著者らは、資金調達のために理研に感謝。我々はまた、遺伝的および免疫組織化学プロトコルに関する議論のためにCagri Yalgin、キャロラインDelandre、とジェイパリッシュに感謝。

試薬の1.Preparation <ol><li>のCa + + -フリーHL3.1生理食塩水28を準備する。 <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は7.2。滅菌濾過し、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>ポリ- L -リジン（PLL）カバースリップを行います。 <ol><li> -20℃で4.2ミリリットルの水に100mgのPLLを溶解し、エッペンドルフチュー​​ブや凍結で300μlアリコートを作る</li><li>コーティングのカバースリップは、最初の雪解けアリコートを、のddH 2 Oで10ミリリットルにそれを起動してコダックフォト-フローの20μlのを追加する前に、最終的なPLL濃度は0.7 mg / mlのです。 </li><li> PLL溶液中で30分のための水没のカバースリップは、削除して乾燥させ、その後水と乾燥で簡単にすすいでください。二回繰り返します。治療をカバースリップ最後の約1ヶ月。 </li></ol></li></ol> 2。遺伝的交雑<ol><li> ToはMARCMのクローンを生成する。ここでパン- DAのドライバ11,27を使用して例のクロスは、次のとおりです。 FRT2A X hsFLP、Gal4の109（2）80、UAS - mCD8：：GFP、浴槽- Gal80 FRT2A/SM5-TM6B </li><li> FLPアウトのクローンを生成する 。ここにクラスIV固有のドライバ8を使用して例のクロスは、次のとおりです。 PPK - Gal4のX YW、hsFLP、UAS - FRT - CD2、Y +ワンストップFRT - mCD8：：GFP </li></ol> 3。胚のコレクション<ol><li> 25℃コレクションの瓶の中にショウジョウバエの交配を維持° Cと酵母ペースト30,31の薄い層でリンゴジュース寒天プレート29スプレッドに胚を収集する。 </li></ol> 4。胚の熱ショックの治療<ol><li>胚が敷設されている時にりんごジュースの寒天プレートに反対して空の皿を置きます。パラフィルム（図2）これらの二つのプレートの周囲に封をします。 </li><li>熱ショック時に、水没の目eの水浴中でプレートとは、金属の重量を使用して、それを押したままにします。 </li><li> MARCMの胚の<ol><li> 2Hのための胚を収集し、囲まれた10センチメートルペトリ皿で​​培養するには、追加の2時間25℃で組織を湿らせた。 注：熱ショックプロトコルを調整するには、クローンが生成される頻度を変更します。 </li><li>単一の孤立したニューロンを目指すクローンの数が少なく、、38で1時間、水浴で沈めると熱ショック℃での</li><li>クローンの数が多いほど、38〜45分の熱ショック° Cの場合は、追加の30分のためにもう一度、RT 30分で熱ショックを回復する。 </li></ol></li><li> 38で1HのためにFLPアウト24時間のための胚、および熱ショックを収集℃に</li></ol> 5。クローンのスクリーニング<ol><li>熱ショック後は、防水配置と場所湿らせたティッシュに囲まれた10cmのペトリ皿にリンゴジュース寒天プレートの蓋を取り外します。文化ワンダまでの胚および25℃で、その後の幼虫° CING第3齢幼虫。 注：特に培養条件、栄養は、DA樹状アーバ形態32,33を変更することが示されている。成長する幼虫は常に酵母ペーストへのアクセス権を持っていることを確認してください。必要に応じて貼り付け、酵母を監視し、補充する。 </li><li>プロトコルの以降で、この点から、ゆっくりと昆虫鉗子を使用して幼虫を操作。 </li><li>簡単に言うと、ゆっくりと水道水の幼虫をリンスし、寒天プレート上に置きます。 注：このステップは、バックグラウンド幼虫の表面上に食べ物や汚れから自家蛍光を減少させる。 </li><li>強力な蛍光解剖顕微鏡下で幼虫を調べます。体壁のGFP陽性ニューロン/細胞と幼虫を識別します。 </li></ol> （6）樹状突起のin vivoイメージング <ol><li> 80％グリセロールの小滴とうつ病のスライドグラスに幼虫を置きます。幼虫を固定するためにスライドにカバースリップを配置。空気が幼虫とcoversliの間に残っていないことを確認してください頁</li><li>静かに関心のニューロンの可視化を可能にする幼虫をロールバックするためにカバースリップを押してください。イメージmCD8：：共焦点顕微鏡を介してGFPで標識された樹状アーバー。 </li></ol> 7。幼虫の解剖 開始する前に注意してください：DAニューロンの樹状突起は、解剖の開始後に急激に低下する。良好な樹状突起の形態を確保するために以下の5分で、個々の幼虫を解剖。 <ol><li>次の変更で以前に34に記載のようにSylgard板の上に第3齢幼虫をさまよう解剖： <ol><li> DAニューロン軸索末端のイメージングのための、CNSが損なわれていないと分節神経が壊れていないことを確認してください。幼虫を開いた後、最初に慎重に体壁に気管の接続を切断してから、ゆっくりと全体の腸を取り除く。 </li><li>単独でのイメージング樹状突起の場合、平坦に取り付け可能にするためにCNSを取り除く。 </li></ol></li></ol> 8。 Fixat幼虫のフィレットのイオンとブロッキング<ol><li>それでもSylgardプレートに固定幼虫で、そっと回転シェーカー上で室温で20分のためにPBS中4％PFAで固定して下さい。 </li><li>固定した後、幼虫組織の痕跡を（脂肪体、気管など）を取り外します。 </li><li> Sylgardプレートとシェーカー上で3倍の10分（0.1％トリトンX - 100を含むPBS）をPBSTで洗浄する。軸索の末端を調べる場合、Sylgardのプレート上にフィレットを保持します。 （染色の樹状突起の場合には、このステップ34で、0.5 mlチューブに幼虫のフィレットを転送することが可能です） </li><li>シェーカーでPBSTで5％正常ロバ血清（NDS）で、室温で20分のための幼虫をブロックする。 </li></ol> 9。幼虫のフィレットの染色<ol><li>一次抗体のブロッキング溶液とインキュベートすることにより囲まれた小さなタッパーウェアの容器に° Cがティッシュを湿らせ一晩4（NDS / PBST 5％）を取り外します。 </li><li> 4℃からチューブを外し、室温でさらに1時間インキュベートする。 </li><li> PBSTで6倍の10分を洗ってください。二次antibを追加odyは、（5％でNDS / PBST）、およびカバーは、フルオロフォアの光退色34を防止する。 </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、室温で一時間が続く。 </li><li> PBSTで幼虫6倍の10分を洗浄し、取り付けに進みます。 </li></ol> 10。樹状突起の検査のために幼虫のフィレットの取付け<ol><li>解剖ハサミや頭部（口のフックを含む）、および34（気門を含めて）後部を切断する外科用メスを用いて可能な限り平坦各幼虫のフィレットをマウントします。 </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、そして&quot;速い&quot;マウント34に対してマニキュアとカバーの両側をシール。 </li><li>永久的な取付けと鮮明な画像のための<ol><li> PLLのカバースリップ上でPBSのドロップに解剖幼虫のフィレット筋肉側を下に置く、それはすぐにカバースリップを遵守します。 </li><li> howeve、実装後できるだけ大量の液体を削除します。rはそれが完全に乾燥することはできません。 </li><li>エタノール系列を通してカバースリップを（添付の幼生フィレット付き）を取る：35％エタノール、50％、70％、95％、続いて、最後に10分間ごとに100％エタノールの2倍（2番目の100パーセントEtOH溶液を頻繁に変更する必要があります。 ）、最終的に、キシレンの2〜3倍に10分を洗浄する。 </li><li>洗浄したスライド上でDPX封入（Distyreneの可塑剤をキシレン ）のドロップを入れて、慎重に上にフィレットサイドダウンカバースリップを置く。暗所で保管し、イメージングの前に設定してDPXに1日待つ。 </li></ol></li></ol> 11。軸索末端の検査のために幼虫のフィレットの取付け<ol><li>分節性神経は解剖、固定時にそのままPNSとCNSとの間で実行されている、と末梢感覚神経の細胞体からVNCへの軸索のトレースを可能にするために染色してください。 </li><li>うつ病のスライドの80％グリセロール中でそれぞれの幼虫のフィレットキューティクル側を下にマウントします。 DAの細胞体から軸索へのトレース共焦点顕微鏡下でVNC。注：VNCの同族セグメントにそれぞれ体壁のセグメントのプロジェクトでPNSニューロン。 </li></ol> 12。代表的な結果： 代表的な結果は、図3-5に示します。図。図3は、共焦点顕微鏡下で生体内でキャプチャされたクラスIVダニューロン、全体のアーバーを示しています。図。 4は正しく形態を維持するために修正されている免疫組織化学的に標識されたクラスIIIのニューロンの樹状突起の部分のクローズアップを示しています。関連するはめ込みショー失敗解剖と固定（抗GFP抗体で染色の両方）の後に発生する可能性が低下。図。図5にCNS内の単一のクラスIV軸索の末端（抗GFP、緑色）、すべてのクラスIVの末端は、抗CD2（マゼンタ）と共染色される。 <img alt="図1" src="/files/ftp_upload/3111/3111fig1.jpg" /> 1プロトコルの概要を示します 。 A）を収集し、熱ショック胚。 B）<html> GFP陽性DAニューロンのクローンと幼虫を選択し、[画像のライブ幼虫で樹状突起をGFPで標識した。 c）の幼虫を摘出し、免疫組織化学的にフィレットを染色。 D）染色したフィレットをマウントし、イメージ樹あずまやDAニューロンのクローンの軸索投射を。 <img alt="図2" src="/files/ftp_upload/3111/3111fig2.jpg" /> 熱ショック用リンゴジュース寒天プレートの2】図 。プレートを取る（白い矢印、AB）、（赤の矢印B）上に別​​のペトリ皿を追加し、パラフィルム（青矢印、B）の周りシール。 <img alt="図3" src="/files/ftp_upload/3111/3111fig3.jpg" /> mCD8の樹状突起の in vivo画像で 図3：：第3齢幼虫のクラスIV（v&#39;ada）ニューロンを示すGFP標識MARCMのクローン（2.1のように遺伝子型）。スケールバーは50μmのです。 d/3111/3111fig4.jpg&quot;/&gt; 図4 mCD8：：成功郭清と固定（緑）の後に良好な形態を示す、抗GFP抗体で染色されたクラスIIIニューロン（ddaA）のGFP標識MARCMのクローン。失敗した解剖と固定（マゼンタ）の後に発生する挿入図の樹状突起の劣化（白矢印）。スケールバーは25μmのです。 <img alt="図5" src="/files/ftp_upload/3111/3111fig5.jpg" /> 図5第3齢幼虫幼虫VNC。単一のクラスVI（VDAB）軸索の末端（2.2のような遺伝子型）のFLPアウトクローンを抗GFP抗体（緑）を使用して検出される。抗CD2は、すべてのクラスIV末端（マゼンタ）のラベルを付けます。スケールバーは25μmのです。

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projections+of+Drosophila+multidendritic+neurons+in+the+central+nervous+system:+links+with+peripheral+dendrite+morphology.">Projections of Drosophila multidendritic neurons in the central nervous system: links with peripheral dendrite morphology.</a> Development. 134, 55-64 (2007).</li><li>Merritt, D. J., Whitington, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+projections+of+sensory+neurons+in+the+Drosophila+embryo+correlate+with+sensory+modality,+soma+position,+and+proneural+gene+function.">Central projections of sensory neurons in the Drosophila embryo correlate with sensory modality, soma position, and proneural gene function.</a> J Neurosci. 15, 1755-1767 (1995).</li><li>Blair, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+mosaic+techniques+for+studying+Drosophila+development.">Genetic mosaic techniques for studying Drosophila development.</a> Development. 130, 5065-5072 (2003).</li><li>Lee, T., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+analysis+with+a+repressible+cell+marker+for+studies+of+gene+function+in+neuronal+morphogenesis.">Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis.</a> Neuron. 22, 451-461 (1999).</li><li>Wong, A. M., Wang, J. W., Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+representation+of+the+glomerular+map+in+the+Drosophila+protocerebrum.">Spatial representation of the glomerular map in the Drosophila protocerebrum.</a> Cell. 109, 229-241 (2002).</li><li>Shimono, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidendritic+sensory+neurons+in+the+adult+Drosophila+abdomen:+origins,+dendritic+morphology,+and+segment-+and+age-dependent+programmed+cell+death.">Multidendritic sensory neurons in the adult Drosophila abdomen: origins, dendritic morphology, and segment- and age-dependent programmed cell death.</a> Neural Dev. 4, 37-37 (2009).</li><li>Feng, Y., Ueda, A., 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=A+modified+minimal+hemolymph-like+solution,+HL3.1,+for+physiological+recordings+at+the+neuromuscular+junctions+of+normal+and+mutant+Drosophila+larvae.">A modified minimal hemolymph-like solution, HL3.1, for physiological recordings at the neuromuscular junctions of normal and mutant Drosophila larvae.</a> J Neurogenet. 18, 377-402 (2004).</li><li>Sullivan, W., Ashburner, M., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila Protocols. , (2000).</li><li>Kaczynski, T. J., Gunawardena, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+the+Embryonic+Nervous+System+in+Whole-mount+Drosophila+Embryos.">Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos.</a> J. Vis. Exp. (46), e2150-e2150 (2010).</li><li>Featherstone, D. E., Chen, K., Broadie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harvesting+and+preparing+Drosophila+embryos+for+electrophysiological+recording+and+other+procedures.">Harvesting and preparing Drosophila embryos for electrophysiological recording and other procedures.</a> J Vis Exp. , (2009).</li><li>Medina, P. M., Swick, L. L., Andersen, R., Blalock, Z., Brenman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+forward+genetic+screen+for+identifying+mutations+affecting+larval+neuronal+dendrite+development+in+Drosophila+melanogaster.">A novel forward genetic screen for identifying mutations affecting larval neuronal dendrite development in Drosophila melanogaster.</a> Genetics. 172, 2325-2335 (2006).</li><li>Mirouse, V., Swick, L. L., Kazgan, N., St Johnston, D., Brenman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LKB1+and+AMPK+maintain+epithelial+cell+polarity+under+energetic+stress.">LKB1 and AMPK maintain epithelial cell polarity under energetic stress.</a> J Cell Biol. 177, 387-392 (2007).</li><li>Brent, J., Werner, K., 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+Immunohistochemistry.">Drosophila Larval NMJ Immunohistochemistry.</a> J. Vis. Exp. 25, e1108-e1108 (2009).</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>Sugimura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+developmental+modes+and+lesion-induced+reactions+of+dendrites+of+two+classes+of+Drosophila+sensory+neurons.">Distinct developmental modes and lesion-induced reactions of dendrites of two classes of Drosophila sensory neurons.</a> J Neurosci. 23, 3752-3760 (2003).</li><li>Zito, K., Parnas, D., Fetter, R. D., Isacoff, E. Y., Goodman, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Watching+a+synapse+grow:+noninvasive+confocal+imaging+of+synaptic+growth+in+Drosophila.">Watching a synapse grow: noninvasive confocal imaging of synaptic growth in Drosophila.</a> Neuron. 22, 719-729 (1999).</li><li>Landgraf, M., Sanchez-Soriano, N., Technau, G. M., Urban, J., Prokop, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charting+the+Drosophila+neuropile:+a+strategy+for+the+standardised+characterisation+of+genetically+amenable+neurites.">Charting the Drosophila neuropile: a strategy for the standardised characterisation of genetically amenable neurites.</a> Dev Biol. 260, 207-225 (2003).</li></ol>

利害の衝突は宣言されません。

ショウジョウバエ幼虫のDAニューロンモデルは、制御のニューロンの形態と回路形成そのメカニズムを調べる一つの優れた遺伝システムを提供します。 MARCMは、一般的に標識のためにと体DAニューロンのクローンを生成するために使用されます。 MARCMのために私達はどちらか汎神経（例えば、Gal4の C155）またはDAニューロン固有のドライバを使用してください。汎?...

<table border="1"><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（省略可能） </th></tr><tr><td> SZX16蛍光解剖顕微鏡（GFPHQフィルター付） </td><td>オリンポス</td><td> SZX16 </td><td></td></tr><tr><td>昆虫鉗子を生きる</td><td> FST </td><td> 26030〜10 </td><td></td></tr><tr><td> 26ミリメートルXの76ミリメートルうつ病のスライドガラス</td><td> Toshinriko（株） </td><td> T8 - R004 </td><td></td></tr><tr><td> Sylgard 184（またはSilpot 184） </td><td>ダウコーニング</td><td> 3097358-1004 </td><td></td></tr><tr><td>ポリ- L -リジン</td><td>シグマ</td><td> P - 1524 </td><td>この製品は、最も効果的であることが証明され</td></tr><tr><td> DPX封入剤</td><td>シグマ</td><td> 44581 </td><td></td></tr><tr><td>ウサギ抗GFP </td><td>インビトロジェン</td><td> - 11122 </td><td>希釈1:500 </td></tr><tr><td>ラット抗CD8 </td><td> Caltag </td><td> 5H10 </td><td>希釈1:200 </td></tr><tr><td>マウス抗CD2 </td><td> ABD serotec </td><td> MCA443R </td><td>希釈1:700 </td></tr><tr><td>マウス抗Fasciclin2 </td><td> DSHB </td><td> 1D4 </td><td>希釈午前1時10分</td></tr></table>

morphological analysis of drosophila larval peripheral sensory neuron dendrites and axons using genetic mosaics

神経系の発達には、正確なニューロンのクラス特有の樹状突起の発達と軸索の配線が続くニューロンの位置と同一の正確な仕様が必要です。最近ショウジョウバエの幼虫の末梢神経系（PNS）の樹状分枝（DA）の感覚ニューロンは、ニューロンの分化の両方の一般とクラス固有のメカニズムを解明するための強力な遺伝的モデルとなっている。 4つの主要なDAニューロンのクラス（I - IV）1があります。彼らは、増加する樹状突起の複雑さの順に名前が付けられ、それらの分化2-10の遺伝的制御のクラス固有の相違点を持っています。 1）それが2）、DAニューロン樹状突起が広がる、ショウジョウバエで利用できる強力な遺伝学的ツールを活用することができますので、DA感覚システムは、樹枝状形態の制御11から13の背後にある分子メカニズムを調べるために実用的なモデルです。光学的にCLEの下にだけ2次元のそれは容易にin vivoでの高分解能で可視化することAR幼虫のクチクラ、3）樹枝状形態におけるクラス固有の多様性は、単純な比較高度に分岐した樹枝状の木の形成を制御する重要な要素を見つけるために比較分析を容易に、そして4）樹状あずまや固定観念別のDAニューロンの形状は、形態学的統計分析を促進する。 DAニューロンの活動は、幼虫の運動中枢パターン発生器14から16の出力を変更します。別のDAニューロンのクラスは、異なる感覚モダリティを持っており、それらの活性化は、14,16-20異なる行動反応を誘発する。さらに別のクラスには、腹側神経索のショウジョウバエ幼虫の中枢神経系（VNC）21に型通りに軸索投射を送る。これらの予測は、樹状フィールド7,22の体壁のDAニューロンの感覚モダリティと位置の両方の地形表現で終了、23。 DA軸索突起のよって検査は、地形図7,22,23、だけでなく、簡単な回路を変調幼虫の運動14から17までの配線の基礎となるメカニズムを解明するために使用することができます。 ここではMARCM（抑制性細胞マーカーを持つモザイク解析）1,10,25とFLPアウト22,26,27技術（図1に要約）を介して24マーキングDAニューロンを生成し、遺伝的モザイクを分析する実用的なガイドを提示する。

Watch this Scientific Journal Video about Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics at JoVE.com

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

の樹状分枝感覚ニューロン<em&gt;ショウジョウバエ</em&gt;幼虫の末梢神経系は、一般とニューロンの分化のニューロンクラス特有のメカニズムの両方を解明するために有用なモデルです。我々は樹状突起分枝の神経細胞遺伝学的モザイクを生成して解析するための実用的なガイドを提示する。

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 ...

morphological-analysis-drosophila-larval-peripheral-sensory-neuron

Research

JoVE Journal

Neuroscience

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

15.2K ビュー.  RIKEN Brain Science Institute. の樹状分枝感覚ニューロンショウジョウバエ幼虫の末梢神経系は、一般とニューロンの分化のニューロンクラス特有のメカニズムの両方を解明するために有用なモデルです。我々は樹状突起分枝の神経細胞遺伝学的モザイクを生成して解析するための実用的なガイドを提示する。

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

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

ショウジョウバエ幼虫分節軸索内ではシナプス小胞の in vivo可視化で

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

神経科学

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

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval body wall contains several cell types that are models to study cell and tissue morphogenesis. For example tracheae are used to examine tube morphogenesis1, and the dendritic arborization (DA) sensory neurons of the Drosophila larva have become a primary system for the elucidation of general and neuron-class-specific mechanisms of dendritic differentiation2-5 and degeneration6.
The shape of dendrite branches can vary significantly between neuron classes, and even among different branches of a single neuron7,8. Genetic studies in DA neurons suggest that differential cytoskeletal organization can underlie morphological differences in dendritic branch shape4,9-11. We provide a robust immunological labeling method to assay in vivo microtubule organization in DA sensory neuron dendrite arbor (Figures 1, 2, Movie 1). This protocol illustrates the dissection and immunostaining of first instar larva, a stage when active sensory neuron dendrite outgrowth and branching organization is occurring 12,13.
In addition to staining sensory neurons, this method achieves robust labeling of microtubule organization in muscles (Movies 2, 3), trachea (Figure 3, Movie 3), and other body wall tissues. It is valuable for investigators wishing to analyze microtubule organization in situ in the body wall when investigating mechanisms that control tissue and cell shape.

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

To understand how complex cell shapes, such as neuronal dendrites, are achieved during development, it is important to be able to accurately assay microtubule organization. Here we describe a robust immunohistological labeling method to examine microtubule organization of dendritic arborization neuron sensory dendrites, trachea, muscle, and other Drosophila larva body wall tissues.

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 ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

使用して、マウス大脳皮質および海馬における可視化と樹状突起と棘の遺伝子操作<em&gt;子宮内で</em&gt;エレクトロポレーション

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

軸索、樹状突起および脳スライスにおける個々のニューロンの樹状突起棘から電位感受性色素記録

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

幼虫給電回路の機能解析ショウジョウバエ</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during 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 ...

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.

This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.

多孔質フィルタを使用した生化学分析のための感覚ニューロンからの軸索の産生および単離

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in communication with the epidermal cells they innervate and thus their analysis benefits from in situ visualization of both neuronally and epidermally expressed proteins by immunofluorescence. Traditional methods of preparing larval fillets for immunofluorescence experiments leave intact the muscle tissue that covers most of the body wall, presenting several challenges to imaging neuronal and epidermal proteins. Here we describe a method for removing muscle tissue from Drosophila larval fillets. This protocol enables imaging of proteins that are otherwise obscured by muscle tissue, improves signal to noise ratio, and facilitates the use of super-resolution microscopy to study da neuron development.

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Studies of neuronal morphogenesis using Drosophila larval dendritic arborization (da) neurons benefit from in situ visualization of neuronal and epidermal proteins by immunofluorescence. We describe a procedure that improves immunofluorescence analysis of da neurons and surrounding epidermal cells by removing muscle tissue from the larval body wall.

の除去<em&gt;ショウジョウバエ</em感覚ニューロンおよび表皮細胞の免疫蛍光分析のための幼虫の切り身から&gt;筋肉組織

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

勉強するリニアアガロースチャネルを使用します<em&gt;ショウジョウバエ</em&gt;幼虫クロールの動作

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

キイロショウジョウバエ幼虫と大人の脳の代謝解析

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

の形態素解析ショウジョウバエ幼虫末梢感覚ニューロンの樹状突起と軸索