我々は、資金調達のために理研に感謝。 P10 - Gal4のはアランヴィンセント（大学ポールサバティエ、トゥールーズ、フランス）の一種の贈り物だった。

1。試薬の調製 開始前の注意：解剖と免疫組織化学的染色は、磁気チャンバー内で実施され、幼虫は、特殊形状の昆虫ピンを使用して釘付けされる。磁気室の建設、及びこれらのピンの準備手順の詳細については、関連する参照14,15に記載されています。簡単に言うと、1x1cmの正方形の穴が磁気シートと小さなチャンバーを作るために、シートの背面に貼付されているカバースリップに切断される。チャンバーの側面は、エポキシ接着剤で密封され、この接着剤は、チャンバが使用前に70％エタノールで数回洗浄している設定された後。解剖の昆虫ピンは、必要な形状に曲げ加工し、金属製のタブ14,15の上に接着することによって調製される。また金属製のタブに、我々は、カットオフ黄色の先端から作られたハンドルとピンを描く反転のフラットヘッド鋼を使用している。この磁気チェンバーの配置の使用は厳密に制御がオベできますRピンの位置と組織は、解剖中にストレッチ。 DAニューロンの研究者の異なるサブセットにレポーター遺伝子の発現を駆動するためには、いくつかの異なるGal4の行を（下野し、同僚16で要約）使用することができます。これらの行の多くは、株式公開センターから入手できます。この代表的なプロトコルでは、我々はDAニューロンの2つの対照的なクラスが共同で標識されている行の免疫染色を行う：最もシンプルなクラスIおよび最も複雑なクラスIV（P10 - Gal4の17,18、UAS - mCD8：： Kusabira -オレンジ（KO））。 <ol><li>のCa + + -フリーHL3.1生理食塩水を準備<ol><li> mMで：70のNaCl、5のKCl、20のMgCl 2、10飽和NaHCO 3、5 HEPES、115スクロース、および5トレハロース、pH 7.2の19。ろ過滅菌し、4℃で保存します。注：CA + + - free溶液は、解剖時の筋収縮を防ぎます。 </li></ol></li><li> 2倍PHEMバッファを準備します<ol><li> mMで：130 PIPES、60 HEPES、20 EGTA、4マグネシウム4、pHは7.0。ろ過滅菌し、4℃で保存します。 注：pHが7.0に近づくまで、これらの材料は溶解しません。 </li></ol></li><li>固定の直前に新鮮な固定液を準備します。 <ol><li> 25ミリリットルソリューション、Mixの最初の2グラムのパラホルムアルデヒド、50ミリリットルファルコンチューブに100μlの1M NaOHおよび10mlの水を準備するために。 </li><li>溶液が透明になるまで振とう機で55℃の水浴中で固定して振る。 （総容積は約11.5ミリリットルとなります。） </li><li>氷上で冷却固定液。 </li><li> 12.5ミリリットル2倍PHEMバッファを追加。 </li><li> 1M HClでpHを7.0に調整する。 </li><li>ボリュームが25ミリリットルになるまで水で解決策を埋める。 </li><li>ワットマン濾紙を使用してソリューションをフィルタリングする。 </li></ol></li></ol> 2。幼虫の解剖解剖の開始後に急速にブレークダウン、微小管ネットワーク、そして特に感覚樹状突起のものになります：開始する前に注意してください 。 AC直接固定に続いて5分足らずで高速の郭清をhievingは、このプロトコルの成功の鍵となる要因です。 <ol><li>水中で幼虫を洗うとすぐに髪のループを使用して、このドロップにそれらを移動する。 </li><li>オリエント幼虫背側の上とガラス上の腹側。注：この向きは、腹側クラスタのニューロンを調べることです。背側クラスタのニューロンを調べるには、向きを反転。 </li><li>口のフックの近くに前端を固定する中央の虫ピンを使用してください。最良の結果を得るための最後の近くにピンを置きます。 </li><li>解剖室でHL3.1生理食塩水の低下を置きます。 </li><li> microscissorsと幼虫の非常に後部先端を切り取ります。注：このステップはmicroscissors（ステップ2.7）へのアクセスを許可する幼虫の後端に開口部​​を開く。 </li><li>鉗子で今幼虫の後端に開口部​​から突っついている腸の領域をつかむ。ゆっくりと全体の腸を引き出します。 </li><li>置き前方に向かって腹側正中線に沿って開口部とカットスルーmicroscissorsの一つ刃の先端。 </li><li>コー​​ナーピンを使用して、最初に、次に前方後方今すぐ無料のコーナーを、ピン。同時にそっと幼虫フィレットを開くストレッチ。 </li></ol> 3。固定、ブロッキング染色、および幼虫のフィレットの取り付け 開始前の注意：すべての固定と染色の手順は、解剖室で実施されています。このプロセスの間、これは組織の損傷につながる可能性があるため、幼虫を保持している虫ピンをノックしないように注意してください。乾燥から実験を防ぐために、湿らせたティッシュに囲まれた小さなタッパーウェアの容器内のすべての染色の手順を実行します。 <ol><li>黄色のチップを用いてCa + +のフリーHL3.1バッファーを吸引除去する。すぐに別のpipettemanを使用して固定液を加える。 </li><li>ゆっくりとCa + +のフリーHL3.1バッファの残りのトレースで固定液を混合するために上下にピペッティングし、チャンバーインチすぐにそれを吸引し、チャンバー内に新鮮な固定液を加える。 </li><li> 20分間室温でインキュベートする。 </li><li> PBSTで6倍速10分（0.1％トリトンX - 100 PBSで）を洗う。 </li><li>室温で20分間PBSTで5％ヤギ血清でブロック。 </li><li> PBSTで5％ヤギ血清で希釈した一次抗体とブロッキング溶液を交換してください。使用する一次抗体は1 / 1000希釈の両方のマウス抗α-チューブリン（DM1A）及びラット抗CD8（5H10）です。 注：調査員は、マウス抗Futsch（22C10）いくつかの事情で20,21希薄化後1 / 1000（議論を参照）とマウス抗α-チューブリンを（DM1A）置き換えることができます。 </li><li> 4℃（少なくとも16時間）一晩インキュベート℃を</li><li> PBSTで6倍の10分を洗ってください。 </li><li> PBSTで5％ヤギ血清で希釈した二次抗体溶液を加える。使用される二次抗体はAlexa Fluor 488ヤギ抗マウスIgGおよびCy3ロバ抗ラットIgGです。蛍光団を防ぐためにカバーのサンプルを保持する光退色22。 </li><li> 2時間室温でどちらインキュベート、または一晩4℃で、室温で1時間が続く。 </li><li> PBSTで6倍の10分を洗ってください。 </li><li>スライドダウンキューティクル側での幼虫のフィレットを配置し、80％グリセロールでマウントして、&quot;迅速な&quot;マウント用マニキュアとカバーの両側をシール。 注：改良された組織のクリア、および恒久的な安定したサンプルの場合は、DPXのマウントは、以前に23を説明するように。 </li></ol> 4。代表的な結果： 蛍光染色法は、共焦点顕微鏡下で調べた。図では樹状突起内1-2複数のブランチをそれぞれ異なる細胞骨格の組織を持っている。図1は、第1齢幼虫の段階でクラスIV DAニューロンのアーバーの領域を示しています。 KOは、と二次抗CD8抗体と蛍光（Cy3標識）を用いて検出：：全体のあずまやがmCD8でマークされます。チューブリン、抗α-チューブリン抗体とフッ素を使用して検出される自己消費の二次（のAlexa Fluor ® 488）。主枝はチューブリン陽性の、いくつかの薄い側の枝は、チューブリン陰性です。ムービー1は、クラスI DAニューロンの同様の染色を通じて、連続切片のセットです。図2は、第1齢幼虫の段階でFutschおよびCD8に対する抗体で染色したクラスIV DAニューロンのアーバーの領域を示しています。主枝がFutsch陽性である、いくつかの薄い側の枝はFutsch陰性です。図3。幼虫体壁の気管は、複雑な微小管の組織を示す。作品2と3は、体壁の筋肉と気管を通して染色の連続切片を示す。 <img alt="図1" src="/files/ftp_upload/3662/3662fig1.jpg" /> 図1。図。 1は第1齢幼虫の段階でクラスIV DAニューロンのアーバーの領域を示しています。 ：全体のアーバーはmCD8でマークされています：KOと抗CD8抗体と蛍光二次（Cy3標識）を用いて検出。チューブリン、抗＆-細管を用いて検出されるn個の抗体および二次蛍光（アレクサ488）。パネルACシーケンシャル共焦点のz断面（0.5μmの）、C&#39;- C&quot;、と（黄色の矢印）または微小管が強調されています（紫矢印）のない枝のパネルC.の例から、単一の抗体染色では、基本となるの赤矢印のハイライトの微小管上皮細胞。 <img alt="図2" src="/files/ftp_upload/3662/3662fig2.jpg" /> 図2。図。 2は、第1齢幼虫の段階でFutschおよびCD8に対する抗体で染色したクラスIV DAニューロンの東屋の類似領域を示しています。 ：全体のアーバーはmCD8でマークされています：KOと抗CD8抗体と蛍光二次（Cy3標識）を用いて検出。 Futschは抗Futschの抗体と（アレクサ488）二次蛍光を用いて検出される。主枝がFutsch陽性である、いくつかの薄い側の枝はFutsch陰性です。と（黄色の矢印）または（紫矢印）Futschのない枝の例は、強調表示されます。 図3。気管は、上述の抗チューブリンのプロトコルを用いて染色。この幼虫は三齢であり、前述した23,24。ムービー3として解剖正方形でマークされたフィールドから拡大した連続切片を示しています。 ムービー1。クラスIニューロンの樹状アーバを通してチューブリン染色をトレースシリアルのセクションでは、。 KO（マゼンタ）と二次抗CD8抗体と蛍光（Cy3標識）を用いて検出：：全体のあずまやがmCD8でマークされます。チューブリン（緑）は抗α-チューブリン抗体と（のAlexa Fluor ® 488）の二次蛍光を用いて検出される。スケール：ビデオ画像の片側がセクションで46.88μmに対応する。 <a href="http://www.jove.com/files/ftp_upload/3662/YalginMooreMovie1.avi">ムービーを表示するにはここをクリック。</a> ムービー2。BODのチューブリン染色をトレースシリアルのセクションでは、三齢幼虫のyの壁の筋肉。チューブリン、抗α-チューブリン抗体と蛍光二次（のAlexa Fluor 488）を使用して検出される。スケール：ビデオ画像の片側がセクションで46.88μmに対応する。 <a href="http://www.jove.com/files/ftp_upload/3662/YalginMooreMovie2.avi">ムービーを表示するにはここをクリック。</a> 動画3。Figure.3の正方形でマークされた3番目の幼虫の体壁気管内チューブリンの染色を、トレースシリアルのセクションでは、。チューブリン、抗α-チューブリン抗体と蛍光二次（のAlexa Fluor 488）を使用して検出される。スケール：ビデオ画像の片側がセクションで46.88μmに対応する。 <a href="http://www.jove.com/files/ftp_upload/3662/YalginMooreMovie3.avi">ムービーを表示するにはここをクリック。</a>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+Analysis+of+Drosophila+Larval+Peripheral+Sensory+Neuron+Dendrites+and+Axons+Using+Genetic+Mosaics.">Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics.</a> J. Vis. Exp. (57), e3111-e3111 (2011).</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-e1107 (2009).</li><li>Tao, J., Rolls, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendrites+have+a+rapid+program+of+injury-induced+degeneration+that+is+molecularly+distinct+from+developmental+pruning.">Dendrites have a rapid program of injury-induced degeneration that is molecularly distinct from developmental pruning.</a> J. Neurosci. 31, 5398-5405 (2011).</li><li>Yamamoto, M., Ueda, R., Takahashi, K., Saigo, K., Uemura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+axonal+sprouting+and+dendrite+branching+by+the+Nrg-Ank+complex+at+the+neuron-glia+interface.">Control of axonal sprouting and dendrite branching by the Nrg-Ank complex at the neuron-glia interface.</a> Curr. Biol. 16, 1678-1683 (2006).</li><li>Mattie, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+microtubule+growth,++TIPs,+and+kinesin-2+are+required+for+uniform+microtubule+polarity+in+dendrites.">Directed microtubule growth, +TIPs, and kinesin-2 are required for uniform microtubule polarity in dendrites.</a> Curr. Biol. 20, 2169-2177 (2010).</li><li>Pawson, C., Eaton, B. A., Davis, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formin-dependent+synaptic+growth:+evidence+that+Dlar+signals+via+Diaphanous+to+modulate+synaptic+actin+and+dynamic+pioneer+microtubules.">Formin-dependent synaptic growth: evidence that Dlar signals via Diaphanous to modulate synaptic actin and dynamic pioneer microtubules.</a> J. Neurosci. 28, 11111-11123 (2008).</li><li>Williams, D. W., Tyrer, M., Shepherd, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tau+and+tau+reporters+disrupt+central+projections+of+sensory+neurons+in+Drosophila.">Tau and tau reporters disrupt central projections of sensory neurons in Drosophila.</a> J. Comp. Neurol. 428, 630-640 (2000).</li></ol>

我々は、開示することは何もない。

理解するために複雑な細胞の形状が達成される方法、それは正確にアッセイの微小管の組織にできることが重要です。ここでは、樹状突起分枝のニューロン感覚樹状突起のアッセイの微小管の組織に強固な免疫標識法を説明します。感覚ニューロンを染色に加えて、この方法では気管、筋肉や他の体壁組織の強固な免疫組織染色を実現しています。 我々は、DAニューロン...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ 数は</th><th>コメント （省略可能） </th></tr><tr><td>鉗子</td><td>デュモン</td><td> 11251〜20 </td><td></td></tr><tr><td> Microscissors </td><td> FST </td><td> 15000〜08 </td><td></td></tr><tr><td>マウス抗α-チューブリン（クローン：DM1A） </td><td>シグマ</td><td> T9026 </td><td>希釈1000分の1 </td></tr><tr><td>マウス抗Futsch（クローン：22C10）、 上清</td><td>発達 研究 ハイブリドーマバンク</td><td> 22C10 </td><td>希釈1000分の1 </td></tr><tr><td>ラット抗CD8（クローン：5H10） </td><td> Caltag </td><td> MCD0800 </td><td>希釈1000分の1 </td></tr><tr><td>のAlexa Fluor ® 488抗マウスIgG </td><td>インビトロジェン</td><td> - 11001 </td><td>希釈500分の1 </td></tr><tr><td> Cy3標識抗ラットIgG </td><td>ジャクソンイムノ</td><td> 712-166-150 </td><td>希釈200分の1 </td></tr></tbody></table>

immunohistological labeling of microtubules in sensory neuron dendrites, tracheae, and muscles in the drosophila larva body wall

複雑なセル形状の違いが達成されるかを理解するために、それは正確に微小管の組織に従うことが重要です。 ショウジョウバエ幼虫の体壁は、細胞や組織の形態形成を研究するモデルであるいくつかの細胞型を含んでいます。例えば、気管はチューブの形態形成1、 ショウジョウバエの幼虫の感覚ニューロンは、一般の解明と樹状突起分化2-5と変性6のニューロンクラス特有のメカニズムを、プライマリシステムとなっている樹状分枝（DA）を調べるために使用されます。 。 樹状突起の枝の形状は、ニューロンのクラス間、そしてさらに、単一ニューロン7,8の異なるブランチ間で著しく異なることがあります。 DAニューロンにおける遺伝学的研究では、差動細胞骨格の組織が ​​樹枝形状4,9-11の形態学的相違の根底にあることを示唆している。我々は、堅牢な免疫標識法を提供するDA感覚ニューロン樹状突起（図1、2、動画1） の in vivo微小管の組織で SSAY。このプロトコルは、最初の幼虫の解剖と免疫染色、アクティブな感覚ニューロンの樹状突起の伸長と分岐組織は12,13を発生している段階を示しています。 感覚ニューロンを染色することに加えて、この方法では、堅牢な筋肉の微小管の組織のラベリング（作品2本、3）、気管（図3、ムービー3）、およびその他の体壁の組織を実現しています。それは、メカニズムをコントロールする組織および細胞の形状を調査するときに体壁のin situで微小管の組織を分析することを希望する研究者のために貴重です。

Watch this Scientific Journal Video about Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall at JoVE.com

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

このようなニューロンの樹状突起のような複雑な細胞の形状は、開発中に達成されるかを理解するために、それは正確にアッセイの微小管の組織にできることが重要です。ここでは、樹状突起分枝のニューロン感覚樹状突起の微小管の組織、気管、筋肉を調べるために堅牢な免疫標識法を説明し、他の<em&gt;ショウジョウバエ</em&gt;幼虫の体壁の組織。

免疫組織学的な感覚ニューロンの樹状突起における微小管の標識、気管、との筋肉ショウジョウバエ幼虫の体壁

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval ...

immunohistological-labeling-microtubules-sensory-neuron-dendrites

Research

JoVE Journal

Neuroscience

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

12.3K ビュー.  RIKEN Brain Science Institute. このようなニューロンの樹状突起のような複雑な細胞の形状は、開発中に達成されるかを理解するために、それは正確にアッセイの微小管の組織にできることが重要です。ここでは、樹状突起分枝のニューロン感覚樹状突起の微小管の組織、気管、筋肉を調べるために堅牢な免疫標識法を説明し、他のショウジョウバエ幼虫の体壁の組織。

ビデオ: Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

無傷の感覚器官の前駆細胞の生細胞イメージングショウジョウバエ蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

神経科学

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

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

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

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

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

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

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 ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

の中枢神経系における単一細胞のラベリング<em&gt;キイロショウジョウバエ</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

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 ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

のコンピュータ追跡することによってアッセイ嗅覚行動<em&gt;ショウジョウバエ</em&gt; 4象限オルファクトメーターで

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

脳スライスと一次細胞培養における錐体ニューロンの弾道標識

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased stereological counting is the standard for quantification of these cells, but it remains a laborious and time-consuming process, which may not be feasible for all projects. Here, we describe the use of an image analysis platform, which can accurately estimate the quantity of labeled cells in a pre-defined region of interest. We describe a step-by-step protocol for this method of analysis in rat brain and demonstrate it can identify a significant reduction in tyrosine hydroxylase positive neurons due to expression of mutant &#945;-synuclein in the substantia nigra. We validated this methodology by comparing with results obtained by unbiased stereology. Taken together, this method provides a time-efficient and accurate process for detecting changes in dopaminergic neuron number, and thus is suitable for efficient determination of the effect of interventions on cell survival.

Here we present an automated method for semi-quantitative determination of dopaminergic neuron number in the rat substantia nigra pars compacta.

自動画像解析を用いたげっ歯類モデルのサブスタチングニグラにおけるドーパミン作動性ニューロン密度の半定量的決定

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased ...

ショウジョウバエの神経筋接合部および筋細胞における微小管ネットワークの表示

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of &#945;-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster,&#160;and then examining the microtubule networks in the neuromuscular junction and muscle cells, we&#160;accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila&#160;melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

著者スポットライト:ショウジョウバエの神経筋接合部における微小管ネットワークの理解 

Here, we present a detailed protocol to visualize the microtubule networks in neuromuscular junctions and muscle cells. Combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule-dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.