私たちは、批判的に原稿を読み取るためのフライ株式、およびGalkoラボメンバーのために、ブルーミントンショウジョウバエストックセンターを冷たいプローブアッセイの初期段階を開発するためのサラ・ウーとカミラ・グラハム感謝します。この作品はHNTに、およびNIH R01NS069828、R21NS087360や基礎研究でテキサス大学MDアンダーソンクラークフェローシップによってMJGにNIH NRSA（NIH F31NS083306）によってサポートされていました。

幼虫の調製<ol><li> 25ºCインキュベーターで株式や遺伝的交雑を上げます。 <ol><li>クロスを培養する場合は、20-25処女雌と通常のコーンミールフライメディアを含むバイアルあたり15〜20人の男性を使用しています。女性は、食品の新しいバイアルにそれらを転送する前に、約48時間産卵させます。 </li></ol></li><li> 4-5日産卵後、穏やかにどろどろ食品及び幼虫への水の流れを噴出することにより、所望の遺伝子型の第3齢幼虫を収集し、中型、清潔なペトリ皿に内容物を注ぎ出す（60ミリメートル×15ミリメートル）。 </li><li>次に、穏やかに鉗子を使用して、サイズまたは試験されることから、より大きな「放浪第3齢」又は小さな病弱幼虫を排除する絵筆によって幼虫を並べ替えます。望ましくない遺伝子マーカーやバランサーを含む幼虫を捨てます。 </li><li>実験者が元に「ブラインド」になるようにソートされた幼虫が配置される仲間のラボのメンバーのラベルの皿や容器を持っていますperimental状態または該当する場合は幼虫の遺伝子型。実験者は、実験室部材によって生成された鍵を使用して実験した後に収集されたデータに復号化されたラベルを適用することができます。 </li><li>薬さじを用いて、（34ミリメートル×10ミリメートルまたは60ミリメートル×15ミリメートル）を室温の水できれいに、標識されたペトリ皿充填途中に生鮮食品のダイムサイズの量を転送します。 </li><li>穏やかに鉗子またはペイントブラシを使用して（テストは20分以上かかると予想される場合飢餓や乾燥を防止するために）食品にソートされた幼虫を移動させます。 </li></ol> 2.コールドプローブアッセイ<ol><li>それが所望の温度に冷却するためのいくつかの分を可能にコールドプローブユニットをオンにします。より低い温度に設定した場合、結露はおそらくプローブに沿って形成することになります。 </li><li>実験室での余分な水分を拭き取り幼虫への適用前に拭きます。ない時はすぐに使用で、場所は絶縁するように、両方の、きれいな先端を保つために、損傷を防ぐため、プローブ上のキャップ絶縁。 </li><li>暗い可動ビニルの薄片上に中間第3齢幼虫を置き、明視野顕微鏡下（典型的には、ノートブック・バインダーから切断小片を使用）。コントラスト視覚化およびプローブと適切に整列させるために、それに触れることなく、幼虫の移動の黒ビニルエイズ。 </li><li>顕微鏡を調整し、コントラストを提供し、早すぎる乾燥から幼虫を防止し、光ユニット（ 材料の表を参照）培地明るさ（50から75パーセントの最大輝度）（ 材料の表を参照のこと）。 </li><li>彼らは結果を混乱させる可能性があるので最初に通常の蠕動運動を示さない任意の幼虫を捨てます。幼虫は、そうでなければ幼虫は、正常歩行を阻害ビニルに付着する水のペトリ皿から湿ったなければなりません。水は幼虫の周りにパドルべきではありません。 </li><li> CORに幼虫を移動するビニルを使用して、前後体軸に対して90°の角度で幼虫に向かって手でプローブを前進RECT位置（ 図1A参照 ）。 </li><li>東洋は、プローブの先端を穏やかプローブ顕微鏡ステージに対して約45°の角度を有する幼虫の中間背側表面を横切って敷設します。 </li><li>プローブ接触すると、実験室のタイマーを起動します。依然として前方または後方に移動可能にしながら、わずかに幼虫キューティクルの表面にインデントするのに十分な下向きの圧力を加えます。 </li><li>最大10秒間またはコールド誘発行動応答が観察されるまで、所定の位置にプローブを保持する - いずれか早い方。その後、プローブを削除します。 </li><li>観察行動応答と応答待ち時間を記録します。 10秒以内に応答しない幼虫は、「非応答者」とみなされます。待ち時間はまた、応答のために記録および応答ロバスト性/振幅の変化を測定するために使用することができます。 </li><li>幼虫を破棄し、次の準備。 </li><li>試験幼虫の所望の数が（N 3組= 20-40幼虫がUた達するまで繰り返し2.3から2.11までステップ）ここでのsed。 </li></ol>

<ol>
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米国特許出願（CL）は冷たいプローブの設計に保留されています。冷たいプローブ部品番号と構成の詳細に関する追加情報だけでなく、ツールの設計上の追加的なコンサルティングは、要求に応じて提供されます。

ここに記載のアッセイは、定性的および定量的に様々な遺伝的背景、環境の影響、および/または損傷誘導条件の幼虫に痛覚または侵害受容感を評価するために使用することができます。このアッセイは冷刺激の焦点アプリケーションを可能にするため、このツールを使用すると、ワンは、特に低温に応答して、末梢感覚ニューロンのサブセットの機能を評価することができます。興味深いこ...

ショウジョウバエは、新規保存された遺伝子と複合行動の根底にある神経回路の同定のため非常に有用であることが証明されています。ハエは、洗練された遺伝的ツールキットおよび痛覚5、6、7の細胞および分子の塩基を分析するために、正確な遺伝的および神経操作1、2、3、4を可能にする簡略化し、神経系を提供します。幼虫は、優しい肌触り8、9、10、有害な熱11、12、13及び侵害刺激4の機械的感覚のための行動アッセイことを考えると、これらの分析に特に有用です</sup&gt; 11は 、既に確立されている、透明幼虫キューティクルは、表皮とその下にある感覚ニューロンのライブまたは固定のイメージングが可能になります。最近では、有害な寒さのためのアッセイはまた、我々は、ここでより詳細に説明7を 、開発されました。 罰金、円錐先端が冷たいプローブを使用して、我々は、 ショウジョウバエの幼虫が展示優しいタッチ、次の通常の歩行運動中に観察された行動とは別個の冷特有の反応行動のセットを、その、または過酷な機械的または高温刺激7、8、11の後に示して。冷たい固有の動作は、堅牢なフルボディの収縮（CT）、後部セグメント（PR）の45-90ºレイズとU字型（US）への前部と後部セグメントの同時昇給が含まれています。これらの行動の有病率は減少温度が、Sの各ピークに増加します軽く異なる低温。最近の研究は、CT応答が有害な熱や過酷な機械的刺激7に対応するものとは異なる末梢感覚ニューロンによって媒介されることを示唆しています。 多くの脊椎動物の侵害受容器のように、 ショウジョウバエの複数の樹状（MD）末梢感覚ニューロンは、表皮1の上にarborize複雑な樹枝状構造を有しています。 MDニューロンは、腹側神経索14への軸索を投射する、すべての幼虫の身体セグメント中に存在します。 MD感覚ニューロンは、樹状形態に基づいて、4つの異なるクラス（I-IV）に分離され、感覚機能4、9、10、15、16、17を変化しています。クラスIVニューロンは、幼虫の横車体ロール応答のために必要とされる一方で高温や過酷な機械的刺激4、クラスIIIニューロンは穏やかタッチ応答9、10のために必要とされるのみ冷によって活性化されていないだけでなく、冷誘発行動応答7のために必要とされます。両方のクラスIIIおよびクラスIVニューロンが刺激9、10、17、19 7、11、18侵害および非侵害に対する行動反応を促進するために、離散的一過性受容体電位（TRP）チャネルを利用します。さらに、幼虫侵害受容は、細胞20および行動レベル12、21で、次の傷害を感作されています。 ここに記載されるアッセイはquantificatioすることができます通常の、又は潜在的に周囲温度（18〜22ºC）に有害な冷（≤10ºC）の範囲の低温、無害クール（11-17ºC）に対する行動応答の改変のいずれかのN。このアッセイで使用される低温は、堅牢で再現性のカルシウムが増加し、定性的および定量7を分析することができる冷誘発行動応答を誘発する、直接クラスIII感覚ニューロンを活性化することができます。このアッセイは、実質的に任意の遺伝子型、ならびに冷痛覚、侵害受容感または侵害受容可塑性に影響を与える遺伝的および環境的双方の因子を決定するために、多様な環境条件（変更された栄養、傷害、薬理学的物質）に曝露された幼虫の幼虫に適用することができます。 thermosensationは、多くの種間でユビキタスであることを考えると、このアッセイは、痛覚の研究のための貴重なツールを提供し、新たな遺伝子標的または改善される神経細胞の相互作用を明らかにします脊椎動物痛覚の我々の理解。 特注コールドプローブは、（コールドプローブ、 材料の表を参照）、熱伝導を介してアルミニウム軸と円錐形先端を冷却する閉ループ温度制御されたペルチェ素子を利用します。アルミニウム円錐形先端は、制御ユニットにリアルタイムの温度を報告内部サーミスタが埋め込まれています。ヒートシンクとファン（熱制御ユニット、 材料の表を参照のこと）を達成することができるペルチェ効果の熱負荷（QC）は（22-0℃）での所望の温度範囲を調節するために、熱電モジュールに取り付けられています。コールドプローブ先端の有害な冷刺激が前方および後方端部から等距離セグメント（単数または複数）に、背側正中線に手で適用される幼虫の（おおよそセグメントA4、 図1Aを参照）。フルボディ：冷たい刺激に応答して、幼虫は、一般的に10秒のカットオフ内の3つの冷誘発行動の1を生産します収縮（CT）、U字状に前方および後方セグメントの45-90º昇給（US）、または（ 結果を参照）後部セグメント（PR）の昇給。これらの行動はいずれも正常な蠕動運動や採餌行動の間に実行されません。これらの挙動も優しいタッチレスポンスと高い温度や有害な機械的刺激に対する嫌悪ローリング応答とは区別されます。

<table><tbody><tr><td>Cold Probe</td><td>Pro-Dev Engineering</td><td>Custom-built on demand</td><td>Part numbers and construction details can be provided on request</td></tr><tr><td>Thermal Control Unit</td><td>TE Technology</td><td>Custom Built enclosure</td><td>Part numbers and construction details can be provided on request</td></tr><tr><td>Zeiss Stemi 2000 microscope</td><td>Zeiss</td><td>NT55-605</td><td></td></tr><tr><td>Fiber-Lite MI-150 High Intensity Illuminator</td><td>Dolan-Jenner Industries.</td><td>A20500</td><td></td></tr><tr><td>Schott Dual Gooseneck 23 inch Fiber Optic Light Guide</td><td>Schott North America, Inc.</td><td>Schott A08575</td><td></td></tr><tr><td>Forceps</td><td>FST</td><td>FS-1670</td><td>Used to sort and handle larvae. Be sure to smooth and blunt forceps tips slightly to lower the risk of accidently puncturing or injuring the larvae</td></tr><tr><td>Paintbrush</td><td>Dick Blick Art Materials</td><td>06762-1002</td><td>Used to sort and handle larvae. It is helpful if the paintbrush is damp during use.</td></tr><tr><td>35 mm x 10 mm Polystyrene Petri dish</td><td>Falcon</td><td>351008</td><td></td></tr><tr><td>60mm x 10 mm Polystyrene Petri dish</td><td>Falcon</td><td>351007</td><td></td></tr><tr><td>Piece of black vinyl (at least 2 inches x 2 inches)</td><td></td><td></td><td>Used to provide contrast and orient larvae to the cold probe</td></tr><tr><td>Fisherbrand Scoopula Spatula</td><td>Fisher Scientific</td><td>14-357Q</td><td>Used to move food</td></tr><tr><td>Kimtech Science Kimwipes</td><td>Fisher Scientific</td><td>06-666A</td><td>Used to dry the larvae and cold probe if there is excess moisture</td></tr></tbody></table>

novel assay for cold nociception in drosophila larvae

どのように生物が感知し、まだよく理解されている有害な温度に反応します。さらに、そのような末梢神経障害または損傷誘発性感を経験する患者のような感覚機械の感作を、基礎となるメカニズムは、十分に特徴付けられていません。遺伝的に扱いやすいショウジョウバエモデルは、有害な熱の検出に必要な細胞および遺伝子を研究するために使用されています これは、関心のある複数の保存された遺伝子が得られました。リトルは、細胞および有害な寒さの検出のための重要な受容体についてはしかし、知られています。 ショウジョウバエは、彼らが感知し、多分有害な冷たい刺激を避ける方法を、低温（≤10ºC）への長期暴露に耐えられないので、涼しい避けるだろう、行動好みアッセイにおける気温の上昇を好む、が、ごく最近研究されています。 ここでは、説明との最初の有害な寒さ（≤10ºC）の行動分析を特徴付けますショウジョウバエ。このツールおよびアッセイを使用して、我々は、定性的および定量的に冷たい侵害受容行動を評価するための方法を研究者を示しています。これは、通常の/健康な培養条件の下で、あるいはおそらく疾病、傷害または感作のコンテキストで行うことができます。さらに、このアッセイはthermosensation、疼痛、または侵害受容感に影響を与える可能性があり、所望の遺伝子型のために選択された幼虫に適用することができます。痛みはさらにそう脊椎動物を含む他の種の痛みプロセスの重要な理解を収集します熱痛覚を研究するために、このアッセイを用いて、高度に保存されたプロセスであることを考えます。

時折ポーズ、ヘッドターン、および方向22の変化を含む蠕動運動とショウジョウバエの幼虫移動します。しかし、有害な寒冷刺激の焦点用途に応じて、幼虫は、有害な熱および機械的刺激に対する嫌悪横ロールとは異なり、独自の行動の集合を示します。これらの行動は優しいタッチ8、9、10...

Watch this Scientific Journal Video about Novel Assay for Cold Nociception in Drosophila Larvae at JoVE.com

Novel Assay for Cold Nociception in Drosophila Larvae

ここでは、 ショウジョウバエの幼虫に冷たい痛覚を研究するための新規なアッセイを示しています。このアッセイは、焦点有害冷たい刺激を印加することが可能な特注のペルティエプローブを利用し、定量化可能な冷たい固有の動作になります。この技術は冷たい痛覚の更なる細胞および分子解剖を許可します。

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 ...

novel-assay-for-cold-nociception-in-drosophila-larvae

Research

JoVE Journal

Neuroscience

Novel Assay for Cold Nociception in Drosophila Larvae

7.7K ビュー.  UT MD Anderson Cancer Center. ここでは、 ショウジョウバエの幼虫に冷たい痛覚を研究するための新規なアッセイを示しています。このアッセイは、焦点有害冷たい刺激を印加することが可能な特注のペルティエプローブを利用し、定量化可能な冷たい固有の動作になります。この技術は冷たい痛覚の更なる細胞および分子解剖を許可します。 

ビデオ: Novel Assay for Cold Nociception in Drosophila Larvae

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 ...

神経科学

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 ...

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an intricate network of inducers, detectors, and activators rapidly responds to neuron damage, infection or other immune infractions. This inflammation of immune cells is intimately associated with the pathology of neurodegenerative disorders, such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease and ALS. Under compromised disease states, chronic inflammation, intended to minimize neuron damage, may lead to an over-excitation of the immune cells, ultimately resulting in the exacerbation of disease progression. For example, loss of dopaminergic neurons in the midbrain, a hallmark of PD, is accelerated by the excessive activation of the inflammatory response. Though the cause of PD is largely unknown, exposure to environmental toxins has been implicated in the onset of sporadic cases. The herbicide paraquat, for example, has been shown to induce Parkinsonian-like pathology in several animal models, including Drosophila melanogaster. Here, we have used the conserved innate immune response in Drosophila to develop an assay capable of detecting varying levels of nitric oxide, a cell-signaling molecule critical to the activation of the inflammatory response cascade and targeted neuron death. Using paraquat-induced neuronal damage, we assess the impact of these immune insults on neuroinflammatory stimulation through the use of a novel, quantitative assay. Whole brains are fully extracted from flies either exposed to neurotoxins or of genotypes that elevate susceptibility to neurodegeneration then incubated in cell-culture media. Then, using the principles of the Griess reagent reaction, we are able to detect minor changes in the secretion of nitric oxide into cell-culture media, essentially creating a primary live-tissue model in a simple procedure. The utility of this model is amplified by the robust genetic and molecular complexity of Drosophila melanogaster, and this assay can be modified to be applicable to other Drosophila tissues or even other small, whole-organism inflammation models.

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Levels of the inflammatory cell-signaling molecule nitric oxide (NO) are commonly assayed using Griess reagent. In this protocol, we have created a modified Griess assay utilizing live Drosophila brain tissue in order to detect the secretion of NO in a simple, quantifiable and highly repeatable method.

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an ...

免疫・感染学

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 ...

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

での採餌行動のためのシングル·フライアッセイ<em&gt;ショウジョウバエ</em

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila larvae, customized metal nickel-titanium alloy (nitinol) filaments. These mechanical probes are similar to the von Frey filaments used in vertebrates to measure mechanical nociception. Here, we demonstrate how to make and calibrate these mechanical probes and how to generate a full behavioral dose-response from subthreshold (innocuous or non-noxious range) to suprathreshold (low to high noxious range) stimuli. To demonstrate the utility of the probes, we investigated tissue damage-induced hypersensitivity in Drosophila larvae. Mechanical allodynia (hypersensitivity to a normally innocuous mechanical stimulus) and hyperalgesia (exaggerated responsiveness to a noxious mechanical stimulus) have not yet been established in Drosophila larvae. Using mechanical probes that are normally innocuous or probes that typically elicit an aversive behavior, we found that Drosophila larvae develop mechanical hypersensitization (both allodynia and hyperalgesia) after tissue damage. Thus, the mechanical probes and assay that we illustrate here will likely be important tools to dissect the fundamental molecular/genetic mechanisms of mechanical hypersensitivity.

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

The goal of this protocol is to show how to perform an improved assay for mechanical nociception in Drosophila larvae. We use the assay here to demonstrate that mechanical hypersensitivity (allodynia and hyperalgesia) exists in Drosophila larvae.

ショウジョウバエ幼虫における機械的な結知を測定するための改善されたアッセイとツール

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila ...

行動学

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 ...

Behavioral Assays for Optogenetic Manipulation of Neural Circuits in Drosophila melanogaster

Optogenetics has become a fundamental technique in neuroscience, enabling precise control of neuronal activity through light stimulation. This study introduces easy-to-implement setups for applying optogenetic methods in Drosophila melanogaster. Two optogenetic tools, CsChrimson, a red-light-activated cation channel, and GtACR2, a blue-light-activated anion channel, were employed in four experimental approaches. Three of these approaches involve single-fly experiments: (1) a blue-light optogenetic thermotactic positional preference assay targeting temperature-sensitive heating cells, (2) a red-light optogenetic positional preference assay activating bitter sensing neurons, and (3) a proboscis extension response assay activating the sweet-sensing neurons. The fourth approach (4) is a fly maze setup to assess avoidance behaviors using multiple flies. The ability to manipulate neural activity temporally and spatially offers powerful insights into sensory processing and decision-making, underscoring the potential of optogenetics to advance our knowledge of neural function. These methods provide an accessible and robust framework for future research in neuroscience to enhance the understanding of specific neural pathways and their behavioral outcomes.

This paper presents methods for optogenetic manipulation in Drosophila melanogaster, utilizing CsChrimson and GtACR2 to activate and silence specific neurons. Four experiments are described to utilize optogenetics to explore thermotactic and gustatory behaviors, providing insights into the underlying neural mechanisms governing these processes.

Drosophila melanogasterにおける神経回路の光遺伝学的操作のための行動アッセイ

Optogenetics has become a fundamental technique in neuroscience, enabling precise control of neuronal activity through light stimulation. This study ...

Local Heat Probe Assay: A Method to Assess Nociception of High Temperatures in Drosophila Larvae

Source: Chattopadhyay, A., et al. <a href="https://www.jove.com/video/3837/" target="_blank">Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae</a>. J. Vis. Exp. (2012).
Drosophila larvae have dedicated sensory neurons&#8211;nociceptors&#8211;whose response to potentially harmful thermal stimuli triggers aversive&#8211;or so-called &#34;nocifensive&#34;&#8211;behaviors in the animal. This video describes a method to assess thermal nociception by recording larval behavior in response to local application of a heat probe onto the cuticle. This method is, therefore, called the local heat probe assay.

局所熱プローブアッセイ:ショウジョウバエ幼虫の高温の侵害受容を評価する方法

Source: Chattopadhyay, A., et al. Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae. J. Vis. Exp. (2012).
Drosophila larvae ...

冷痛覚のための新規アッセイ

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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

でProprioceptorsの可視化

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

で食欲連想嗅覚学習

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

での採餌行動のためのシングル·フライアッセイ

ショウジョウバエ幼虫における機械的な結知を測定するための改善されたアッセイとツール

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

Drosophila melanogasterにおける神経回路の光遺伝学的操作のための行動アッセイ

局所熱プローブアッセイ:ショウジョウバエ幼虫の高温の侵害受容を評価する方法