私たちは、原稿の重要な読書のための熱プレートアッセイ、フライ株式のブルーミントンのショウジョウバエストックセンター、Galkoラボのメンバーを提案して幼虫のヒートプローブアッセイ、ショーン·スウィーニーを開発するための熱プローブ設計、ダニエル·バブコックのためにキリスト教のランドリーに感謝します。この作品は、MJG、研究キャリア（AVG）に少数派にアクセスするためのNIH MARC U-STARトレーニンググラント（ヒューストン - ダウンタウン学者アカデミーの大学へのT34GM079088）にNIH R01 NS069828によってサポートされていました。

<ol>
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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+regulating+dendritic+outgrowth,+branching,+and+routing+in+Drosophila.">Genes regulating dendritic outgrowth, branching, and routing in Drosophila.</a> Genes Dev. 13, 2549-2561 (1999).</li><li>Walters, E. T., Illich, P. A., Weeks, J. C., Lewin, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defensive+responses+of+larval+Manduca+sexta+and+their+sensitization+by+noxious+stimuli+in+the+laboratory+and+field.">Defensive responses of larval Manduca sexta and their sensitization by noxious stimuli in the laboratory and field.</a> J. Exp. Biol. 204, 457-469 (2001).</li><li>Woolf, C. 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我々は、開示することは何もありません。

ここで説明したアッセイは、定性的かつ定量的に有害な熱刺激に対する反応性の異なる遺伝子型の幼虫を評価するために使用することができます。ヒートプローブアッセイの主な機能は、刺激が単一の遺伝子座でしか与えられていることです。これはおそらく、おそらく唯一の小さなプローブが接触セグメントのクラスIVニューロン-もののサブセットと、すぐに隣接するセグメント11のものの発火につながる。ため局所的刺激により、ヒートプローブアッセイは、地域のような熱いストーブに接触する手など、特定の体に局在している有害な刺激を検出する一般的な感覚体験を模倣しています。ヒートプローブアッセイの欠点は、おそらく三つの要素に起因することができますいくつかのユーザ間のばらつきを持っているということです：私は）ユーザーがプローブⅱ）正確な位置、幼虫にプローブを適用すると圧力基本的な侵害受容ニューロン、および、iii）の正確なANGへの相対的な幼虫で幼虫の表面プローブ接点でル。 我々は以前に与えられた温度を1に、非レスポンダー、遅い応答し、その撤退の待ち時間に基づいて、高速応答に分類幼虫の定量的な戦略を報告した。ここではさらに高い温度に幼虫の反応性について報告する。興味深いことに、我々は、幼虫の熱侵害受容応答に天井があることを見つけて、この上限は52と54との間にあることを℃にこれは52よりも高い温度でのゲーティングが可能な一過性受容体電位（TRP）チャネル℃を有していないその幼虫が示している可能性がありますまた、それが神経細胞や筋肉が彼らも嫌悪撤退で機能する前に、破損しモータの応答を開始または遂行するために使用されることを示唆している可能性があります。また、撤退の振幅の異なる分析応答を使用して応答の &quot;堅牢性&quot;の指標としてロールの番号のどちらかを報告します。単純に、1これらのパラメータは刺激の温度の上昇や時間の経過とともに増加することが期待されます。驚くべきことに、我々はこのようなケースではないことを見つける。有害な範囲の下限（42℃）の温度で長い時間刺激した幼虫は多くのロールと、より多くの時間は、幼虫がより高い温度（48から52°C）でプローブしたときよりも、ローリング過ごし表示されます。これは有害な温度のウィンドウ内では、主に応答の振幅を決定する露出の期間であることを示唆している。幼虫は非常に有害な温度にさらされているので（48から52°C）非常に迅速に平均的に反応するの幼虫などの多くのロールは時間の長い長さの少ない有害な温度にさらされるとして、彼らは示さない。ここで報告された応答振幅の分析は、異なる遺伝子型や環境の操作を比較することができるとともに、別の定量的な次元を追加します。 熱プレートアッセイの主な特徴は、それが有害にグローバルな露出を伴うことです。熱。このように、それは熱いストーブに触れてより加熱釜に座っている動物に似ています。幼虫は、野生でグローバルに侵害刺激が発生する可能性がありますときには明らかではないが、ラボではこのグローバル曝露に対する行動応答は局所刺激によって観察されたものよりも複雑です。また、他の3で述べた熱プレートアッセイの強さは、幼虫に触れると、プロトコルのコンポーネントではありませんので、それは少しユーザ間のばらつきがあるということです。唯一の実質的な差異は、各動作が開始し、これが繰り返される表示/親しみやすさを最小限に抑えることができたときに定義することであるように思われる。アッセイの間に興味深い相違点は、嫌悪行動が開始する温度である。これらは、ヒートプローブに比べて熱プレートアッセイではるかに低い。ヒートプローブ（ヘッドとテールの昇給）と接触幼虫によって示された予備的な動作は、°Cの熱プラットフォームで〜27で観測された頭のスラッシュの相関かもしれません電子アッセイ。それは、この応答は、 &quot;痛み&quot;よりも &quot;不快感&quot;を反映している可能性があります。我々は、高温下でも、発作を打つの相関、および麻痺が観察されていない（最大48°C）熱プローブアッセイの温度、それは身体の複数の領域から発射感覚ニューロンのクリティカルマスがあるのか​​もしれないこれらの行動にもたらすために必要としていました。そのグローバルな刺激は、ローカルでの動作をトリガするためには不十分であるニューロンの応答の加算含むことができることを示すヒートプローブで観察された侵害受容閾値の下端以下 - 興味深いことに、発作と麻痺の動作は、温度（37℃〜34）で観測されているヒートプローブのアプリケーション。これらの温度は、実際に幼虫に有害として認識されていることは、ほとんどのケースではありません麻痺の動作を開始し、続いてフライ食品に回復させ、幼虫が（図5C）生き残るという観察によってサポートされています。さらに熱PLという主張を支持する食べたアッセイは、侵害受容性応答を読んでいると知られている侵害受容性感覚ニューロンにシナプス伝達をブロックして観測された行動（図6）のほとんどのレイテンシを増加させるという事実である。高温度ホイップ動作のレイテンシの増加はありませんという観察は、MD-GAL4を発現しない他の感覚ニューロンは、この動作のために必要とされることを示唆している。 グローバルなアッセイで急速に加熱水滴のローカルアッセイと液浸の小さな金属製のプローブのホットチップ - 和では、両方のアッセイは、定義された温度の有害な熱刺激に個々の幼虫を露出伴う。遺伝的背景および/ ​​または環境条件（例えば、プラスまたはマイナスの組織の損傷のために）変化にさらされるの変化のショウジョウバエの幼虫の行動応答は、学び、これらのアッセイを用いて定量することができます。最終的に、これらのアッセイの結果から、私たちはより良いNOCを制御する遺伝子ネットワークを理解するのに役立ちますショウジョウバエや他の近縁種でiception。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>温度プローブ</td><td>プロ-Devにエンジニアリング</td><td>オンデマンドカスタムビルド</td><td>連絡先情報は、リクエストに応じて提供することができ</td></tr><tr><td>乾いたバスインキュベーター</td><td>フィッシャー·サイエンティフィック</td><td> 11から718 </td><td> 1固体の加熱ブロックと16mmの井戸1加熱ブロック</td></tr><tr><td>ライカDFC290 12v/400mA カラーカメラ</td><td>ライカマイクロシステムズ</td><td> 12730080 </td><td>それと同等の カメラが行います。 </td></tr><tr><td>ライカMZ6顕微鏡</td><td>ライカマイクロシステムズ</td><td> MZ6ズーム本体の部品番号（光学キャリア）は10445614です。 </td><td></td></tr><tr><td>ショットエースModulampユニット</td><td>ショットノースアメリカ株式会社</td><td> A20500 </td><td></td></tr><tr><td>ショットデュアルグースネック23インチ光ファイバライトガイド</td><td>ショットノースアメリカ株式会社</td><td>ショットA08575 </td><td></td></tr><tr><td>温度制御ユニット</td><td> TSCI株式会社。 </td><td>カスタムビルド</td><td>詳細はご要望に応じて提供することができ</td></tr><tr><td>ツァイスSTEMI 2000顕微鏡</td><td>ツァイス</td><td> NT55-605 </td><td>それと同等の 顕微鏡を行います。 </td></tr><tr><td>鉗子</td><td> FST </td><td> FS-1670 </td><td></td></tr><tr><td> 1ミリメートルのメッシュ</td><td>ジェネシー科学</td><td> 57から101 </td><td></td></tr><tr><td>絵筆</td><td>ディックブリックアート材料</td><td> 06762-1002 </td><td></td></tr><tr><td> UV架橋</td><td>フィッシャー·サイエンティフィック</td><td> 1199289 </td><td></td></tr><tr><td>コプリンジャー</td><td>フィッシャー·サイエンティフィック</td><td> 08から816 </td><td></td></tr><tr><td> 10mlのビーカー</td><td>フィッシャー·サイエンティフィック</td><td> 02-540C </td><td></td></tr><tr><td>ジエチルエーテル</td><td>フィッシャー·サイエンティフィック</td><td> E138-500 </td><td></td></tr><tr><td> 35 X 10 mmのポリスチレンシャーレ</td><td>ファルコン</td><td> 351008 </td><td>我々はテストしていません 別の料理をお楽しみいただけます。 </td></tr><tr><td>ガラス顕微鏡スライド</td><td>コー​​ニング</td><td> 26003 </td><td></td></tr><tr><td>熱電対</td><td>オメガエンジニアリング株式会社</td><td> HH802U </td><td></td></tr><tr><td>ビニールのワンピース</td><td>オフィス·デポ</td><td> 480009 </td><td></td></tr><tr><td>微小遠心管</td><td>デンヴィル科学（株） </td><td> C-2170 </td><td></td></tr></tbody></table>

local and global methods of assessing thermal nociception in drosophila larvae

本稿では、 ショウジョウバエの幼虫の熱痛覚を勉強するためのアッセイを示しています。第二は、ほとんどの卸売（グローバル）アクティベーションまたはすべてのそのようなニューロンの3が含まれている間1つのアッセイは、熱侵害受容器1,2の（ローカル）空間的に制限された刺激が含まれます。一緒に、これらの技術は、 ショウジョウバエの侵害受容感覚ニューロンの行動機能の可視化と定量化を可能にします。 ショウジョウバエの幼虫は、熱痛覚、進化種の1,2を越えて保存されている潜在的に有害な温度に感覚応答を研究するために確立されたモデルシステムです。そのような研究のためのショウジョウバエの利点は、すべての後生動物のように、responsその神経系やショウジョウバエでは基礎生物学4-6の分子的基礎を分析するために使用することができ遺伝学的手法の高度化の相対的なシンプルさです。有害な熱刺激への電子は、一般的に提示刺激7 &quot;nocifensive&quot;嫌悪撤退を伴います。このような刺激は自由神経終末または侵害受容器を介して検出され、生物体の応答の振幅は、侵害刺激8を受信した侵害受容器の数に依存しています。 ショウジョウバエでは、感光体10のような彼らの最近発見された役割に加えて有害な熱的、機械的刺激9を検出するクラスIV樹状分枝の感覚ニューロンである。非常によく発達レベルで研究されているこれらのニューロンは、バリアの表皮シートの上に樹枝状になると11,12のほぼすべての表皮細胞との接触を行います。彼らは二次ニューロンに接続することが中枢神経系11の腹側神経索に各クラスIVニューロンのプロジェクトの一つの軸索は、脳へのプロジェクト。 ベースライン条件の下で、侵害受容性知覚神経相対的に高いしきい値に達するまで、nsは発生しません。アッセイは、研究者はベースラインの行動応答または、おそらく、次の組織の損傷をすさまじい感を定量化することができますここで説明する。各アッセイは、有害な熱刺激に異なるが、関連する運動器官の行動反応を引き起こし、 ショウジョウバエの幼虫の熱痛覚のさまざまな側面を可視化し定量化する研究を可能にします。アッセイは、所望の遺伝子型の幼虫や痛覚に影響を与える可能性があり、異なる環境条件下で飼育幼虫​​に適用することができます。熱痛覚が種間で保存されているので、 ショウジョウバエの遺伝的解剖から集められた知見は、おそらく脊椎動物を含む他の種で熱痛覚の我々の理解をお知らせします。

Watch this Scientific Journal Video about Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae at JoVE.com

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae | Neuroscience | Video

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

本稿では、熱痛覚を勉強するためのアッセイを実証<em&gt;ショウジョウバエ</em&gt;幼虫。 1つのアッセイは、熱侵害受容器の（ローカル）空間的に制限された刺激を伴う<sup&gt; 1,2</sup&gt;中に2番目のは、ほとんどの卸売（グローバル）、アクティベーションまたはそのようなすべてのニューロンを含む<sup&gt; 3</sup&gt;。一緒に、これらの技術は、行動機能の可視化と定量化を可能にする<em&gt;ショウジョウバエ</em&gt;侵害受容感覚ニューロン。

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 ...

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Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

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15.7K ビュー.  The University of Texas MD Anderson Cancer Center. 本稿では、熱痛覚を勉強するためのアッセイを実証ショウジョウバエ幼虫。 1つのアッセイは、熱侵害受容器の（ローカル）空間的に制限された刺激を伴う 1,2中に2番目のは、ほとんどの卸売（グローバル）、アクティベーションまたはそのようなすべてのニューロンを含む 3。一緒に、これらの技術は、行動機能の可?...

ビデオ: Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

Electrophysiological Measurements and Analysis of Nociception in Human Infants

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are based on behavioural and physiological body reactions, such as crying, body movements or changes in facial expression. While these measures demonstrate that infants mount a response following noxious stimulation, they are limited: they are based on activation of subcortical somatic and autonomic motor pathways that may not be reliably linked to central sensory processing in the brain. Knowledge of how the central nervous system responds to noxious events could provide an insight to how nociceptive information and pain is processed in newborns.
The heel lancing procedure used to extract blood from hospitalised infants offers a unique opportunity to study pain in infancy. In this video we describe how electroencephalography (EEG) and electromyography (EMG) time-locked to this procedure can be used to investigate nociceptive activity in the brain and spinal cord.
This integrative approach to the measurement of infant pain has the potential to pave the way for an effective and sensitive clinical measurement tool.

The assessment and treatment of pain in infants is difficult because infants cannot verbally report their experience. In this video we describe quantitative electrophysiological methods and analysis techniques that can be used to measure the response to noxious events from the infant nervous system.

電気生理学的測定とヒト幼児における痛覚の分析

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are ...

神経科学

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

アッセイのメソッドショウジョウバエ行動

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

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 ...

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 ...

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is initiated by photon absorption-inducing isomerization of the chromophore, promoting conformational changes of the opsin and resulting in a second dark-stable photopigment state (metarhodopsin, M). Investigation of this bi-stable photopigment using random mutagenesis requires simple and robust methods for screening mutant flies. Therefore, several methods for measuring reductions in functional photopigment levels have been designed. One such method exploits the charge displacements within the photopigment following photon absorption and the huge amounts of photopigment molecules expressed in the photoreceptors. This electrical signal, named the early receptor potential (or early receptor current), is measured by a variety of electrophysiological methods (e.g., electroretinogram and whole-cell recordings) and is linearly proportional to functional photopigment levels. The advantages of this method are the high signal-to-noise ratio, direct linear measurement of photopigment levels, and independence of phototransduction mechanisms downstream to rhodopsin or metarhodopsin activation. An additional electrophysiological method called prolonged depolarizing afterpotential (PDA) exploits the bi-stability of Drosophila photopigment and the absorption-spectral differences of fly R and M pigment states. The PDA is induced by intense blue light, converting saturating amounts of rhodopsin to metarhodopsin, resulting in the failure of light-response termination for an extended time in darkness, but it can be terminated by metarhodopsin to rhodopsin conversion using intense orange light. Since the PDA is a robust signal that requires massive photopigment conversion, even small defects in the biogenesis of the photopigment lead to readily detected abnormal PDA. Indeed, defective PDA mutants led to the identification of novel signaling proteins important for phototransduction.

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

We present a protocol to electrophysiologically characterize bi-stable photopigments: (i) exploiting the charge displacements within the photopigment molecules following photon-absorption and their huge amount in the photoreceptors, and (ii) exploiting the absorption-spectra differences of rhodopsin and metarhodopsin photopigment states. These protocols are useful to screen for mutations affecting bi-stable photopigment systems.&#160;

ショウジョウバエ光受容体の光色素レベルを測定するための電気生理学的方法

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is ...