAlexaのヒドラジドの同時注入ではないため、GF（BoernerとGodenschwegeから図4C、、2010 5）に成功した注射を監視するために使用することができます。 <img src="/files/ftp_upload/3080/3080fig1.jpg" alt="図1" /> 図1。フライの模式図が横方向に表示。胸部は内部構造を可視化するために縦切開で表示されます。解剖時には、背側縦飛翔筋（DLMS、青）は飛翔筋の腹側にある腹側神経索（VNC、深い赤）を、損なうことなく、カットされた。腸（緑）はVNCの上に位置しています。 <img src="/files/ftp_upload/3080/3080fig2.jpg" alt="図2" /> 図2。解剖ハエの背ビュー。 2本のピンをフライの位置を頭の後ろに配置されます。飛翔筋（DLM）を介して貼付端子は胸部が開いて保持する。内臓は、腹側神経索（VNC）を公開する削除されました。 <img src="/files/ftp_upload/3080/3080fig3.jpg" alt="図3" /> 図3。色素充填セットアップ。 <img src="/files/ftp_upload/3080/3080fig4.jpg" alt="図4" /> ルシファーイエローを充填した1 GFを示すVNCの後部の4）Z -射影図 。画像は、染料がスポットカメラで充填した後、すぐに買収された。野生型マウスでは胸部にある端末（楕円）がはっきりと見える。 B）GF色素の共焦点画像は、野生型のハエにNeurobiotinを詰めます。 GFS（矢印）とGFS（矢印の頭）にシナプス後ニューロンの両方が標識されている。 Neurobiotinは、ストレプトアビジンは、Cy3のために結合を持つ可視化した。 C）野生型GFSの共焦点画像は、Alexa555でいっぱい。

<ol>
	<li>Power, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thoracico-abdominal+nervous+system+of+an+adult+insect+Drosophila+melanogaster.">The thoracico-abdominal nervous system of an adult insect Drosophila melanogaster.</a> J. Comp. Neurol. 88, 347-409 (1948).</li><li>Allen, M. J., Godenschwege, T. A., Tanouye, T. A., Phelan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+an+escape:+development+and+function+of+the+Drosophila+giant+fibre+system.">Making an escape: development and function of the Drosophila giant fibre system.</a> Semin. Cell Dev. Biol. 17, 31-41 (2006).</li><li>Boerner, J., Duch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Average+shape+standard+atlas+for+the+adult+Drosophila+ventral+nerve+cord.">Average shape standard atlas for the adult Drosophila ventral nerve cord.</a> J. Comp. Neurol. 518, 2437-2455 (2010).</li><li>Gu, H., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+synaptic+transmission+in+adult+Drosophila+Kenyon+cells+in+sity.">Cholinergic synaptic transmission in adult Drosophila Kenyon cells in sity.</a> J. Neurosci. 26, 265-272 (2006).</li><li>Boerner, J., Godenschwege, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+for+the+Drosophila+ventral+nerve+cord+standard+in+neuronal+circuit+reconstruction+and+in-depth+analysis+of+mutant+morphology.">Application for the Drosophila ventral nerve cord standard in neuronal circuit reconstruction and in-depth analysis of mutant morphology.</a> J. Neurogent. 24, 158-1567 (2010).</li><li>Godenschwege, T. A., Kristiansen, L. V., Uthaman, S. B., Hortsch, M., Murphey, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conderved+role+for+Drosophila+Neuroglian+and+human+L1-CAM+in+central-synapse+formation.">A conderved role for Drosophila Neuroglian and human L1-CAM in central-synapse formation.</a> Curr. Biol. 16, 12-23 (2006).</li><li>Uthaman, S. B., Godenschwege, T. A., Murphey, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechanism+distinct+from+highwire+for+the+Drosophila+ubiquitin+conjugase+bendless+in+synaptic+growth+and+maturation.">A mechanism distinct from highwire for the Drosophila ubiquitin conjugase bendless in synaptic growth and maturation.</a> J. Neurosci. 28, 8615-8623 (2008).</li><li>Storkebaum, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dominant+mutations+in+the+tyrosyl-tRNA+synthetase+gene+recapitulate+in+Drosophila+features+of+human+Charcot-Marie-Tooth+neuropathy.">Dominant mutations in the tyrosyl-tRNA synthetase gene recapitulate in Drosophila features of human Charcot-Marie-Tooth neuropathy.</a> Proc. Natl. Acad. Sci. USA. 106, 11782-11787 (2009).</li><li>Allen, M. J., Godenschwege, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+recordings+from+the+Drosophila+giant+fiber+system+(GFS).">Electrophysiological recordings from the Drosophila giant fiber system (GFS).</a> Cold Spring Harb. Protoc. 2010, pdb.prot5453-pdb.prot5453 (2010).</li><li>Augustin, H., Allen, M. J., Partridge, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+Recordings+from+the+Giant+Fiber+Pathway+of+D.+melanogaster.">Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster.</a> J. Vis. Exp. , (2011).</li></ol>

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神経系は、ハエの体からそれを抽出することなく解剖することができます。これは、最初、次の2つの利点が、解剖は神経系へのダメージが少ない、そして第二に、ほとんどの神経は、筋肉や感覚器官に付着したままでいる。解剖を行って、説明されているように、まっすぐ前方に色素標識GFSのためのサンプルを準備します。便利に、GFSにシナプス後ニューロンは、筋肉に付着したままでいる...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント</th></tr><tr><td> Sylgard 184シリコーンエラストマーキット</td><td>ダウコーニング</td><td></td><td></td></tr><tr><td>解剖顕微鏡</td><td> AmScope </td><td> SM - 2TZ </td><td></td></tr><tr><td> Vannasはさみスーパーファイン</td><td>アカデミックインスツルメンツ</td><td> VS1023 </td><td></td></tr><tr><td>デュモン鉗子Dumontstar 55 </td><td>ファイン科学ツール</td><td> 11295〜51 </td><td></td></tr><tr><td>アウステルリッツの昆虫ピン</td><td>ファイン科学ツール</td><td> 26002〜10 </td><td> Ø 0.1ミリメートル</td></tr><tr><td>ホウケイ酸ガラス電極</td><td>世界の精密機器</td><td> 1B100F - 4 </td><td></td></tr><tr><td> 700C垂直ピペットプラー</td><td>デビッドKopfは楽器</td><td> 700Cモデル</td><td></td></tr><tr><td>ルシファーイエローCH二リチウム塩</td><td>シグマ</td><td> L0259 </td><td> H 2 Oの1％ </td></tr><tr><td>固定ステージ正立顕微鏡とカメラ</td><td>ニコン</td><td> FN - 1＆DS - U2カメラ</td><td></td></tr><tr><td>計画フルーア10Xエア目的</td><td>ニコン</td><td> CFIプランフルーア16ミリメートル10倍、NA 0.3 WD </td><td></td></tr><tr><td>フッ素40X水浸対物</td><td>ニコン</td><td> CFIフルーア40X W NA 0.80 WD 2.0ミリメートル</td><td></td></tr><tr><td> 3D油圧マニピュレーター</td><td>ナリシゲインターナショナル</td><td>モデルMW0 - 3 </td><td></td></tr><tr><td>アンプ</td><td>楽器をはじめ、（株） </td><td>モデル5A </td><td></td></tr></tbody></table>

whole mount preparation of the adult drosophila ventral nerve cord for giant fiber dye injection

ニューロンの軸索と樹枝状形態を分析するために、それはニューロン構造の正確なラベル付けを得るために不可欠です。ない組織の損傷に少しでも標識サンプルを準備するので変異体異常の同定を可能にする、私たちは細胞の形態を分析し、お互いに個々のサンプルを比較することができます。 実証解剖の方法では神経系は成人フライの内側主に残ります。背側を切開して、腹部と胸部が開かれ、ほとんどの内臓のが削除されます。 GFの細胞体と樹状突起を含む脳がそのまま頭に2のまま腹神経索（VNC）と巨大な繊維（GFS）1の大きい軸索を含む子宮頸結合（CVC）の唯一の背側は、公開されています。この準備ではVNCのほとんどの神経はそれらの筋肉に接続されたままにしてください。 解剖の後、蛍光色素を持つ巨大な繊維（GF）の細胞内の充填が実証される。 CVCにGFの軸索は、背面に配置されていますので、微分干渉コントラスト（DIC）光学系と顕微鏡下で容易に可視化することができる。これは、このサイトでは染料とGFの軸索の注入は、VNCの軸索とその端子を含む全体のGFにラベルを付けることができます。ニューロンは、落射蛍光顕微鏡で充填した後、直ちに画像化できるようにGFSの信頼性と強力な染色では、このメソッドの結果。また、蛍光シグナルは高分解能共焦点顕微鏡に適した標準的な免疫組織化学の手順3を使用して拡張することができます。

Watch this Scientific Journal Video about Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection at JoVE.com

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

アン<em&gt; in vivoで</em成人の&gt;解剖<em&gt;ショウジョウバエ</em&gt;腹神経索（VNC）が示されています。この特定の解剖法は、高分解能のイメージングのための蛍光染料と巨大な繊維の神経細胞のその後のラベル付けを可能にするVNCへのダメージが少ない。

成人の全体のマウントの準備ショウジョウバエ</emジャイアント繊維染料注入のための>腹神経索

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

whole-mount-preparation-adult-drosophila-ventral-nerve-cord-for-giant

Research

JoVE Journal

Neuroscience

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

18.9K ビュー.  Florida Atlantic University. アン in vivoで解剖ショウジョウバエ腹神経索（VNC）が示されています。この特定の解剖法は、高分解能のイメージングのための蛍光染料と巨大な繊維の神経細胞のその後のラベル付けを可能にするVNCへのダメージが少ない。

ビデオ: Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

ホールマウントの胎児神経系の可視化ショウジョウバエ胚

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

神経科学

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

の巨大線維経路からの電気生理学的記録 D.キイロ</em

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

用いて、化合物の生理活性をスクリーニングするためのペアNanoinjectionと電気生理学的アッセイキイロショウジョウバエジャイアントファイバシステム

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

ホールマウントマッスル分析による再現性のあるマウスの坐骨神経クラッシュと再生のその後の評価

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1).
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however, other options have been discussed as well5.
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.

マウス胚の脊髄の全載標本での分岐軸索勉強するDRGニューロンのDII -標識

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

全マウント神経筋接合解析のための トランス対アブドミン筋 の解剖

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

における嗅覚受容ニューロンのホールマウント免疫標識<em&gt;ショウジョウバエ</em&gt;アンテナ

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional afferent fibers monitor other characteristics of the muscle environment, including metabolite buildup, temperature, and nociceptive stimuli. Overall, abnormal activation of sensory neurons can lead to movement disorders or chronic pain syndromes. We describe the isolation of the extensor digitorum longus (EDL) muscle and nerve for in vitro study of stretch-evoked afferent responses in the adult mouse. Sensory activity is recorded from the nerve with a suction electrode and individual afferents can be analyzed using spike sorting software. In vitro preparations allow for well controlled studies on sensory afferents without the potential confounds of anesthesia or altered muscle perfusion. Here we describe a protocol to identify and test the response of muscle spindle afferents to stretch. Importantly, this preparation also supports the study of other subtypes of muscle afferents, response properties following drug application and the incorporation of powerful genetic approaches and disease models in mice.

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons are involved in proprioceptor signaling and also report on metabolic state and injury related events. We describe an adult mouse in vitro muscle-nerve preparation for studies on stretch-activated muscle afferents.

アン<em&gt;体外</em筋求心性神経の焼成特性を研究するための&gt;成体マウス筋肉神経の準備

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

成体マウス蝸牛のホールマウント解剖および免疫蛍光

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to network properties. We show how to prepare viable oblique slices of the spinal cord of young mice (P2 - P11). In this preparation, the motoneurons retain&#160;their axons coming out from the ventral roots of the&#160;spinal cord. Stimulation of these axons elicits back-propagating action potentials invading the motoneuron&#160;somas and exciting the motoneuron&#160;collaterals within the spinal cord. Recording of antidromic action potentials is an immediate, definitive and elegant way to characterize motoneuron identity, which surpasses&#160;other identification methods. Furthermore, stimulating the motoneuron collaterals is a simple and reliable way to excite the collateral targets of the motoneurons within the spinal cord, such as other motoneurons or Renshaw cells. In this protocol, we present antidromic recordings from the motoneuron somas as well as Renshaw cell excitation, resulting from ventral root stimulation.

We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

前根刺激のための斜め脊髄スライスの調製

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...

成人の全体のマウントの準備ショウジョウバエ腹神経索