著者は私達の抗体を送信するためにアイオワ大学の発達研究ハイブリドーマバンクに感謝します。この作品は、イスラエル科学財団からの助成金（第29号/ 08）によってサポートされていました。 ASにもDFG（ドイツ学術振興）から研究助成金によってサポートされています。

始める前に<ol><li>希望するフライの文化を育てる。 （50mlのバイアルあたり約30ハエ）空いてバイアルを保持します。ハエは、それぞれのバイアルに一日だけのために卵を産みましょう。これは幼虫十分な食糧供給を提供し、彼らが食べ物から出クロールする前に、最大サイズに到達することができます。 3齢幼虫は、バイアルの壁にさまようし始めるまで、適切な温度でバイアルを保持します。 </li><li>生理食塩水（PBS）とPBT（0.1％PBSでTween）のリン酸緩衝新鮮な株式を準備します。氷上で10 mlのPBSを保持します。 </li><li>新鮮な固定液中1-5ミリリットル（PBTの4％ホルムアルデヒド）を準備し、それを氷上で保管してください。我々は、ストック溶液としてボトル37から38パーセントのホルムアルデヒドを使用しています。 </li></ol> 1。 3齢幼虫の解剖と固定<ol><li>バイアル壁から放浪3齢幼虫を選び、Sylgard清板（にSylgard 184シリコーンエラストマーキット、ダウコーニング社製の氷冷PBS50μlのドロップに入れてください組織培養皿）。 </li><li>微細な鉗子を（デュモン＃5鉗子）を使用して、口のフックの近くに、幼虫、背側を保持し、幼虫の脳を介して虫ピン（minutien、0.1ミリメートル、ステンレス鋼）を貼り付けます。 </li><li>ピンセットで幼虫の後端をつかみ、慎重に引き抜いて、ゆっくりと縦に幼虫を伸ばす。 2後部気門の間に別の虫ピンを貼り付けます。 </li><li>春のはさみを使用すると、前部と後部のピンに近づけ体の壁に2つの水平方向の切開を（垂直前後軸まで）、カット。 </li><li>春のはさみを使用すると、前方から後方に切開背側正中線に沿って幼虫の体壁を切った。 </li><li>微細な鉗子を使用して、内部器官（腸、唾液腺、マルピーギ管など）や気管を削除します。 PBSで一度か二度優しく洗ってください。 </li><li>それらを伸ばし、各サイドに2つの虫ピンを使用してプレートにピン、ピンセットを持つ2つの体壁フラッ​​プをつかむ。 </li><li> Pを削除します。BSと定着液（PBTの4％ホルムアルデヒド）の50μlを加える。室温で20分間インキュベートします。 </li><li>固定を外します。 PBSで2回洗浄します。虫ピンを引き抜きます。春のはさみを使用すると、長方形のフィレットを残して幼虫の頭と尾を切った。 PBSで冷却したエッペンドルフチュー​​ブに固定した組織を転送します。 </li><li>幼虫の十分な数が固定されると、一つは抗体染色に直接続けることができます。即時の染色が必要でない場合は、固定の幼虫は、95％エタノールで3回、各5分間洗浄し、そして-20℃で95％エタノールに格納する必要があります</li></ol> 2。解剖および蛹の固定パートI <ol><li> 3齢幼虫がpupariate始めるまで、幼虫の固定用としてバイアルに参加しています。バイアルにpupariatedた時間ごとやマークの幼虫を調べます。マーク蛹は29℃で24℃、または24から27時間で30から40時間を開発することができ℃の</li><li>微細な鉗子を使用すると、目の20-30蛹を選ぶ電子適切な年齢と暗い磁器マルチウェル解剖皿に入れます。興味のある組織を傷つけないように注意してください。 </li><li>蛹のケースから蛹を抽出します。蓋を剥離することから始めると蛹が解放されるまで継続する。よくPBTでいっぱいに蛹を入れてください。すべての蛹を抽出し続けています。ステップ2.4​​から2.6までは、一度に5蛹の小さなバッチで実行する必要があります。 </li><li>解剖皿のウェル間の平らな面に5蛹を置きます。ミクロ解剖ナイフを使用すると、頭部と腹部の後端（あるいは、それは蛹の両端に穴を引き裂くように鉗子の2ペアを使用することが可能である）の先端を切った。 </li><li>微細な鉗子を使ってその場で蛹を保持し、前部の開口部を通してPBTを注入することにより、蛹の内臓を洗浄するために1 mlのシリンジを使用しています。 </li><li>ウェルのPBSを充填し、氷上で保存エッペンドルフチュー​​ブに冷たい固定に移動にそれを浸漬することにより解剖蛹手短に洗います。 </li><li> 4時（ON）で一晩インキュベートします。 </li></ol>パートII <ol start="8"><li>固定液を捨て、PBTで蛹三回、各5分間に、洗ってください。氷の上で洗浄した蛹を保持します。 </li><li> PBTと解剖皿の二つの井戸を埋める。井戸のいずれかにポリエチレンパスツールピペット転送いくつかの蛹を使用します。 </li><li>第2ウェルに一度蛹を移動します。翼の透明な蛹の表皮を剥がし、完全に一致ヒントをシャープ鉗子の2ペアを使用して：鉗子をよく使用して1つのペアの底に蛹を確保し、ゆっくりと鉗子の第二のペアを使用してキューティクルを引き裂く。一度キューティクルは、その皮翼から、引き裂かれています。蛹から羽を切断しないように注意してください。ウイングブレードからキューティクルを剥離した後、翼ヒンジ（ここで、翼ChOsが配置されている）のキューティクルの剥離を続ける。 </li><li>翼キューティクルを剥離した後、人は同様の方法で足のキューティクルを剥離試みることができます。多くの足がプロセスで失われる可能性がありますが、それは非常に脚ChOsの染色を向上させるため、低収率にもかかわらず、この手順を強くお勧めします。平均棍と腹部のChOsはさらに表皮を剥離することなく可視化することができる。 </li><li>メタノールを充填したエッペンドルフチュー​​ブに &quot;剥離&quot;蛹を置き、室温で保持します。すべての蛹の表皮を剥離し続け、同じチューブに追加します。少なくとも10うまく剥離蛹は、それぞれの染色のために収集する必要があります。 </li><li>メタノールを除去し、95％エタノールで3回、各5分間に、洗ってください。固定の蛹は、-20℃で95％エタノールに長時間保つことができます</li></ol> 3。幼虫および蛹の免疫染色<ol><li>室温でPBT 3回、各30分、で固定した組織を洗浄する。幼虫は回転板上で穏やかに振盪しながら洗浄することができる。蛹は、振とうせずに洗浄される。 </li><li>ブロッキングバッファー（PBTを交換PBT + 5％、Normalヤギ血清）と4℃でインキュベート℃、 </li><li>ブロッキングバッファーを除去し、4℃でONに一次抗体（ブロッキングバッファーで希釈した）でインキュベート</li><li>ステップ3.1で説明したようにPBTで4回洗浄します。 </li><li> 4℃での二次抗体を用いたPBTとインキュベートする（ブロッキングバッファーで希釈した）を削除します。 </li><li> PBTで2回洗浄し、PBSで1回、ステップ3.1で説明します。 </li><li>マウンティング培地（Dako社製蛍光マウンティングメディウム（DAKO社サイトメーション、グロストラップ、デンマーク）と4℃でインキュベートONでPBSを交換してください。 </li></ol> 4。幼虫の取り付け<ol><li>幼虫は、その下に向けてキューティクルと上向きに体壁の筋肉を使って取り付け、メディアのドロップ顕微鏡スライド上にマウントされています。きれいなカバースリップ上で封入剤の小滴を入れて準備をカバーするために使用します。マウントされた幼虫上の任意の圧力を加えないでください。 </li></ol> 5。蛹の取り付け<ol><li> 2顕微鏡スライドを準備します。マウンティング培地、解剖用と実装用番目のドロップダウンである。 </li><li>スライドのいずれかにいくつかの蛹を入れてください。 </li><li>使用して、春のはさみは、頭部と腹部の後方部分を削除します。 </li><li>翼と脚の間に切断して腹側の半分から胸部の背側半分を分離します。 </li><li>マウンティング溶液の液滴第二顕微鏡スライド上に蛹の解剖体の部分を配置します。翼と脚を伸ばしていることを確認し、組織のオーバーレイを可能な限り最小限に抑えるようにしてください。 </li><li>カバースリップ上に取り付け培地のドロップを配置し、試料上に置きます。マウントされたサンプルに圧力を加えないでください。 </li></ol> 6。代表的な結果 3齢幼虫の免疫染色ChOsの例を図1に示されています。この例では、8 ChOsの7がはっきりと見えるこれでうまく伸ばして腹部のセグメントを示します。ニューロンは、実験室です。MAb22C10（1:20、アイオワ大学の発達研究ハイブリドーマバンクから得られる）とeled、キャップ、靭帯、キャップアタッチメントおよび靭帯付着細胞は抗αTub85E（1:10、Kleinらが付いています。 2010）。蛍光染色のための二次抗体はCy3、またはCy5標識anti-mouse/rabbit（1:200、ジャクソンイムノ研究所）であった。サンプルは共焦点顕微鏡（LSM 510、Zeiss）を用いて観察した。 35時間齢の蛹の腹部の半径方向の静脈で翼ChOsのクラスタを図2に示します。 <img alt="図1" src="/files/ftp_upload/3846/3846fig1.jpg" /> 図1単一Chの臓器を構成する6つの細胞型の（A）の模式図。キャップアタッチメント（CA）、キャップ（C）、scolopale（S）、ニューロン（N）、靭帯（L）および靭帯付着（LA 。） 5 ChOsがクラスタ化されているLCh5臓器は、（LT横横の筋肉のグループ全体に引き伸ばされ1-4）。横縦筋（LL1）、腹側縦筋（VL1-4）と横方向の腹斜筋（LO1）も示されている。腱細胞は茶色の球体た（Klein et al。、2010年から撮影した）として示されている。 （B）3齢幼虫の一つは腹部のセグメントは10倍の倍率で見た。それぞれの腹部のセグメント内に存在する8 ChOsのセブンは明白です：一緒にpentascolopidial臓器（LCh5）、単一横臓器（LCh1）と二つの腹ChOs（VChB）のいずれかを形成する5外側の臓器。 ChOsのニューロン（N）は神経細胞のマーカーモノクローナル抗体22C10（赤）で標識されています。靭帯（L）、キャップ（C）と添付ファイルの細胞（LA、CA）（青）抗αTub-85Eが付いています。キャップと靱帯細胞は、さらにデイ座（Nachman ら 、未発表データ）からの調節要素を保有するCHO-特定のGFPレポーターが付いています。 <img alt="図2" src="/files/ftp_upload/3846/3846fig2.jpg" /> 図2。 </strong&gt;腹ラジアル静脈における翼振動や音響に反応する器官は40X倍率で表示されています。ニューロン（N）は神経細胞のマーカー22C10（赤、B）が付いています。靭帯（L）とキャップ（C）細胞は、抗αTub85E抗体（ブルー、C）とCHO-特定のGFPレポーター遺伝子（Nachman ら 、未発表データ）と共同でラベル付けされます。

<ol>
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利害の衝突が宣言されません。

このビデオで説明されたプロトコルでは、幼虫および蛹の段階で自己受容ChOsを可視化する方法を提供します。自己受容ChOsの構造と開発に関する研究はこれまで主に胚の段階へと幼虫の成虫に制限されている。したがって、幼虫と成虫の両方ChOsの開発の後期段階の多くの側面は、大部分は不明のままである。 ショウジョウバエの共通の遺伝的ツールと組み合わせて記述されたプロトコルは、ChOs開発の後の段階で役割を果たす新しい遺伝子および経路を特定し、研究に利用することができます。このプロトコルは、CHOの可視化のために最適化されたが、そのような蛹の翼6のような他の組織の染色に適合させることができます。

<table border="1"><tbody><tr><td> 試薬/ツールの名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>デュモン＃5（または＃55）鉗子、biologieヒント</td><td> FST </td><td> 11252から20または11252から10 （または類似の鉗子） </td><td></td></tr><tr><td>アウステルリッツのステンレス製の昆虫ピン、minutiens 0.1ミリメートル</td><td> Roboz手術器具株式会社</td><td> RS-6083から10 </td><td></td></tr><tr><td> Sylgard 184シリコーンエラストマーキット</td><td>ダウコーニングコーポレーション</td><td> 240.4019862 </td><td></td></tr><tr><td> Vannasマイクロハサミ（ストレート、7.5センチ、刃3mm）の</td><td> Mdeizintechnik社AS </td><td> 11-590-00 </td><td>同一仕様のVannas春のはさみはRoboz手術器具（株）から購入することができます</td></tr><tr><td>オービタルシェーカー - Rotamax-120 </td><tD&gt; Heidolph </td><td> N / A </td><td></td></tr><tr><td> Dako社製蛍光マウンティングメディウム</td><td>ダコサイトメーション、グロストラップ、デンマーク</td><td> DK-5302392 </td><td></td></tr><tr><td> X10 PBS製剤</td><td colspan="2"> 2 GR /点灯のKCl、2 GR /点灯KH 2 PO 4、80グラム/点灯のNaCl、 21.7グラム/点灯のNa 2 HPO4.7H 2 O </td><td> PBSの品質は、このプロトコルを成功させるために重要です。 </td></tr><tr><td> PBT </td><td colspan="2"> X1 PBS + 0.1％Tween 20を</td><td></td></tr></tbody></table>

visualization of proprioceptors in drosophila larvae and pupae

固有感覚は、身体内の生じる刺激に応答することによって、身体の部分の動き、または位置を感知する能力である。ショウジョウバエや他の昆虫に固有感覚は、振動や音響に反応する器官（ChOs 2）と呼ばれる特殊な感覚器官で提供されています。 ショウジョウバエの他の多くの臓器と同様に、ChOsはハエのライフサイクル中に2回開発しています。まず、幼虫ChOsは、胚形成の間に開発しています。その後、成人ChOsは、幼虫成虫に開発し、変態中に区別するために継続して開始します。 胚発生時の幼虫のChOsの開発が広く10,11,13,15,16検討されている。各CHOの目玉は、ニューロンとscolopaleセルで構成される感覚のユニットです。感覚のユニットは特殊な表皮添付ファイル細胞1,9,14を経由してキューティクルに接続するアクセサリー2種類の細胞の間に張られています。時ハエの幼虫の動きは、電子の相対変位pidermal付着細胞は樹状突起8,12の外側のセグメントで特定の一過性受容器電位バニロイド（TRPV）チャネルの感覚装置とその結果としての開口部の延伸につながる。信号は、中枢神経系における運動中枢パターン発生回路に転送される誘発した。 複数ChOsは、大人のフライ7に記載されている。これらは成人のフライ付属器（足、翼とホルター）の関節の近くにあり、胸部と腹部に位置しています。さらに、ChOs数百は、総称して機械的エネルギー3,5,17,4、その伝達し音響大人のアンテナでジョンストン器官を形成しています。 胚の段階でChOsの開発に関する広範な知識とは対照的に、ほとんどは幼虫の段階でこれらの器官の形態について知られている。また、大腿ChOsの例外18、ジョンストン器官で、私たちのknowled大人のフライでChOsの開発と構造についてGEは​​、非常に断片的です。 ここでは、3齢幼虫と蛹のChOsを染色し、可視化する方法について説明します。このメソッドは、優れた形態を特徴づけるとフライの様々なChOsの発展を理解するために遺伝的ツールと一緒に適用することができます。

Watch this Scientific Journal Video about Visualization of Proprioceptors in Drosophila Larvae and Pupae at JoVE.com

Visualization of Proprioceptors in Drosophila Larvae and Pupae

の幼虫や蛹に振動や音響に反応する臓器を免疫染色し、可視化する方法<em&gt;キイロショウジョウバエ</em&gt;記載されています。

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 ...

visualization-of-proprioceptors-in-drosophila-larvae-and-pupae

Research

JoVE Journal

Neuroscience

Visualization of Proprioceptors in Drosophila Larvae and Pupae

11.9K ビュー.  Technion-Israel Institute of Technology. の幼虫や蛹に振動や音響に反応する臓器を免疫染色し、可視化する方法キイロショウジョウバエ記載されています。

ビデオ: Visualization of Proprioceptors in Drosophila Larvae and Pupae

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

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.
The Drosophila larva is an established model system to study thermal nociception, a sensory response to potentially harmful temperatures that is evolutionarily conserved across species1,2. The advantages of Drosophila for such studies are the relative simplicity of its nervous system and the sophistication of the genetic techniques that can be used to dissect the molecular basis of the underlying biology4-6 In Drosophila, as in all metazoans, the response to noxious thermal stimuli generally involves a "nocifensive" aversive withdrawal to the presented stimulus7. Such stimuli are detected through free nerve endings or nociceptors and the amplitude of the organismal response depends on the number of nociceptors receiving the noxious stimulus8. In Drosophila, it is the class IV dendritic arborization sensory neurons that detect noxious thermal and mechanical stimuli9 in addition to their recently discovered role as photoreceptors10. These neurons, which have been very well studied at the developmental level, arborize over the barrier epidermal sheet and make contacts with nearly all epidermal cells11,12. The single axon of each class IV neuron projects into the ventral nerve cord of the central nervous system11 where they may connect to second-order neurons that project to the brain.
Under baseline conditions, nociceptive sensory neurons will not fire until a relatively high threshold is reached. The assays described here allow the investigator to quantify baseline behavioral responses or, presumably, the sensitization that ensues following tissue damage. Each assay provokes distinct but related locomotory behavioral responses to noxious thermal stimuli and permits the researcher to visualize and quantify various aspects of thermal nociception in Drosophila larvae. The assays can be applied to larvae of desired genotypes or to larvae raised under different environmental conditions that might impact nociception. Since thermal nociception is conserved across species, the findings gleaned from genetic dissection in Drosophila will likely inform our understanding of thermal nociception in other species, including vertebrates.

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.

に温痛覚を評価するためのローカルおよびグローバルメソッド<em&gt;ショウジョウバエ</em&gt;幼虫

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

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

Appetitive Associative Olfactory Learning in Drosophila Larvae

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

で食欲連想嗅覚学習<em&gt;ショウジョウバエ</em&gt;幼虫

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

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer basic questions on how light information is processed and shared between rapid and circadian behaviors. Drosophila larvae display a stereotypical avoidance behavior when exposed to light. To investigate light dependent behaviors comparably simple light-dark preference tests can be applied. In vertebrates and arthropods the neural pathways involved in sensing and processing visual inputs partially overlap with those processing photic circadian information. The fascinating question of how the light sensing system and the circadian system interact to keep behavioral outputs coordinated remains largely unexplored. Drosophila is an impacting biological model to approach these questions, due to a small number of neurons in the brain and the availability of genetic tools for neuronal manipulation. The presented light-dark preference assay allows the investigation of a range of visual behaviors including circadian control of phototaxis.

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Here we describe a light-dark preference test for Drosophila larva. This assay provides information about innate and circadian regulation of light sensing and processing photobehavior.

に自然免疫とサーカディアン規制Photobehaviorを勉強する光プアッセイ<em&gt;ショウジョウバエ</em&gt;幼虫

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer ...

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

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the underlying neuronal components in the circuits1-6. Application of optogenetics7,8 in the larval neural circuit allows us to manipulate neuronal activity in spatially and temporally patterned ways9-13. Typically, specimens are broadly illuminated with a mercury lamp or LED, so specificity of the target neurons is controlled by binary gene expression systems such as the Gal4-UAS system14,15. In this work, to improve the spatial resolution to "sub-genetic resolution", we locally illuminated a subset of neurons in the ventral nerve cord using lasers implemented in a conventional confocal microscope. While monitoring the motion of the body wall of the semi-intact larvae, we interactively activated or inhibited neural activity with channelrhodopsin16,17 or halorhodopsin18-20, respectively. By spatially and temporally restricted illumination of the neural tissue, we can manipulate the activity of specific neurons in the circuit at a specific phase of behavior. This method is useful for studying the relationship between the activities of a local neural assembly in the ventral nerve cord and the spatiotemporal pattern of motor output.

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

Here we describe a protocol for optogenetic manipulation of motoneuronal activity while monitoring changes in motor output (muscle contraction) in semi-intact Drosophila larvae using lasers within a conventional confocal microscope. This technique enables researchers to achieve local perturbation of neural activity in a few neuromeres to elucidate the dynamics of motor circuits.

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

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

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

パスの長さのプロトコルを採餌<em&gt;キイロショウジョウバエ</em&gt;幼虫

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...

Novel Assay for Cold Nociception in Drosophila Larvae

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

Novel Assay for Cold Nociception in Drosophila Larvae

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

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

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

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which enables them to seek out their preferred thermal landscape. To define the temperature preferences of larvae over a defined linear range, we developed an assay using a temperature gradient. To establish a single-directional gradient, two aluminum blocks are connected to independent water baths, each of which controls the temperature of individual blocks. The two blocks set the lower and upper limits of the gradient. The temperature gradient is established by placing an agarose-coated aluminum plate over the two water-controlled blocks so that the plate spans the distance between them. The ends of the aluminum plate that is set on the top of the water blocks defines the minimum and maximum temperatures, and the regions in-between the two blocks form a linear temperature gradient. The gradient assay can be applied to&#160;larvae of different ages and can be used to identify mutants that exhibit phenotypes, such as those with mutations affecting genes encoding TRP channels and opsins, which are required for temperature discrimination.

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Here, we present a protocol to determine the preferred environmental temperature of Drosophila larvae using a continuous thermal gradient.

ショウジョウバエ幼虫の温度設定を決定する温度勾配試験

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at the Drosophila NMJ enables researchers to acquire synaptic information, encompassing both quantitative data on synapse abundance and detailed insights into their morphology. However, the diffuse distribution and limited visual range of the TEM present challenges for the ultrastructural analysis. This study introduces an innovative and efficient sample preparation method that surpasses the conventional approach. The procedure begins by placing a metal mesh at the base of a flat-bottomed bottle or test tube, followed by positioning fixed larvae samples onto the mesh. An additional mesh is placed over the samples, ensuring that they are positioned between the two meshes. The fixed samples are thoroughly dehydrated and infiltrated before proceeding with the embedding procedure. Then embedding of the samples in epoxy resin is performed in a flat sheet manner, which allows for the preparation of muscles for positioning and sectioning. Benefiting from these steps, all the muscles of Drosophila larvae can be visualized under light microscopy, therey facilitating subsequent positioning and sectioning. Excess resin is removed after locating the 6th and 7th muscles of body segments A2 and A3. Serial ultra-thin sectioning of the 6th or 7th muscle is performed.

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

This report provides a new sample preparation procedure for visualizing neuromuscular junctions in Drosophila Larvae. This method is more effective in preventing the curling of the samples compared to the traditional method and is particularly useful for Drosophila neuromuscular junction ultrastructural analysis.

ショウジョウバエ幼虫の神経筋接合部の透過型電子顕微鏡法のためのサンプル調製プロセスの最適化

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at ...

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.