私たちは、幼虫のクロールデータを生成するためAstha Maltareに感謝します。私たちは、原稿上の彼のコメントを与えるために博士はニコラス·ランソン·ジュニアに感謝します。この作品は、ジョンズホプキンス大学でALSのためにロバートパッカードセンター（UBP）および筋萎縮性側索硬化症協会（UBP）がサポートされていました。と国立精神衛生研究所（CDN）からR01MH083689。

A.幼虫クロールアッセイ 1。幼虫コレクション<ol><li>ハエの8オンスボトル（10-15男性+女性10月15日）を設定します。 </li><li>ハエの明確な瓶その後、ハエは24時間の卵を産むしましょう​​。 （新しいボトルに大人を転送し、必要に応じて繰り返します）。 </li><li> 3-4日のためにボトルをインキュベートし、または3齢幼虫になるまで表示されます。 </li><li>幼虫のボトルに20％ショ糖の100 mlを、20分間放置 - 50を追加します。幼虫は上にフロートします。 </li><li>チップと25ミリリットル血清ピペットを用いて収集した幼虫は、カットオフ、およびメッシュバスケットに場所。 </li><li> 2倍の脱イオンH 2 Oでメッシュバスケットに幼虫を洗う幼虫は現在の実験の準備が整いました。 </li></ol> 2。薬で幼虫を治療する<ol><li>溶液5％スクロース+薬剤を含む5 mlのビーカーに幼虫の希望数を輸送するためにブラシを使用しています。 </li><li>少なくとも15分間幼虫してみましょう。</li><li>メッシュバスケットに薬剤処理した幼虫を入れ、すすいでください。彼らは今、使用する準備が整いました。 </li></ol> 3。運動アッセイ（測定総走行距離や体壁の収縮） <ol><li>に個々の幼虫を輸送するためにブラシを使用します。 <ol><li> 0.2センチメートル2グリッドとグラフ用紙の上に、2％アガロース（以前に注ぎ、硬化さ）を含む15センチメートルペトリ皿。 <ol><li> 1分で交差するグリッド線の数をカウントします。 </li></ol></li><li>希薄酵母糊液を含有するガラス解剖皿のよく。 <ol><li>解剖顕微鏡下で観察しながら1分で蠕動収縮を（後方移動= 1収縮への完全な前方）カウントされます。 </li></ol></li></ol></li><li>幼虫の希望する番号がカウントされるまで繰り返します。 </li></ol> B.ラピッド負の走地性（RING）プロトコルを反復このアッセイは、もともとドでしたガルガーノら 5によってスクライブ。 <ol><li>食品（または食品+薬）を含む標準バイアルに光CO 2麻酔と場所の下に新たに浮上して成人男性のハエを収集します。 </li><li> CO 2（定常状態薬物濃度の蓄積が適切であれば）からの回復を可能にするために2〜3日室温（ベンチの上に。〜22°C）でハエを維持しています。 </li><li>準備されたポリスチレンバイアルに麻酔なしで 25ハエについて転送します。 </li><li>リング装置（図1）にハエのバイアルを組み立てる。 </li><li>ハエは15〜20分間、邪魔されず、環境に順応することができます。 </li><li>この時間の場所にデジタルカメラの間に装置の前に〜1メートル（プラットフォームバイアルの中間の高さでレンズの中心を揃えるために必要な場合に）、フォーカス、装置の上にカメラをズームし、タイマーを設定する3.0秒。 </li><li>慎重にdistにならないように左手でリング装置のホールドを取るURBは、ハエ、そして右手でタイマーを保持します。 </li><li>急激にタップが難しいバイアルの底にすべてのハエをノックダウンするのに十分であることを保証する、ベンチの表面に三回に装置をシャットダウン]をタップします。 </li><li>第三タップの完成と同時に、3秒のカウントダウンタイマーを起動します。 </li><li> 3秒で写真を撮る。 </li><li> 1分間タイマーをリセットして起動します。この時間の間にカメラの電源をリセットして装置に焦点を当て、3秒のタイマーの別のチャネルを設定します。 </li><li> 1分は、手順1.7から1.10の後に</li><li> 5月6日の試験の合計した後、コンピュータに画像をアップロードして開くようにお気に入りの画像ビューアを使用して、平均身長のスコアは各バイアルのため登った。 </li><li>登った高さの平均を比較して、異なるグループの統計分析を実行します。 </li></ol> Cの求愛と交尾アッセイ<ol><li>午前中にまず最初に、FLIの明確なだけでなく生産のボトルESは、使用することができます。 </li><li>一日のコース（3〜4時間毎）を介して、新たに浮上して性的にナイーブな雄と雌を収集します。 <ol><li>媒体とバイアルまたはチューブ内で個別に男性を配置します。 </li><li>バイアル/チューブあたり一緒に行われ5-6の女性。 </li><li> 5日間暗12時間の明/下25°Cで収集されたハエを分離します。 </li><li>交配ホイールの室に一人の女性を転送します。 </li><li>交配ホイールのチャンバーに1男性を転送します。 </li><li>次の動作のために解剖顕微鏡下でペアを守ってください。 <ol><li>オリエンテーション（女性に向かって男性向き） </li><li> （男性が女性をタップする）をタップ</li><li>翼の歌（男性が拡張し、片翼を振動させる） </li><li> （男性が女性の性器を舐める）ブーツ</li><li>カーリング（オス自体は下で腹部をカール） </li><li>交尾の試み（女性をマウントしようとしたときに、アクティビティをカーリング） </li></ol></li></ol></li><li> 1の観察0分または成功した交尾まで、交尾（求愛指数を計算するための）と同様に、首尾よく与えられた動作を実行ペアの数になるまで求愛行動に従事し、それぞれの現象が発生した時刻（待ち時間）、合計時間を記録（周波数）。野生型のペアの100％が一般的に5分以内に交尾します。 </li><li>計算する 交尾までの合計時間で割った求愛で費やされた時間を割って求愛指数（CI）。野生型のペアの場合、これは通常0.6から0.8の間の範囲。 </li></ol> D.描写結果アッセイをクロール通常の野生型幼虫は約3センチメートル/分、展示〜1分間に40から50体壁の収縮をさまよいます。我々は最近、障害6を登る幼虫のクロール欠陥の減少寿命と成人を示しています。FUS / TLS関連の筋萎縮性側索硬化症のショウジョウバエモデルを開発した。我々は表現oを対象とし運動ニューロン（OK-371-GAL4ドライバー）へとf野生型とFUS / TLSの変異体は、幼虫のクロールアッセイを行った。以下に示すように、野生型のFUSの発現が約6cmの幼虫のクロール能力が低下し、一方、野生型幼虫は、最大12 cmまでクロールします。 FUS / TLSのALSの原因となる変異R521Cを発現している動物は約1 cm /分をクロールし、それらのクロールの動き（図1）は非常に重度の障害を示しています。 負の走地性リングアッセイ若い野生型の成虫は、3秒の時間で〜4,5センチ（時間が与えられたために与えられた平均の高さを定義するには、異なる株または活性レベルに対応するために3秒から調整することができ、平均登山高さを持つべきひずみ/処理）。下部に残っているハエは​​、0の値が割り当てられています。それがその後の高さを測定するために、個々のフライの位置を決定することが困難になるので、バイアルあたり25以上のハエを使用することをお勧めされていません。 NOの脱感作は、我々が採用していることを離れて1分間隔で6つの連続した​​試験までに観察されている。それは、使用されるバイアルに入れ、新しいハエは新鮮なバイアルと同程度に上昇しないので、収集されたデータの初期セットの後に、このアッセイにおけるポリスチレンのテストバイアルを再使用しないことが重要です。 <img alt="図1" src="/files/ftp_upload/3795/3795fig1.jpg" /> 図1。リングアッセイのセットアップ。デジタルカメラは、ポリスチレンバイアル中にハエを含む装置の前に〜1メートルに配置されています。装置の上にカメラをフォーカスとズーム、および3.0秒にタイマーを設定します。 <img alt="図2" src="/files/ftp_upload/3795/3795fig2.jpg" /> 図2。を使用して幼虫のクロールアッセイからの代表的なデータは、異所性運動ニューロンドライバ（OK371-GAL4）の制御下にUAS-FUS WTと、UAS-FUS R521Cを表現飛ぶ。 <pクラス= &quot;jove_content&quot;&gt;

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我々は、開示することは何もありません。

ショウジョウバエの行動は密接に遺伝的要因と環境要因によって調節される。我々は、他のものは、以前の行動やショウジョウバエ 5-19にモデル化する人間の神経変性疾患への飛行に関連する遺伝子を調べるためにデータを収集するためにここで説明するアッセイを使用しています。クロールアッセイのために、第3齢幼虫の慎重な選択が重要なステップです。?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>スクロース</td><td>フィッシャー·サイエンティフィック</td><td> S5-500 </td></tr><tr><td>アガロース</td><td>インビトロジェン</td><td> 16500-500 </td></tr><tr><td> 6オンスショウジョウバエのボトル</td><td>ジェネシー科学</td><td> 32から130 </td></tr><tr><td>ペイントブラシ（＃1） </td><td>テッド·ペラ社</td><td> 11859 </td></tr><tr><td> 食品成分を飛ぶ </td><td></td><td></td></tr><tr><td>コー​​ンミール</td><td>フィッシャー·サイエンティフィック</td><td> NC9109741 </td></tr><tr><td>寒天</td><td>ジェネシー科学</td><td> 66から104 </td></tr><tr><td>糖蜜</td><td>フィッシャー·サイエンティフィック</td><td> NC9349176 </td></tr><tr><td>プロピオン酸</td><td>アクロス</td><td> 14930-0010 </td></tr><tr><td> Tegosept </td><td>頂点</td><td> 20から258 </td></tr><tr><td>エタノール</td><td>フィッシャー科学的な</td><td> BP2818-4 </td></tr><tr><td>酵母</td><td>ジェネシー科学</td><td> 62から107 </td></tr></tbody></table>

methods to assay drosophila behavior

キイロショウジョウバエ 、ショウジョウバエは、例えば癌、心血管疾患や様々な神経疾患1などのヒト疾患の広い範囲の分子機構を研究するために使用されています。私たちは、幼虫の移動、大人のクライミング能力（リングアッセイ）、 ショウジョウバエの求愛行動を決定するためのシンプルかつ堅牢な行動アッセイを最適化した。これらの行動のアッセイは、ハエの行動に関する遺伝的要因と環境要因の役割を研究するために広く適用されます。幼虫のクロール能力は確実にショウジョウバエの幼虫のクロール能力の早期の変化を決定するため、また、それらの運動に薬やヒト疾患遺伝子の効果を（トランスジェニックハエの）を調べるために使用することができます。彼らがそうでなければ評価することができたところこれらのハエが大人に生きていけないように遺伝子の発現または廃止は、蛹の大人の段階で致死性が発生した場合に幼虫のクロールアッセイは、より適切になります。この基本的なssayは、 ショウジョウバエの幼虫で追加行動反応を調べるために、明るい光やストレスと組み合わせて使用することもできます。求愛行動は広く性的行動の遺伝的基礎を調査するために使用されており、また同様に学習や記憶など、活動との調整を調べるために使用することができます。 ショウジョウバエの求愛行動は、視覚、聴覚、および嗅覚信号を含む様々な感覚刺激の交換を伴う成功した交尾で絶頂に達する十分に特徴付けられ、モータの動作の複雑な一連につながる男女の間に。伝統的な成人登山のアッセイは、（負の走地性）は、異なる試験2月4日の間には有意な変化で、退屈な、労働集約的で、時間がかかります。急速（RING）負の走地性を反復アッセイ図5に示すように 、再現性に敏感な、高スループットのアプローチ大人の運動を定量化すると負の走地性bを提供し、より広く用いられてプロトコルを介して多くの利点がありますehaviors。リングアッセイでは、いくつかの遺伝子または薬物治療は、ハイスループットスクリーニングのアプローチは、実験のほうが従順することで、同時に動物の多数を使用してテストすることができます。

Watch this Scientific Journal Video about Methods to Assay Drosophila Behavior at JoVE.com

Methods to Assay Drosophila Behavior

キイロショウジョウバエでは、世紀の1以上にわたり、多くの重要な生物学的過程の分子および細胞基盤を理解するために使用されている遺伝的に行動的に扱いやすいモデル系である。 ショウジョウバエがよく飛ぶ行動の遺伝的洞察を得るために利用されてきた。

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 ...

Research

JoVE Journal

Neuroscience

Methods to Assay Drosophila Behavior

42.6K ビュー.  Louisiana State University Health Sciences Center. キイロショウジョウバエでは、世紀の1以上にわたり、多くの重要な生物学的過程の分子および細胞基盤を理解するために使用されている遺伝的に行動的に扱いやすいモデル系である。 ショウジョウバエがよく飛ぶ行動の遺伝的洞察を得るために利用されてきた。

ビデオ: Methods to Assay Drosophila Behavior

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

昆虫における単一感覚子録音キイロショウジョウバエとハマダラカ</em

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

神経科学

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

で逃避行動のOptogenetic刺激<em&gt;キイロショウジョウバエ</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

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 ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

勉強するリニアアガロースチャネルを使用します<em&gt;ショウジョウバエ</em&gt;幼虫クロールの動作

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

使用して報酬を求める行動の変化を測定するための簡単な方法<em&gt;キイロショウジョウバエ</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Quantification of Drosophila Grooming Behavior

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a grooming assay protocol and novel chamber design that is cost-efficient and scalable for either small or large-scale studies of Drosophila grooming. Flies are dusted all over their body with Brilliant Yellow dye and given time to remove the dye from their bodies within the chamber. Flies are then deposited in a set volume of ethanol to solubilize the dye. The relative spectral absorbance of dye-ethanol samples for groomed versus ungroomed animals are measured and recorded. The protocol yields quantitative data of dye accumulation for individual flies, which can be easily averaged and compared across samples. This allows experimental designs to easily evaluate grooming ability for mutant animal studies or circuit manipulations. This efficient procedure is both versatile and scalable. We show work-flow of the protocol and comparative data between WT animals and mutant animals for the Drosophila type I Dopamine Receptor (DopR).

This protocol describes a scalable individual grooming assay technique in Drosophila that yields robust, quantitative data to measure grooming behavior. The method is based on comparing the difference in dye accumulation on the bodies of un-groomed versus groomed animals over a set period of time.

ショウジョウバエのグルーミング行動の定量

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

ショウジョウバエ島アッセイにおける歩行行動の高スループット解析

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.

嗅覚ニューロンの光刺激に対する応答のショウジョウバエ幼虫の行動を追跡

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

ショウジョウバエメラメラノガスターにおける学習誘発シナプス可塑性の生体内光学カルシウムイメージング

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using reflexive tests that allow inferences regarding pain's nociceptive component but provide little information about the affective or motivational component of pain. Developing tests that capture these components of pain are therefore translationally important. Hence, researchers need to use non-reflexive behavioral assays to study pain perception at that level. Mechanical conflict-avoidance (MCA) is an established voluntary non-reflexive behavior assay, for studying motivational responses to a noxious mechanical stimulus in a 3 chamber paradigm. A change in a mouse's location preference, when faced with competing noxious stimuli, is used to infer the perceived unpleasantness of bright light versus tactile stimulation of the paws. This protocol outlines a modified version of the MCA assay which pain researchers can use to understand affective-motivational responses in a variety of mouse pain models. Though not specifically described here, our example MCA data use the intraplantar complete Freund's adjuvant (CFA), spared nerve injury (SNI), and a fracture/casting model as pain models to illustrate the MCA procedure.

The mechanical conflict-avoidance assay is used as a non-reflexive readout of pain sensitivity in mice which can be used to better understand affective-motivational responses in a variety of mouse pain models.

マウスの疼痛行動を測定するための機械的衝突回避アッセイ

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using ...