著者は、WSNに授与セントルイス大学の学長の研究基金を承認したいと思います

1。人口ケージのメンテナンス<ol><li> 12時間の明暗サイクルで25℃、人口のケージを維持する。限り、対照群と実験群は、同一の照明条件にさらされているように、この技術は、標準的な実験室の設定で行うことができる。 </li><li>女性はリンゴジュース寒天プレート上で一晩卵を産むことができます。 </li><li> 24時間25℃で、新たに堆積した卵でプレートを維持することにより、新たに孵化した幼虫を収集します。孵化した幼虫を引き付けるために、プレートの中央に酵母の小さなほんの少し加え配置します。 </li><li>リンゴジュースプレートの中央に酵母ペーストに移行した1 回目の幼虫を収集します。新鮮なリンゴジュースプレートに転送する金属ヘラを使用してください。付加的な酵母ペーストはアッセイを実施するまで十分な食料があることを確認するために添加することができる。グレープジュースプレートは、リンゴジュース板の代わりに使用することができる。 </li><li>後半2 番目の早第3齢Lを得る1 STは 40〜48時間熟成させることによって、齢arvae。この範囲内での供給率は、13の定数である。各幼虫の脱皮と、この構造体の明確な変化があるように、年齢は、口のフックを調べることにより確認することができる。 </li><li>後半2 番目の早第3齢幼虫を静かにかつ広範囲に水とリンゴジュースのプレートを洗浄し、メッシュフィルターに幼虫を収集することにより、行動分析のために収集されます。幼虫は、その後、寒天プレートに移す。 </li><li>すべての分析は、対照および実験動物を用いて並行して行われる。 </li></ol> 2。行動パラダイム - ロコモーション<ol><li> 100mmの組織培養皿の中で2％寒天基板上に単一の第3齢幼虫を置き、幼虫を30秒間順応することを可能にする。幼虫は、寒天、基板の表面全体に自分の体を推進するために彼らの咽頭骨片（口フック）を使用します。これは、別のプレート上で行われますので、私tは、動物、酵母とほぼ同じ色であるように酵母溶液に本体の収縮を可視化することはより困難である。 </li><li> 1分の間、基板の上に前方運動に各後部を観察し、記録します。 N =各遺伝子型のための20。これ以上の10以上の動物がプレート当たり、アッセイされるべきである。 </li></ol> 3。行動パラダイム - 給餌<ol><li>鈍イノックス＃5ピンセットを使用して、慎重に2パーセント活性化し、パン酵母溶液5mlを重層した寒天で満たされたプレートの中央に運動寒天プレートから第3齢幼虫を移す。酵母は時間をかけて定着するように溶液が均一であることを確認してください。際に酵母液に、幼虫は主に行動の観察が容易に場所や餌のままになります。口のフック収縮の速度を直接14を摂取した食物の量と相関する。 </li><li>幼虫は、30秒間順応することができます。 </li><li>の数を観察し、記録する1分の間、口のフックの収縮。 N =各遺伝子型のための20。 </li></ol> 4。幼虫ガットの解剖<ol><li> 3ウェルのスポットガラス1Xリン酸緩衝生理食塩水（PBS）で所定の位置に4％のEMグレードホルムアルデヒド固定液を作る。 </li><li>慎重に前胃がそのまま残されていることを確認するたびに作り、1×PBS溶液を使用した3ウェルガラス皿に遅い第3齢幼虫の根性をさまよううち解剖。 1鉗子で、後端を保持し、相互に、口のフックを保持します。後端不動を押さえながら、ゆっくりと根性にアクセスするために口のフックを引っ張る。関連する組織（唾液腺、脳、脂肪体など ） を削除してから、ホルムアルデヒド修正を含む3ウェルディッシュに各腸を転送します。 第3齢幼虫を徘徊しているため、開発のこの段階で、幼虫はpupariationの準備のために供給することを中止使用されており、前胃は酵母のクリアされます。 </li> &lt;李&gt;不透明な組織培養ボックス中で一晩4℃で根性をインキュベートする。 </li><li>井戸からのホルムアルデヒド固定液を除去する前に、胃の盲腸を削除し、突起がはっきりと支障なしで見ることができるように、前胃から〜150μmの2中腸をクリップ。 </li><li>井戸からのホルムアルデヒドの修正を削除し、1X PBT（1X PBS、0.1％のプロテアーゼフリーウシ血清アルブミン、0.1％トリトンX-100）の緩衝液と交換してください。徹底的1X PBT中で10分間勇気を洗う6X。回の洗浄をしている間、機械的回転装置に組織サンプルを置きます。 </li><li>セロトニンシグナリングを強化するために10 -6 Mの5-HTで1時間、4℃でインキュベートする。徹底的1X PBT中で10分間勇気を洗う6X。以前の研究は、外因性5-HTのこの濃度は、免疫組織化学分析におけるニューロンアーキテクチャまたは静脈瘤密度に影響を与えないことを実証し、単に雑音比15-16に信号を増強している。 </li><li> 4でインキュベートする＆＃176;で一晩抗セロトニン一次抗体（マウスまたはウサギで産生されたポリクローナル育ちモノクローナル）であった。徹底的1X PBT中で10分間勇気を洗う6X。回の洗浄をしている間、機械的回転装置に組織サンプルを置きます。 </li><li> （; 1:400希釈のAlexaフルーア568ヤギ抗マウスまたは抗ウサギIgG）を二次抗体での90分間4℃でインキュベートする。徹底的1X PBT中で10分間勇気を洗う6X。回の洗浄をしている間、機械的回転装置に組織サンプルを置きます。 </li><li> 4％n-プロピルgallate/20 10mMの炭酸ナトリウムでマウントし、蛍光の下で表示して、機械的な回転装置上で10分間4 mMの炭酸ナトリウムでインキュベートする。炭酸ナトリウムを封入媒体に使用される緩衝液およびpHにサンプルを変換するために使用される。 </li><li>分析のための400倍の倍率で免疫染色組織サンプルの画像をキャプチャします。 </li></ol> 5。神経回路の解析<ol><li>神経繊維は、静脈瘤の（数とサイズを定量化したと分岐度）NeuroleucidaとNeuroexplorerを使用。しかしながら、これは、手動で達成または簡易神経トレーサ（オンラインでダウンロードすることができる無料のアプリケーション）を使用してすることができる。 </li><li>前胃に脳からの個々の繊維投影をトレースし、静脈瘤番号、支店および単位長さ当たりの大静脈瘤の数を定量化する。脳から突出する軸索繊維がrecurrens神経にバンドルし、彼らは分離前胃と束生のに到達するまで、分析のためにアクセスできません。 </li></ol>

<ol>
	<li>Weiss, E., Maness, P., Lauder, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+do+neurotransmitters+act+like+growth+factors?.">Why do neurotransmitters act like growth factors?.</a> Perspect Dev Neurobiol. 5, 323-335 .</li><li>Herlenius, E., Lagercrantz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotransmitters+and+neuromodulators+during+early+human+development.">Neurotransmitters and neuromodulators during early human development.</a> Early Hum. Dev. 65, 21-37 .</li><li>Budnik, V., Wu, C., White, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+branching+of+serotonin-containing+neurons+in+Drosophila+mutants+unable+to+synthesize+serotonin+and+dopamine.">Altered branching of serotonin-containing neurons in Drosophila mutants unable to synthesize serotonin and dopamine.</a> J. Neurosci. 9, 2866-2877 (1989).</li><li>Sodhi, M., Sanders-Bush, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+and+brain+development.">Serotonin and brain development.</a> International Review of Neurobiol. 59, 111-174 (2004).</li><li>Goldberg, J., Kater, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+function+of+the+neurotransmitter+serotonin+during+development+of+the+Helisoma+nervous+system.">Expression and function of the neurotransmitter serotonin during development of the Helisoma nervous system.</a> Dev. Biol. 131, 483-495 (1989).</li><li>Goldberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+regulation+of+neurite+outgrowth+in+identified+neurons+from+mature+and+embryonic+Helisoma+triyolvis.">Serotonin regulation of neurite outgrowth in identified neurons from mature and embryonic Helisoma triyolvis.</a> Perspect Dev Neurobiol. 5, 373-387 (1998).</li><li>Haydon, P., McCobb, P., Kater, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+selectively+inhibits+growth+cone+motility+and+synaptogenesis+of+specific+identified+neurons.">Serotonin selectively inhibits growth cone motility and synaptogenesis of specific identified neurons.</a> Sci. 226, 561-564 (1984).</li><li>Neckameyer, W. 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+trophic+role+for+serotonin+in+the+development+of+a+simple+feeding+circuit.">A trophic role for serotonin in the development of a simple feeding circuit.</a> Dev. Neurosci. 32, 217-237 .</li><li>De Vry, J., Schreiber, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+selected+serotonin+5-HT+1+and+5-HT+2+receptor+agonists+on+feeding+behavior:+possible+mechanisms+of+action.">Effects of selected serotonin 5-HT 1 and 5-HT 2 receptor agonists on feeding behavior: possible mechanisms of action.</a> Neurosci. Biobehav. Rev. 24, 341-353 (2000).</li><li>Schoofs, A., Niederegger, S., van Ooyen, A., Heinzel, H., Spie&#223;, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+brain+can+eat:+Establishing+the+existence+of+a+central+pattern+generator+for+feeding+in+third+instar+larvae+of+Drosophila+virilis+and+Drosophila+melanogaster.">The brain can eat: Establishing the existence of a central pattern generator for feeding in third instar larvae of Drosophila virilis and Drosophila melanogaster.</a> J. Insect Physiol. 56, 695-705 (2010).</li><li>Spie&#223;, R., Schoofs, A., Heinzel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+the+stomatogastric+nervous+system+associated+with+the+foregut+in+Drosophila+melanogaster+and+Calliphora+vicin+third+instar+larvae.">Anatomy of the stomatogastric nervous system associated with the foregut in Drosophila melanogaster and Calliphora vicin third instar larvae.</a> J. Morphol. 269, 272-282 (2008).</li><li>Neckameyer, W. S., Bhatt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophic+actions+of+dopamine+on+the+development+of+a+serotonergic+feeding+circuit+in+Drosophila+melanogaster.">Neurotrophic actions of dopamine on the development of a serotonergic feeding circuit in Drosophila melanogaster.</a> Biomed Cent NeuroSci. 13, 26 (2012).</li><li>Sewall, D., Burnet, B., Connolly, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+larval+feeding+behavior+in+Drosophila+melanogaste.">Genetic analysis of larval feeding behavior in Drosophila melanogaste.</a> Genet. Res. 24, 163-173 (1975).</li><li>Joshi, A., Mueller, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+higher+feeding+rate+in+Drosophila+due+to+density-dependent+natural+selection.">Evolution of higher feeding rate in Drosophila due to density-dependent natural selection.</a> Evolution. 42, 1090-1093 (1988).</li><li>Budnik, V., Wu, C., White, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+branching+of+serotonin-containing+neurons+in+Drosophila+mutants+unable+to+synthesize+serotonin+and+dopamine.">Altered branching of serotonin-containing neurons in Drosophila mutants unable to synthesize serotonin and dopamine.</a> J. Neurosci. 9, 2866-2877 (1989).</li><li>Sykes, P., Condron, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+sensitivity+to+serotonin+of+Drosophila+varicosities+in+the+central+nervous+system.">Development and sensitivity to serotonin of Drosophila varicosities in the central nervous system.</a> Dev. Biol. 286, 207-216 (2005).</li><li>Garrity, P. A., Goodman, M. B., Samuel, A. D., Sengupta, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Running+hot+and+cold:+behavioral+strategies,+neural+circuits,+and+the+molecular+machinery+for+thermotaxis+inC.+elegansand+Drosophila.">Running hot and cold: behavioral strategies, neural circuits, and the molecular machinery for thermotaxis inC. elegansand Drosophila.</a> Genes Dev. 24, 2365-2382 (2010).</li><li>McKemy, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+sensing+across+species.">Temperature sensing across species.</a> Pflugers Archives. 454, 777-791 (2007).</li></ol>

我々は、開示することは何もありません。

後期胚形成時に発生するセロトニン作動口と胃の回路の異常な発展は、その成熟の機能に影響を与えます。神経突起のアーキテクチャの変更は、腸（酵母を溶液中に口のフック収縮によって測定される）速度を供給される回路の機能出力（図1）と相関させることができる支配する。 ショウジョウバエにおけるUAS-Gal4の二部系を用いることにより、特異的に特定の組織に与?...

ショウジョウバエは、急激な世代時間、低実験コスト、遺伝的要因と環境要因を操作し、制御する能力に神経回路の発達を研究するための非常に強力なモデル系である。神経発生、神経の経路検索やシナプス形成は、ヒトとショウジョウバエの間で保存されるため、作成、維持し、神経回路を変更することでのメカニズムも ​​同様に保存されている。 例えば、セロトニン（5 -ヒドロキシトリプタミンまたは5-HT）のような古典的な神経伝達物質は、成熟した神経回路1-3のシグナル伝達分子としての役割を採用する前に、成長因子として機能することができる 以前の研究は、成熟ニューロン4の接続性を改変胚形成の間に5-HTのレベルを摂動が示されている。その他、培養Helisomaニューロンへの5-HTの異所性アプリケーションが神経突起伸長だけでなく、シナプス形成5-7を抑制することが示されている。 Dではrosophilaは、発達5-HTレベルは逆CNS 8から前腸内に突出した神経突起の長さに沿って、静脈瘤の数およびサイズ、ならびにaborizationの程度に関連している。 セロトニン作動性神経伝達は、 ショウジョウバエ 8-9を含む、多様な種における摂食行動を調節することが示されている。 ショウジョウバエの給電回路は、前腸から脳への軸索突起の発達における変化と機能的出力（摂食）を相関させるためのモデルとして使用することができる比較的簡単な回路である。 Schoofs ら 。 ショウジョウバエ幼虫の摂食を筋肉組織10に影響を与える中枢パターン発生器によって調節されることが示されている。特定の筋肉の解剖学が完全に理解されていないが、それは触角神経、上顎神経、および前胸腺副神経はに関与して筋肉の目標に責任があることが示されている摂食行動。無脊椎動物の供給の筋肉と神経解剖学を含むほとんどのデータはオオクロバエ幼虫に制限されています。 二齢幼虫の供給速度は、咽頭骨片（口フック）の後退によって評価され、再現性があり、高スループットであることができます。咽頭プレートは前頭神経を介して中央5-HTニューロンからの繊維によって神経支配されています。前胃、または腸は、腸内束生のと（ 図1）11 月12日前腸の収縮に関与しているセロトニン作動性線維（神経をrecurrens）によって支配されている。軸索分岐の変化、神経突起の長さに沿った静脈瘤の数及び大きさは、免疫組織化学法を用いて定量することができる。直接的または間接的に、開発中に5-HTニューロンを操作する、morpholoの変化で評価し、相関させることができるこの給電回路の機能的出力を変更することができ神経突起アーキテクチャのGY。

<table border="0" cellpadding="0" cellspacing="0" width="758">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:217px;">Eclipse E-800 Microscope</td>
			<td style="width:175px;">Nikon Instruments</td>
			<td style="width:115px;"></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:217px;">Neuroleucida</td>
			<td style="width:175px;">MBF Biosciences</td>
			<td style="width:115px;">NL-15</td>
			<td style="width:251px;">Used to analyze gut fiber architecture, not necessary to have</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:217px;">Northern Eclipse</td>
			<td style="width:175px;">Empix Inc</td>
			<td style="width:115px;"></td>
			<td style="width:251px;">Imaging software</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:217px;">G-2E/C TRITC EX 528-553</td>
			<td style="width:175px;">Nikon Instruments</td>
			<td style="width:115px;">96312</td>
			<td style="width:251px;">Filter for specific secondary antibody</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:217px;">N.A. 0.75; W.D. 0.72 mm; DIC Prism: 40xI, 40x I-C; Spring loaded</td>
			<td style="width:175px;">Nikon Instruments</td>
			<td style="width:115px;">MRH00400</td>
			<td style="width:251px;">Objective used for imaging</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:217px;">Simple Neurite Tracer</td>
			<td style="width:175px;">NIH Image J</td>
			<td style="width:115px;"></td>
			<td style="width:251px;"><a href="http://fiji.sc/Simple_Neurite_Tracer">http://fiji.sc/Simple_Neurite_Tracer</a></td>
		</tr>
	</tbody>
</table>

functional analysis of the larval feeding circuit in drosophila

キイロショウジョウバエの幼虫におけるセロトニン給電回路は、回路の開発時に非常に重要でのニューロンの基板を調査するために使用することができる。回路の機能出力を用いて、供給は、口と胃の神経系のアーキテクチャの変更を可視化することができる。摂食行動は、脳からの神経支配を受ける口のフックの後退速度を観察することによって記録することができる。幼虫は寒天基板全体横断する彼らの口のフックを使用しているため、運動の様子は、供給のための生理学的対照として使用する。摂食行動の変化は、神経突起の軸索アーキテクチャと相関させることができる腸を支配する。免疫組織化学を使用すると、これらの変化を視覚化し、定量することができる。彼らは操作に非常に敏感であるように行動パラダイム中の幼虫の不適切な取り扱いは、データを変更することができます。神経突起アーキテクチャ神経支配の適切なイメージング腸は、正確な静脈瘤の数とサイズの定量と同様にブランチノードの範囲のために重要である。ほとんどの回路の解析は神経突起のアーキテクチャや行動への影響の視覚化を可能にするが、このモデルでは、1が神経アーキテクチャの障害を持つ回路の機能出力を相関させることができます。

ショウジョウバエの幼虫におけるセロトニン作動性供給回路は、神経系の発達上の特定の要因の影響を観察するには極めて有効なモデルとして機能することができます。供給速度を定量することにより、その機能の出力（図1）と給電回路の軸索アーキテクチャをリンクすることができる。幼虫は、寒天の表面を横切ってそれ自体を推進するために、口のフックを使用する...

Watch this Scientific Journal Video about Functional Analysis of the Larval Feeding Circuit in Drosophila at JoVE.com

Functional Analysis of the Larval Feeding Circuit in Drosophila

キイロショウジョウバエの幼虫での給電回路は、供給速度の変化は口と胃の神経回路の変化と相関することを可能にする、シンプルでありながら強力なモデルを提供しています。この回路は、口のフックに予測だけでなく、腸を送信中枢セロトニン作動性神経細胞で構成されている。

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 ...

functional-analysis-of-the-larval-feeding-circuit-in-drosophila

Research

JoVE Journal

Neuroscience

Functional Analysis of the Larval Feeding Circuit in Drosophila

10.2K ビュー.  Saint Louis University School of Medicine.  キイロショウジョウバエの幼虫での給電回路は、供給速度の変化は口と胃の神経回路の変化と相関することを可能にする、シンプルでありながら強力なモデルを提供しています。この回路は、口のフックに予測だけでなく、腸を送信中枢セロトニン作動性神経細胞で構成されている。 

ビデオ: Functional Analysis of the Larval Feeding Circuit in Drosophila

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of its convenient genetics and fully sequenced genome. Additionally, the larval nervous system is an ideal model system to study mechanisms of axonal transport as the larval segmental nerves contain bundles of axons with their cell bodies located within the brain and their nerve terminals ending along the length of the body. Here we describe the procedure for visualization of synaptic vesicle proteins within larval segmental nerves. If done correctly, all components of the nervous system, along with associated tissues such as muscles and NMJs, remain intact, undamaged, and ready to be visualized. 3rd instar larvae carrying various mutations are dissected, fixed, incubated with synaptic vesicle antibodies, visualized and compared to wild type larvae. This procedure can be adapted for several different synaptic or neuronal antibodies and changes in the distribution of a variety of proteins can be easily observed within larval segmental nerves.

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster larvae provide an ideal model system to investigate the mechanisms of axonal transport within larval segmental nerves. Using this procedure, 3rd instar larvae carrying various mutations can be compared to wild type larvae.

3の幼虫分節神経の可視化 RD齢ショウジョウバエ幼虫準備

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of ...

神経科学

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 ...

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 ...

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 2. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 3 4. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes5: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s 6 7 8.
Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc9 and initiates the assembly of undifferentiated cells into ommatidia10-11. The 'founder cell' R8 is specified first and recruits R1-6 and then R7 12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes 15, including rhabdomere formation, synaptogenesis and eventually rh expression.
In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.

幼虫、蛹および成人の解剖と免疫組織化学<em&gt;ショウジョウバエ</em&gt;網膜

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

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 ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

のライブイメージング<em&gt;ショウジョウバエ</em&gt;幼虫の神経芽細胞

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

なぜ定量事項：での表現型のキャラクタリゼーション<em&gt;ショウジョウバエ</em&gt;幼虫の神経筋接合部

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

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 ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

ショウジョウバエ幼虫神経筋接合部のシナプス電流の焦点 Macropatch 録音

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...