私たちは、原稿を改善するために自分の仕事のために4の匿名査読に感謝します。我々は、MMSにMBGとYG、および大学·ド·ブルゴーニュ·研究のフランスの省への財政支援のためのセンター国立デラルシェルシュ科学研究に感謝します。 YGの実験室での研究は、欧州研究会議（ERCの開始グラント、GliSFCo-311403）、通信社国立·デ·ラ·ルシェルシュ（ANR-JCJC、GGCB-2010）、Conseilの地域·ド·ブルゴーニュ（フェーバー）、およびCNRSによって資金を供給される。

1。始める前に<ol><li>安定した強固な行動の表現型を持つisogenized参照ストックを使用してください。すべての潜在的なコントロールは、異種背景対立遺伝子を運ぶことができるので、この株を選択するための一般的な規則はありません。 </li><li>後の工程のために必要な他のすべての株式を戻し交配するために、この対照株を使用してください。この戻し交配工程は、通常、2〜3同質遺伝子リファレンス男性5に（相同染色体間の可能な交差を可能にするために）単一の処女の女性の少なくとも5連続した×印で表されます。この手順は異なるフライ銘柄間の遺伝的背景を均一化することが重要です。 </li><li> 25℃で12時間の明/暗サイクルで、標準的なコーンフラワー（9％）、酵母（10％）、および寒天培地（1.5％）、抗生物質（0.4％メチルパラヒドロキシベンゾエート）で補完上ショウジョウバエストックを維持する℃、 </li><li>遠赤色光の下で恒温室（25℃）での化学感覚の実験を実現（視覚的な手がかりの寄与を排除するため、および化学感覚信号に集中する）。定期的に各実験の間に十分な換気をお部屋の空気を更新。 </li></ol> Y字迷路アッセイを使用して2。嗅覚応答<ol><li>試験前に湿ったペーパータオルを含むガラス管中で25℃で16〜18時間のためにハエを飢え。 </li><li> 2ガラスバイアルに小さいプラスチックバイアル（積載バイアル）にY字コネクターに参加。 3バイアルにコネクタをリンクするために、しっかりと密封されたY字型迷路を取得するために、発泡栓を通過する1ミリリットルピペットチップを使用してください。 「読み込み」を形成するために、二つの &quot;罠&quot;のバイアルを形成するために、（それがその決定をした後にその場のいずれかのリターンを避けるために〜2ミリメートルの直径）2ピペットチップの狭い端をカットし、1ピペットチップの大端チューブ（ 図1A）。 </li><li>単に「トラップ」バイアル（ 図1B）を接続する前に、1個〜直径6mmの濾紙を置き各バイアル。 1濾紙上の着臭剤溶液40μlを追加し、第二の濾紙上の対応する溶媒を40μl。 </li><li> 「ローディング」バイアルに10 4から9日齢のハエをご紹介します。それは行動10に強い影響を持つため、この転送中のCO 2の麻酔を使用しないでください。むしろ氷の上に簡単な冷却を使用しています。麻酔ハエの適切な操作は、可能な限り多くの対象者へのストレスを制限することが重要である。 <ol><li>視覚刺激を極力回避すること（可能な加熱源を制限するためにLED電球を使用）遠赤色光下で25°CでY字型迷路一連のテストを実行する。 （; 図1C左の、または右の匂い物質の入ったチューブおよびロードチューブフロントや背面に）Y字迷路の向きを交互にするように注意してください。 </li><li>ハエのための数時間は、付臭剤や溶剤を含むトラップバイアル中に入力することができます。 participatiを高めるために24時間後にハエを数える最大80％以上にし、最大嗅覚インデックス値（シモネ、私信）を提供する。 </li><li> - /ロードされたハエの総数（溶媒チューブ内の数字臭気管内の数値）：次の式を使用して結果の嗅覚指標を算出。 </li></ol></li><li>洗浄Y字迷路は、セットアップを次のように一晩RBS 35 MDに解体セットアップを浸す。徹底的に水道水ですすぎます。最終的に脱イオン水で洗浄し、完全に乾くましょう。 </li></ol> 3。データの統計分析を<ol><li>データおよび変数に依存してt検定、一元配置ANOVA又は双方向ANOVAを実行する。 </li></ol>

<ol>
	<li>Mombaerts, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+and+ligands+for+odorant,+vomeronasal+and+taste+receptors.">Genes and ligands for odorant, vomeronasal and taste receptors.</a> Nat. Rev. Neurosci. 5, 263-278 (2004).</li><li>Silbering, A. F., Benton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionotropic+and+metabotropic+mechanisms+in+chemoreception:+'chance+or+design.">Ionotropic and metabotropic mechanisms in chemoreception: 'chance or design.</a> EMBO Rep. 11, 173-179 (2010).</li><li>Ziegler, A. B., Berthelot-Grosjean, M., Grosjean, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+smell+of+love+in+Drosophila.">The smell of love in Drosophila.</a> Front. Physiol. 4, (2013).</li><li>Silbering, A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complementary+function+and+integrated+wiring+of+the+evolutionarily+distinct+Drosophila+olfactory+subsystems.">Complementary function and integrated wiring of the evolutionarily distinct Drosophila olfactory subsystems.</a> J. Neurosci. 31, 13357-13375 (2011).</li><li>Grosjean, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+olfactory+receptor+for+food-derived+odours+promotes+male+courtship+in+Drosophila.">An olfactory receptor for food-derived odours promotes male courtship in Drosophila.</a> Nature. 478, 236-240 (2011).</li><li>Woodard, C., Huang, T., Sun, H., Helfand, S. L., Carlson, J. <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+olfactory+behavior+in+Drosophila:+a+new+screen+yields+the+ota+mutants.">Genetic analysis of olfactory behavior in Drosophila: a new screen yields the ota mutants.</a> Genetics. 123, 315-326 (1989).</li><li>Semmelhack, J. L., Wang, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Select+Drosophila+glomeruli+mediate+innate+olfactory+attraction+and+aversion.">Select Drosophila glomeruli mediate innate olfactory attraction and aversion.</a> Nature. 459, 218-223 (2009).</li><li>Budick, S. A., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free-flight+responses+of+Drosophila+melanogaster+to+attractive+odors.">Free-flight responses of Drosophila melanogaster to attractive odors.</a> The Journal of Experimental Biology. 209, 3001-3017 (2006).</li><li>Martin, F., Charro, M. J., Alcorta, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+affecting+the+cAMP+transduction+pathway+modify+olfaction+in+Drosophila.">Mutations affecting the cAMP transduction pathway modify olfaction in Drosophila.</a> J. Comp. Physiol. A. 187, 359-370 (2001).</li><li>Barron, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaesthetising+Drosophila+for+behavioural+studies.">Anaesthetising Drosophila for behavioural studies.</a> Journal of Insect Physiology. 46, 439-442 (2000).</li><li>Ai, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acid+sensing+by+the+Drosophila+olfactory+system.">Acid sensing by the Drosophila olfactory system.</a> Nature. 468, 691-695 (2010).</li><li>Becher, P. G., Bengtsson, M., Hansson, B. S., Witzgall, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flying+the+fly:+long-range+flight+behavior+of+Drosophila+melanogaster+to+attractive+odors.">Flying the fly: long-range flight behavior of Drosophila melanogaster to attractive odors.</a> J. Chem. Ecol. 36, 599-607 (2010).</li></ol>

著者らは、開示することは何もありません。

当社のY字迷路プロトコルは、先に説明したプロトコル9に基づいています。しかし、我々は2つ​​の大きな違いをご紹介します。まず、我々は、彼らが溶媒または溶媒を加えた匂い物質を含むバイアル中に入ることを決定したら、戻ってからハエを防ぐために、狭いピペットチップを使用しています。これらの狭い先端には、Y字型迷路における匂い物質の拡散を制限するために有用?...

味や嗅覚を通して化学受容は動物の生存のための重要な感覚モダリティである。これは、危険や食料源を検出するためだけでなく、社会的相互作用のために必要不可欠な手がかりを提供します。また、その再生に必要なのセックスパートナーを見つけるために、動物に役立ちます。 20年以上にわたり、「嗅覚受容体と嗅覚系の組織の彼らの発見のための「2004年のリチャード·アクセルとリンダ·バックして、作業を受賞ノーベル賞を含む集中的な研究では、分子および細胞の基盤を明らかにするために行われている嗅覚1,2。 嗅覚を分析する科学者のための好みの動物モデルの一つは、Dであるキイロ 。この昆虫は、哺乳類で同様の細胞および分子匂いコーディング戦略を共有しています。科学界は、このショウジョウバエにおける匂い物質の役割を研究するために、多様な行動パラダイムを使用しています。これらのテストは、courtshiなどのマルチモーダルアッセイが含まれる嗅覚を含む様々な感覚モダリティは、男性の求愛3を引き出すために重要であるPをテストします。他のアッセイはまた、より具体的には臭気物質の役割に取り組むために開発されてきた;これらは、T-迷路、Y字迷路、トラップアッセイ、4フィールドアリーナと風のトンネルの4,5,6,7,8が含まれています。 この記事では、Dを使用して堅牢な嗅覚応答を提供する単純な修正Y字迷路アッセイを提示キイロ 。私たちのセットアップは、前述した方法9とは逆に、エンドチップを使用しています。したがって、我々のY字型迷路は2つの利点がある。ハエが彼女の選択をした後は第一に、それは、システム内の任意のリターンを避けることができます。第二に、Y字迷路のすべての分野で匂い物質の交換に制限されます。 ショウジョウバエは 、多くの場合、付臭飽和を回避するために使用される空気流に非常に敏感であるので、この最後の利点は重要である。空気流と実験設定を調整するには時間とコストを消費するであろう。そのため、私たちのY字迷路アッセイは、EFを表しficientやショウジョウバエの嗅覚の性能をテストするための高速な方法。

<table border="0" cellpadding="0" cellspacing="0" width="835">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td>Drosophila Polystyrene tube</td>
			<td>VWR europe</td>
			<td>734-2255</td>
			<td>30 x 25 mm&#160; 
			Y-maze</td>
		</tr>
		<tr>
			<td>Drosophila Borosilicate tube</td>
			<td>Dijon verre</td>
			<td></td>
			<td>95 X 25 mm 
			Y-maze</td>
		</tr>
		<tr>
			<td>Foam stopper</td>
			<td>Dutscher</td>
			<td>999038</td>
			<td> 
			Y-maze</td>
		</tr>
		<tr>
			<td>Y-shaped connector</td>
			<td>Europrix</td>
			<td>11020605</td>
			<td> 
			Y-maze</td>
		</tr>
		<tr>
			<td>100-1000&#181;l pipet tips</td>
			<td>Corning</td>
			<td>4868</td>
			<td>Join the following pipet tips to the Y-shaped connector. Cut 2 pipet tips at 65 mm from the wide end, and connect the narrow end (with a &#8764;2 mm opening) to 2 test vials. These openings will limit the U-turns once the flies enter the tubes containing the odors. Cut 1 pipet tip at 35 mm from the wide end, and connect it to the loading vial. 
			Y-maze</td>
		</tr>
		<tr>
			<td>Far-Red LED Bulb</td>
			<td>Rubin-Lacaque</td>
			<td>0RB180238</td>
			<td>625-630 nm</td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>45725</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylacetic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>P16621</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast</td>
			<td>Sensient Flavors Strasbourg</td>
			<td>1018880464</td>
			<td></td>
		</tr>
		<tr>
			<td>Cornmeal</td>
			<td>eurogerm</td>
			<td>Farine de ma&#239;s</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Kalys</td>
			<td>HP-697-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl hydroxy 4 benzoate</td>
			<td>VWR international</td>
			<td>25605293</td>
			<td></td>
		</tr>
	</tbody>
</table>

testing drosophila olfaction with a y-maze assay

環境からのシグナルを検出することは、それらの生存を確保するための動物のために必須である。この目的のために、彼らは直接接触を介して、またはより長い範囲で作用する揮発性分子に対する応答を表し嗅覚を通じて、このようなビジョン、機械刺激受容、聴覚、味覚を通じてchemoperception、環境手がかりを使用しています。揮発性の化学分子が危険、食料の供給源、または個人の間で通信するための検出にほとんどの動物のために非常に重要なシグナルである。 キイロショウジョウバエの嗅覚の細胞および分子基盤を探る科学者のための最も一般的な生物学的なモデルの一つです。この小さな昆虫の嗅覚能力を強調するために、我々は古典的にマウスで使用Y字迷路試験に基づいて修正された選択肢のプロトコルを記述します。 Y字型迷路を用いて得られたデータは、より良い動物が彼らの絶え間なく変化する環境に対処する方法を理解するための貴重な情報を提供します。私たちは、勉強するステップバイステップのプロトコルを導入このY字迷路アッセイを使用してその場探索応答に匂い物質の影響。

図1Dは、このY字型迷路アッセイを用いて二つの代表的応答を示す。カントン-Sは、それらが有意に10％フェニル酢酸を回避しないのに対して強く、蒸留水で希釈した10％酢酸を避ける男性が。これらのアッセイは、ローディングバイアルに一緒に入れレプリケートごとに10人の男性に基づいている。このプロトコルは、時々、平均値の大きな標準誤差につながることができます。?...

Watch this Scientific Journal Video about Testing Drosophila Olfaction with a Y-maze Assay at JoVE.com

Testing Drosophila Olfaction with a Y-maze Assay

私たちは、Y字型迷路アッセイを用いてショウジョウバエの行動上の匂い物質の影響を明らかにするために選択試験を提示する。

検査<em&gt;ショウジョウバエ</emY字迷路アッセイで&gt;嗅覚

Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, ...

testing-drosophila-olfaction-with-a-y-maze-assay

Research

JoVE Journal

Neuroscience

Testing Drosophila Olfaction with a Y-maze Assay

14.8K ビュー.  UMR-6265 CNRS. 私たちは、Y字型迷路アッセイを用いてショウジョウバエの行動上の匂い物質の影響を明らかにするために選択試験を提示する。 

ビデオ: Testing Drosophila Olfaction with a Y-maze Assay

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

用いて、化合物の生理活性をスクリーニングするためのペアNanoinjectionと電気生理学的アッセイキイロショウジョウバエジャイアントファイバシステム

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

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 ...

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 ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

テスト視機能のための簡単​​な行動アッセイ<em&gt;アフリカツメガエル</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

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 ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by stressed conspecifics due to an odorant released during stress known as the Drosophila stress odorant (dSO). Through the use of the T-maze apparatus, one can quantify the avoidance of the dSO by responder flies in a very affordable and robust assay. Conditions necessary to obtain a strong performance are presented here. A stressful experience is necessary for the flies to emit dSO, as well as enough emitter flies to cause a robust avoidance response to the presence of dSO. Genetic background, but not their group size, strongly altered the avoidance of the dSO by the responder flies. Canton-S and Elwood display a higher performance in avoiding the dSO than Oregon and Samarkand strains. This behavioral assay will allow identification of mechanisms underlying this social behavior, and the assessment of the influence of genes and environmental conditions on both emission and avoidance of the dSO. Such an assay can be included in batteries of simple diagnostic tests used to identify social deficiencies of mutants or environmental conditions of interest.

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

社会回避での定量化のための簡単​​なアッセイ<em&gt;キイロショウジョウバエ</em

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

でMARCMベースのクローンのMechanosensationための行動アッセイ<em&gt;キイロショウジョウバエ</em

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

Taste Preference Assay for Adult Drosophila

Olfactory and gustatory perception of the environment is vital for animal survival. The most obvious application of these chemosenses is to be able to distinguish good food sources from potentially dangerous food sources. Gustation requires physical contact with a chemical compound which is able to signal through taste receptors that are expressed on the surface of neurons. In insects, these gustatory neurons can be located across the animal's body allowing taste to play an important role in many different behaviors. Insects typically prefer compounds containing sugars, while compounds that are considered bitter tasting are avoided. Given the basic biological importance of taste, there is intense interest in understanding the molecular mechanisms underlying this sensory modality. We describe an adult Drosophila taste assay which reflects the preference of the animals for a given tastant compound. This assay may be applied to animals of any genetic background to examine the taste preference for a desired soluble compound.

Taste Preference Assay for Adult Drosophila

Taste is an important sensory process which facilitates attraction to beneficial substances and avoidance of toxic substances. This protocol describes a simple ingestion assay for determining Drosophila gustatory preference for a given chemical compound.

大人のための味の好みアッセイ<em&gt;ショウジョウバエ</em

Olfactory and gustatory perception of the environment is vital for animal survival.  The most obvious application of these chemosenses is to be able to ...

Dynamic Quantitative Sensory Testing to Characterize Central Pain Processing

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain processing is present in many chronic pain conditions and can influence responses to specific therapies. Thus, the ability to precisely describe the state of central pain processing has profound clinical significance in both prognosis and prediction. Because it is not practical to record neuronal firings directly in the human spinal cord, surrogate behavior tests become an important tool to assess the state of central pain processing. Dynamic QST is one such test, and can probe both the ascending facilitation and descending modulation of incoming nociceptive signals via TS and CPM, respectively. Due to the large between-individual variability in the sensitivity to noxious signals, standardized TS and CPM tests may not yield any meaningful data in up to 50% of the population due to floor or ceiling effects. We present methodologies to individualize TS and CPM so we can capture these measures in a broader range of individuals than previously possible. We have used these methods successfully in several studies at the lab, and data from one ongoing study will be presented to demonstrate feasibility and potential applications of the methods.

By assessing pain in response to repetitive or different types of standardized stimuli, dynamic quantitative sensory testing (QST) can reveal changes in the central processing of pain. We present methods to optimize and individualize two dynamic QST measures: temporal summation (TS) and conditioned pain modulation (CPM).

ダイナミック定量的感覚検査は、中枢性疼痛処理を特徴づけるために、

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain ...

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

キャピラリーフィーダーアッセイにおいて食物摂取を測定します<em&gt;キイロショウジョウバエ</em

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...

Methods Article

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