Name: a behavioral assay for mechanosensation of marcm-based clones in drosophila melanogaster
Uploaded: 2015-12-30T14:00:00.000+00:00
Duration: 5 min 48 s
Description: Watch this Scientific Journal Video about A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster at JoVE.com

著者らは、ハエの株式の寛大な共有のためのブルーミントンストックセンター、Liqun羅、チャールズZuker、とリリーとユウ農月に感謝し、資金調達のために、以下の希望：（JDとSW）はSOMAの-URM、学士フォード学部夏フェローシップを、レニーとアンソニー・M・マーロン、MD &#39;63夏の研究フェローシップ（CLおよびDLに）（SW）は、BD社サマー研究員を経て（TO）ジェームズ・C. &#39;75とジェーンColihan夏の研究フェローシップ（DLに） （TO）にホーリークロス、ストランスキー財団サマー研究員の大学の卒業生/親サマー研究基金。ラボですべての作業をサポートするためのホーリークロスカレッジで生物学科と学部長のオフィスに感謝します。

1.十字MARCMのハエを生成するには<ol><li> 22℃で、または標準のショウジョウバエメディアコーンミール糖蜜培地（0.13％Tegosept; 23.5グラム/ Lの酵母; 60グラム/ Lのコーンミール、60ミリリットル/ Lの糖蜜; 4ミリリットル/ Lの酸ミックス6.5グラム/ Lの寒天）上のハエを維持12時間の明/暗サイクルです。 注：標準温度よりも低い、健康MARCM-株を維持するために使用されます。これは14日に卵と成人期の間の発達の時間を延長します。 </li><li>上流活性化配列（UAS-GFP）、GAL80リプレッサータンパク質、GAL4転写因子の制御下（GFPを必要な遺伝的要素を含むMARCM対応在庫から - （8日齢約1）処女雌を使用してくださいリコンビナーゼ、およびFRT組換え部位を駆動する熱ショック）。 注：私たちのMARCMすぐ銘柄は以下の遺伝子型含まれていますelavGal4、UAS-CD8-GFP、HS-flipaseを。 FRT40A、tubGal80 / CYO。 tubGal4 / TM3Sb。 注：特定のFRT部位は、突然変異の位置に依存しますゲノムインチ例えば、FRT40Aは、NOMPC（MARCMプロトコルの詳細については10を参照のこと）などの第2染色体の左腕、上の変異をテストするために使用されます。 </li><li>目的の変異とそれに対応するFRT部位含ま男性にクロスMARCM対応の処女雌（; NOMPC3、FRT40A / CYO;例えば、W-を+ / +）。効果的なMARCM誘導に制御するために、同じFRT部位を含む男性のハエを使用して第2のクロスを設定したが、突然変異のありません。十字架で1男性：女性5のおおよその比率を使用してください。 </li></ol> 2.時限産卵<ol><li>ハエは、少なくとも一晩交配させた後、新鮮な培地で新しいバイアルに大人を移動します。ハエが再び新しいバイアルに十字架を転送する前に、約4時間、暗い環境で卵を産むようにします。産卵期間の開始時間と終了時間に注意してください。 注意：4時間のintervaの終了前に、メディア上の卵の数が多いがあるように表示されたときに短い間隔が許容されますリットル。 <ol><li>熱が後で衝撃を与えたことを摂氏22度で12時間の明/暗サイクルで十分な卵の数（約20以上）とストアでバイアルを保管してください。 注：メディア上の卵の数が少ないバイアル（約20未満）は行動試験のために飛ぶと効率の利益のためにそれは彼らがさらなる試験のために保持されないことをお勧めします大人の十分な数が得られることはほとんどありません。 注意：私たちのハエは、午前8時から午後8時までにライトで発生します。午後8時と午後8時 - 午前零時 - したがって、我々は、通常4〜時限交配を行い、夕方の時間点が高い卵密度につながる発見しました。ハエが暗いキャビネット内に室温で産卵した後、インキュベーター中で、通常の明暗サイクルを乱さないように、次の日にインキュベーターに戻します。 </li></ol></li></ol> 3.有糸分裂組換えを誘導するために熱ショックを実行します<ol><li>約85 - 産卵後100時間、場所のV1時間37℃で水浴にIALS。水位がバイアル内のメディアの高さの上に達したが、完全に溺れてから幼虫を防ぐためにバイアルを水没しないことを確認してください。 </li><li>水浴からバイアルを取り出し、室温で1時間の回復期間を可能にします。 </li><li>バック1時間、摂氏37度の水浴中にバイアルを置きます。 </li><li>羽化するまで22℃で12時間の明/暗サイクルでインキュベーターで水浴とストアからバイアルを取り外します。 </li></ol>行動試験のための準備4。 <ol><li>羽化後、氷上でハエを麻酔。 MARCMのためのすべての遺伝的要素を含み、GFP発現マーキングされたクローンは、おそらく親銘柄で使用観察可能な表現型マーカーに基づいて観察される示す遺伝子型を有するハエを選択します。虹彩切除術ハサミでハエを斬ります。 注：例えば、私たちは遺伝子マーカーなしでハエのためCYO（カーリー翼）及びSb（無精ひげ毛）同定するために、選択されまし​​たモザイク領域を作成するために必要なすべての遺伝的要素を含むことになる年度ハエは、GFPでマークされました。 注意：斬首が刺激されると飛んから大人を防止する必要があります。 </li><li>閉じられた、湿った環境でのヘッドレスハエを置き、約10可能に - 回復の20時間を。右自身さらなる試験のため乱さハエをのみを使​​用します。テストは断頭の30時間以内に飛びます。 </li></ol> 5.行動試験<ol><li>蛍光解剖顕微鏡下で断頭ハエを観察することによって、毛外部感覚器官のGFPでマークされたホモ接合クローンを同定します。剛毛名および左側または右側を記録します。任意の潜在的なバイアスを排除するために、毛刺激を行うラボメンバーは毛、野生型または変異体の遺伝子型に盲目でなければなりません。 </li><li>硬い髪や細かい鉗子でフライ本体に向かって毛と脚の再を観察マークされたGFPを偏向させることによりグルーミング反射を引き出しますsponse。彼らの足を移動しないハエに刺激し、ゼロのスコアを毛に応じて自分の足を持ち上げハエに1のスコアを与えます。 注：以前の研究6,7は、特定の毛を刺激することによって誘発された足の応答を特徴としています。これは、各剛毛に応答足とその特定の毛の刺激に反応するハエの割合が含まれています。私たちは私たちがVandervorstの分類6に基づいて対応することが期待足の動きを記録しました。 </li><li>離れて2分間隔の5件の試験を実施します。 </li></ol>

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このプロトコルは 、ショウジョウバエでmechanosensationに影響を与える変異をスクリーニングするために、大人の行動アッセイを利用しています。突然変異体のコレクションは、成人としてのスクリーニング不可能に致死P因子の変異が含まれているため、このプロトコルは最初のリーと羅によって記述複雑な遺伝的手法を利用しています、（1999）、成人の致死性を回避するための...

Humans rely on the ability to convert mechanical stimuli from their environment, such as touch, pressure, vibration, or sound waves, into sensory information that can be processed by the nervous system, in a process termed mechanotransduction. Many of the overall mechanistic features of mechanotransduction between humans and invertebrates are the same1, making Drosophila a useful model to study the molecular mechanisms of mechanotransduction. Drosophila melanogaster contain two sets of specialized sensory organs (Type I and II) that are capable of converting mechanical stimuli into action potentials. Type I mechanoreceptors have a neuron with a single dendrite or sensory process, surrounded by three support cells2. The Type I mechanoreceptors include bristle mechanoreceptors, hearing sensitive chordotonal organs (the Johnston's organ), and campaniform sensilla that convey information about wing beats3. The bristles that cover the dorsal side of the fly are the most abundant and easily accessible of the Type I organs. Similar to the extracellular environment of hair cells associated with hearing and balance in vertebrates, the support cells surrounding fly mechanosensitive neurons secrete a high potassium endolymph that creates an unusual concentration gradient for potassium1. Mechanosensitive neurons, of both the mammalian and fly systems, utilize this high extracellular potassium to depolarize the cell. In response to mechanical stimulation of the macrochaete bristles towards the body wall, the sensory neurons respond with a burst of action potentials driven by this potassium depolarization of the cell membrane4. The Drosophila sensory neurons that innervate bristles resemble the mechanosensitive cells of other organisms, including vertebrate hair cells, in both structure and function1,5. The accessibility of Drosophila external sense organs to experimental manipulation and the abundance of genetic techniques available to researchers make Drosophila an excellent model system to investigate the molecular underpinnings of mechanosensation. 
In the fly, stimulation of a single sensory neuron that innervates a bristle leads to an observable behavioral response. Stimulation of different bristles evokes specific, reproducible behavioral responses, depending on the bristle that is stimulated. Upon tactile stimulation, decapitated wild-type flies exhibit a complex grooming reflex wherein they clean the area near the stimulated bristle with a patterned set of leg movements5-7. When homozygous for known single-gene mutations that interfere with learning7 or coordination and locomotor activity5, flies respond abnormally to mechanical stimulation. This grooming reflex is therefore a useful tool to study the effects of single-gene mutations on a specific, replicable behavior.
The robust behavioral response to stimulation of a single macrochaete bristle holds the potential to assist in identifying new genes involved in mechanotransduction. This protocol being used to test a collection of mutant flies for the absence of a behavioral response to indicate that the mutation interferes with mechanosensation. In the mutant collection selected for screening, the mutations cause lethality before adulthood, and therefore would be impossible to test using traditional adult behavior screens. Originally, this collection of lethal p-elements was combined with FRT recombination sites to test cell growth defects in clones. The clones were made specifically in the eye because adult flies can survive in the lab setting without functional vision8. However, removal of all mechanosensation can cause adults to be severely uncoordinated or die before eclosion5. This protocol uses a mosaic approach to circumvent the lethality of the mutations and allow for adult stage testing. A genetic technique called Mosaic Analysis with a Repressible Cell Marker (MARCM)9 is used to generate homozygous mutant cells in a limited number of adult fly sensory organs, while the rest of the organism remains heterozygous. These MARCM flies readily survive until adulthood, yet the bristles are homozygous for the lethal genes of interest.
MARCM allows for regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP), while the rest of the organism remains heterozygous at that particular locus and unmarked9. MARCM combines individual p-element mutations with five common genetic elements: GFP under the control of an upstream-activating sequence (UAS-GFP), Gal80 repressor protein under control of a general promoter (tub-Gal80), Gal4 transcription factor under control of a general promoter (tub-Gal4), the FRT recombinase enzyme expressed through a heat-shock controlled promoter, and a FRT recombination site10. By driving mitotic recombination through a heat-shock activated recombinase, a limited number of cells are made homozygous for the mutation and marked with GFP. GFP expression is repressed in heterozygous cells by the presence of Gal80 on the wild-type copy of the chromosome. 
A heat-shock protocol for MARCM was optimized to induce recombination in the fly bristle external sense organs, while much of the organism remains heterozygous, and thus unmarked with GFP. Mosaic flies generated using this protocol contained homozygous mutations most frequently in the post alar or dorsal central bristles on the surface of the notum. 
We have tested the utility of this combination of MARCM and the grooming behavior screen with a known mechanosensitive mutant, NOMPC. The ion channel, no mechanoreceptor potential C (NOMPC), is an essential component of the mechanotransduction pathway in Drosophila5,11-13. NOMPC belongs to the transient receptor potential (TRP) superfamily of cation channels5 and satisfies all of the criteria to qualify as a mechanosensitive channel in Drosophila14,15: 1) NOMPC is expressed in the ciliate tips of type 1 sensory neurons of Drosophila 13,16-18, 2) NOMPC null larvae do not have an electrical response to tactile stimulation13, 3) Ectopic expression of NOMPC in touch insensitive cells can induce sensitivity to mechanical stimulation13, 4) heterologous expression of NOMPC in Schneider 2 cells yields a mechanosensitive channel13, and 5) NOMPC adult mutants display defects in their response to mechanical stimulation5. Given this evidence, we predicted that NOMPC mutant clones would show an altered or inhibited grooming response in response to mechanical stimulation of bristles.
A MARCM-stock containing the NOMPC mutation was developed for use in a proof of principle experiment of the grooming assay. Mosaic flies were stimulated at macrochaete bristles containing homozygous NOMPC mutant cells. We expected an inhibition of the grooming response following stimulation of the macrochaete bristle. We found that only 2 of 14 mutant bristle flies tested gave a single response to repeated stimulation; most did not respond to stimulation of the homozygous mutant bristle. Having confirmed that this MARCM-based behavioral assay produces an abnormal grooming reflex in a known mechanosensitive mosaic mutant, this technique can be used in a screen for additional mechanosensitive mutations.

<table><tbody><tr><td>Brewers Yeast (25 lb)</td><td>MP Biomedicals&amp;nbsp;</td><td>ICN90331225&amp;nbsp;</td><td>Fly Food</td></tr><tr><td>Corn (25 lb)</td><td>MP Biomedicals&amp;nbsp;</td><td>ICN90141125&amp;nbsp;</td><td>Fly Food</td></tr><tr><td>Agar (1 lb)</td><td>MoorAgar Inc.</td><td>41004</td><td>Fly Food</td></tr><tr><td>Tegosept (5 kg)</td><td>Genesee</td><td>20-259</td><td>Fly Food</td></tr><tr><td>Molasses (1 Gallon)</td><td>Sugarmill Brand - Thomsen Food Service</td><td>0 2625</td><td>Fly Food</td></tr><tr><td>Propionic Acid</td><td>Fisher</td><td>A258-500</td><td>Fly Food</td></tr><tr><td>Phosphoric acid</td><td>Fisher</td><td>A260-500</td><td>Fly Food</td></tr><tr><td>Drosophila Vials, Narrow (PS)</td><td>Genesee</td><td>32-109</td><td>Fly Cultures</td></tr><tr><td>6oz Square Bottom Bottle (PP)</td><td>Genesee</td><td>32-130</td><td>Fly Cultures</td></tr><tr><td>Flugs - Plastic Fly Bottles</td><td>Genesee</td><td>49-100</td><td>Fly Cultures</td></tr><tr><td>Rayon Balls, Large</td><td>Genesee</td><td>51-100</td><td>Fly Cultures</td></tr><tr><td>Droso-Filler, Narrow</td><td>Genesee</td><td>59-168</td><td>Fly Food Preparation</td></tr><tr><td>Droso-Filler, Bottles</td><td>Genesee</td><td>59-170</td><td>Fly Food Preparation</td></tr><tr><td>8A-C / gear driven lab stirrer with c-clamp mount&amp;nbsp; 1/15 HP, 700 rpm variable speed, 115 V, 50/60 Hz</td><td>Cleveland Mixer</td><td>8A-C</td><td>Fly Food Preparation</td></tr><tr><td>Water jacketed Kettle</td><td></td><td></td><td>Fly Food Preparation</td></tr><tr><td>Diurnal Growth Chamber</td><td>Forma Scientific</td><td></td><td>Temperature and light/dark cycle controlled</td></tr><tr><td>Water bath</td><td>VWR</td><td></td><td>For heat shock</td></tr><tr><td>MicroScissors&amp;nbsp;</td><td>Fine Science Tools</td><td>&amp;nbsp;15000-08</td><td>For removing heads</td></tr><tr><td>Fluroscence Dissecting Microscope</td><td>Zeiss</td><td>SteREO Discovery V8&amp;nbsp;</td><td>With GFP cube (KSC295-814D) band pass filter</td></tr><tr><td>Fluroscence Light Source</td><td>Zeiss</td><td>X-Cite 120</td><td>Fiber optic light pipe makes this easy to configure&amp;nbsp;</td></tr><tr><td>Camera for Scope</td><td>Zeiss</td><td>AxioCam ICc1</td><td></td></tr><tr><td>Image acquistion software</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Ice bucket</td><td></td><td></td><td>for cold anthesia</td></tr><tr><td>Homemade cold anthesia tray</td><td></td><td></td><td>for cold anthesia decapitation</td></tr><tr><td>Plastic boxes</td><td></td><td></td><td>for recovery of decaptitated flies in humid environment</td></tr></tbody></table>

a behavioral assay for mechanosensation of marcm-based clones in drosophila melanogaster

そのため哺乳類の内耳の有毛細胞への構造的および機能的相同性のため、 ショウジョウバエの外部感覚器官を神経支配するニューロンはmechanosensationの研究のための優れたモデルシステムを提供します。このプロトコルは、mechanosensationを妨害する変異を同定するために使用することができショウジョウバエでシンプルなタッチの動作について説明します。 macrochaeteの触覚刺激は、ハエの胸郭に毛のいずれかの第1または第3の脚からグルーミング反射を誘発します。 （例えばNOMPCなど）メカノと、または反射弓の他の側面に干渉する突然変異は、グルーミング応答を阻害することができます。大人の行動の伝統的な画面は開発中の重要な役割を持っている変異体を逃したであろう。代わりに、このプロトコルは、exprで生成され、マークされたホモ接合変異体の細胞の限られた領域を可能にするために、抑制性細胞マーカー（MARCM）でモザイク解析でタッチスクリーンを組み合わせました緑色蛍光タンパク質（GFP）のession。異常行動応答についてMARCMクローンを試験することにより、より伝統的な方法では見逃されたであろうmechanosensationに関与する新しい遺伝子を検索するために致死P因子突然変異のコレクションをスクリーニングすることが可能です。

このプロトコルの成功は、主にテスト可能なハエの全収率に依存して、熱ショックの効果は、有糸分裂組換え、2分間隔で刺激されたハエにおける強固な行動反応を得る能力を誘導します。胚の推定12.5％が必要MARCM要素およびG​​FP発現の可能性を有する孵化する成人の推定16.67％を含有することを考えると、それは時限中の卵の高い数を得ることが重要である産みま?...

Watch this Scientific Journal Video about A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster at JoVE.com

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

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

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.

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

a-behavioral-assay-for-mechanosensation-marcm-based-clones-drosophila

Research

JoVE Journal

Neuroscience

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

10.2K ビュー.  College of the Holy Cross. 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.

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

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

ショウジョウバエの幼虫の匂い駆動型の動作の高分解能測定

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

生物学

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

の形態素解析ショウジョウバエ</em遺伝モザイクを使用>幼虫末梢感覚ニューロンの樹状突起と軸索

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

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 ...

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 ...

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

Testing Drosophila Olfaction with a Y-maze Assay

Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, mechanoreception, hearing, and chemoperception through taste, via direct contact or through olfaction, which represents the response to a volatile molecule acting at longer range. Volatile chemical molecules are very important signals for most animals in the detection of danger, a source of food, or to communicate between individuals. Drosophila melanogaster is one of the most common biological models for scientists to explore the cellular and molecular basis of olfaction. In order to highlight olfactory abilities of this small insect, we describe a modified choice protocol based on the Y-maze test classically used with mice. Data obtained with Y-mazes give valuable information to better understand how animals deal with their perpetually changing environment. We introduce a step-by-step protocol to study the impact of odorants on fly exploratory response using this Y-maze assay.

Testing Drosophila Olfaction with a Y-maze Assay

We present a choice test to reveal the influence of odorants on Drosophila behavior using a Y-maze assay.

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, ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

のコンピュータ追跡することによってアッセイ嗅覚行動<em&gt;ショウジョウバエ</em&gt; 4象限オルファクトメーターで

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

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 ...

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 ...

でMARCMベースのクローンのMechanosensationための行動アッセイ

要約

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この動画の章

ショウジョウバエの幼虫の匂い駆動型の動作の高分解能測定

の形態素解析ショウジョウバエ幼虫末梢感覚ニューロンの樹状突起と軸索

アッセイのメソッドショウジョウバエ行動

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

検査

のコンピュータ追跡することによってアッセイ嗅覚行動

冷痛覚のための新規アッセイ

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