私たちは、実りある議論のために生物学、フリブール大学の学科で私たちの仲間に感謝します。私たちは、ハエの株式を提供するためのブルーミントンストックセンターに感謝します。この作品は、財政的にスイス国立科学財団（PP00P3_123339）とSGSにベルックス財団によってサポートされていました

1。幼虫飼育<ol><li> °Cコーンミール媒体に光とタイマーを装備フライインキュベーター内で、12時間の明-12-時間明暗サイクルの下で25℃大衆文化の中で株や遺伝子交配を飛ばしてください。 </li><li>流体ペースト（H 2 Oを蒸留3〜4ミリリットルで希釈し、背の酵母10g）を形成するために水にバッカー酵母を希釈する。コー​​ンミール食品に小滴を追加し、バイアルをカバーしています。酵母ペーストにこだわる大人のハエを避けるために、少なくとも1時間乾燥させます。食品の表面に水で希釈された酵母の小滴を置くことは産卵を高めることができます。あるいはパン酵母ペーストを、20％酢酸（AcOH中）（シグマ·アルドリッチ社、スイスA6283-100ML）を用いることができる。このために、コーンミール食品表面上の解決策と普及上のQ-ティップの先端を浸す。 </li><li>コー​​ンミール食品バイアルに成人ハエを（少なくとも四日間古いが、10日以上経過していない）を入れ。嗜好を繰り返すのに十分な幼虫を得るために、各バイアルに十分な大人に置く何回かテストし、統計的な比較を可能にする。遺伝的交配のために、我々は一般的に20人の女性とバイアルあたり5-10男性の最小値を使用しますが、いくつかの行が十分な幼虫の子孫を得るために、より多くの女性を必要とする。 </li><li>大人が12時間のために卵を産むと新しいバイアルに転送することができます。大人は、朝と夕方に転送することができます。我々は通常、7〜10日のために大人を転送し、必要に応じて新たな大人を取る。 </li><li>インキュベーター内のすべての12時間を取られ、卵のコレクションを保持し、25℃で幼虫を育ててみましょう°C、12時間の光12時間明暗サイクル、60％の湿度。採卵後の定数暗闇に視覚的な動作の動きの幼虫（DD）48時間（二明暗周期に相当する）の概日のコンポーネントをテストする。このため、光のない独立したインキュベーターを使用しますが、温度や湿度などの同一の他のすべての条件を保つ。ただ暗期への切り替え前に、DDインキュベーターにバイアルを転送してください。 anothにバイアルを移動する前にえーインキュベータは段ボール箱にバイアルを置くか、または転送中露光（段ボール箱や包装で梱包がインキュベーター間のシフトの前に &quot;ライトオン段階&quot;の間に行われなければならない）を防ぐために、アルミホイルでバイアルを包む。このポイントの後、実験開始まで、任意の露光から動物を防ぐ。 </li><li> 4日後（産卵84から108時間）（4を参照してください。ライト選好テスト、ポイント。4.4。）テストする時点で初期のL3（給餌の幼虫）を収集。 </li></ol> 2。テストのセットアップ<ol><li>一定の温度と湿度の条件で暗い部屋で実験を行う。 </li><li>暗闇の中でシャーレの2象限を維持するには、黒のテープとアルミホイルでふたをカバーしています。そうするために、蓋円周の中心をマークする。また、それらの間の分離の90°と蓋の境界に4点をマーク。それを簡単にするために、上の10センチの長さの4象限に分割20センチ正方形を描くの紙やコンピュータの助けを借りてシート。アルミ箔と黒テープ（ 図1A）の10センチの正方形をカット。アルミ箔の接着二乗で二つの正反対の象限をカバー、それを覆う第1層と黒テープとして、また蓋の境界線をカバーするように注意してください。 このアッセイの過去の2つのわずかの異なる形態で使用されていた。ここでは、ペトリ皿を10 4つの等しい4分割されている&quot;四半期プレート&quot;アッセイを使用しています。代替&quot;ハーフプレート&quot;アッセイではペトリ皿割った半分（半分光にさらされ、暗闇の中で半分）1,7,8です。今日まで2つのアッセイ間に有意差は公表されていない。 </li><li>接着剤などの右側のテーブル上に光源下蓋のマーキングに使用されたものと象限シート。象限の交差点を中心としたペトリ皿の円周をマーク。光源の下で同じ位置に常に試験板を配置するための基準として符を使用します。 </li><li>インストールランプ、シンプルな白い電球（フィリップス、Softtone 5W）や鉄のサポートスタンド（フィッシャーサイエンティフィック、S47808）の助けを借りて、シャーレ上LEDランプ（LEDランプ、80012白、オスラム）のどちらか。全体の試験板を均一に照明されるようにして高さを調整します。彼らは、従来の電球よりも特定の波長を放射するので、我々は強くLEDランプの使用をお勧めします。またLEDは少ない熱を発する。 </li><li>光度計（環境計PCE EM882）の助けを借りて、光強度を調整します。必要に応じて350から760ルクスに必要な光強度を達成するために、上下にランプを移動します。調整可能な電源にランプを接続すると、ランプ（ 図1B）を移動させずに強度を変更するためのより多くの柔軟性を提供します。 </li></ol> 3。プレートの準備<ol><li>蒸留水（ミリポア）で2.5％の寒天を200ml（Sigma-Aldrich社、スイスA5093-500G）を作る。 </li><li>テーブル（90 mmの直径にペトリ皿を置きます;グライナーバイオワン社、4550 Kremsmeinster、オーストリア）行でホットアガロースを注ぐようにする。 </li><li>ソリューションは完全に透明で流体になるまで電子レンジでアガロースを沸かす。溶液は気泡が含まれていないことを確認してください。注意：慎重にテーブルへの交通ソリューションは非常に熱くなっていますので、！ </li><li>全面を均一溶液の薄層で被覆されるまで、ペトリ皿にホットアガロースを注ぎ、2〜3ミリ十分である。アガロース、コーティングする前に、すべてのプレートを凝固を防ぐために迅速になる。プレートがクールダウンしましょう​​。室温でプレートを保存し、唯一の準備の同じ日にそれらを使用しています。 </li></ol> 4。光嗜好テスト<ol><li> ショウジョウバエは、光の赤色波長を感知することができないので、テストの前に光の影響を防ぐために、実験室での赤色光の条件下で動作します。 Tを照らすランプに取り付けられた赤色電球（フィリップス、PFE712E * 8）を使用して、彼は仕事に配置します。 </li><li> 25ですべての実験を通して温度を維持℃にヒーターや冷却装置によってコントロールルームは、温度、必要に応じて。 </li><li>一定の暗さ（セクション1から）で2日間のサイクルで飼育バイアルからいくつかの食品を取る。幼虫は、通常、食品の表面に掘っているので、スパチュラ（フィッシャー·サイエンティフィック、14 373 25A）の助けを借りて、最上層（約5mm深く）を取る。シャーレのふたの外側に食べ物を広げ、いくつかの水を追加して、へらで軽く混ぜる。 </li><li>食物からのL3幼虫を餌集める。光好みがテストされるように、負の走光性が確保されているので、唯一の供給/初期のL3幼虫を選択することが重要です。食品にクロール後期/放浪L3幼虫や大きな幼虫はおそらくすでにphotobehaviorを切り替えた。その前方気門が開いていて、指状に外側へ突出しているため給電L3幼虫（負の走光）を認識することができる。後部気門sが3つの開口部それぞれ、大分枝毛4つのグループを有する。唾液腺は、第二腹部セグメント11に伸びる。ない白色光を使用しないことに対し、顕微鏡下で幼虫のステージングと便利です。赤色光ランプ、実験が必要となる前に、実体顕微鏡下ステージング場合に幼虫を照明することが必要である。 </li><li>水道水で簡単に幼虫を洗浄し、早期供給サード齢幼虫（まだ赤い光で）を収集。試験板に幼虫を移す前に、ペーパータオルや濾紙で余分な水分を吸収する慎重濡れ絵筆で幼虫を取ると。それは動物に害を与えると、その動作に影響を与える可能性があるので、あまりにも過度に幼虫を乾燥させないでください。 </li><li>ぬれた絵筆を使用して慎重にプレートに幼虫を移す。プレートの中央に約30幼虫のグループを配置。すでに象限で調製蓋付きプレートをカバーし、実験の準備が光源の下でプレートを設定します（秒る2）。 </li><li>白色光ランプをオンにして、タイマーを開始。幼虫は5分間プレートに自由に移動しましょう​​、その後、すぐに蓋を取り外して、暗闇の中で光と象限における幼虫の数を数える。マーカーで各幼虫の位置をマークすると、カウントが容易になります。また、プレートの写真を撮ると事後を数える。幼虫は壁にクロールまたは寒天に穴を掘るように、不明確光選好を示す幼虫は中立好みとして考慮されるべきであるし、ちょうど光選好指数が算出された幼虫の総数に含まれる。 </li><li>カウントした後、幼虫でプレートを破棄し、次の実験のための新しいテストプレートと交換してください。後で取扱い及び処分のためのオートクレーブビニール袋で使用プレートを収集します。 </li><li>たら、新しい遺伝子型をテストできる実験に十分な数を実行した。遺伝子型ごとの10-15試験は、分析のために十分です。 </li></ol> 5。データAnalysiの<ol><li>便宜上エクセル（マイクロソフト）またはさらなる統計分析のために、コンピュータ上の起源（オリジンラボ）などのデータシートにデータを転送。つの列の2番目の列の光象限における動物の数で、プレート（ &quot;中立&quot;の幼虫を含む）における動物の合計3分の暗闇の中で動物の数を手配。 </li><li>次の式を使用して、各実験のために闇の優先指数（PREF）を計算します： PREF（闇）=（暗闇の中で幼虫の数 - 光の中で幼虫の数）/幼虫の総数</li><li>グループのための適切な分析と統計的にデータのセットを比較してください。ここでは、統計的に2つのグループを比較するためにウィルコクソン検定を使用する。正規分布の仮定がマニホールドグループと比較して満たされている場合、Tukeyの多重比較- 事後テストは、行うことができます。とANOVA検定</li><li>日サイクルに沿って線やタイムポイント間で明確に比較を示すグラフを作成します。 W電子使用起源ソフトウェア（オリジンラボ）統計的有意性をテストし、適切なグラフを生成するが、他の統計ソフトウェアが役立ちます。 </li></ol>

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著者らは、開示することは何もありません。

説明光選好テストは幼虫の生得photobehaviorを利用しています。アッセイは、確立することが容易であり、低コストで多くの繰り返しを可能にし、光センシングと処理に関する貴重な情報を提供します。実験的なパラダイムは、個人が光や暗い好むどのように多くの比較的迅速定量化を可能にします。このような好みを粗パーセントとしてあるいは、優先指数（PREF）として表示することができ?...

ここでは、暗闇のための幼虫の選好（または光）に基づいて行動アッセイを記述します。幼虫は餌の段階で強いと常ネガ応答（初期のL3にL1）1と反応する。アッセイは、幼虫の羞明の挙動を評価することを目的とし、寒天でコーティングされたペトリ皿の中で自由に移動する幼虫のグループの光や暗い好みを比較している。この行動のアッセイは視覚系の感度は、統合的、時間的可塑性に関する情報を提供するだけでなく、それがさらなる光感受性とそのプロセスが概日システムによって制御されている方法についてのヒントを提供しています。 ショウジョウバエの幼虫の目（とも呼ばれるBowligオルガン、BO）は、光の知覚のための主要な器官である。それぞれの目にはPRの緑感性rhodopsin6（RH6）と4つのPRを表明12光受容体（PR）、8で構成されている青感性rhodopsin5（RH5）2,3を表現。のPRに加えて、ALS幼虫の体壁を覆うプレーヤークラスIVのmultidendriticニューロンは、4,5有害な光強度に対応するために同定されている。また、中央の幼虫の脳に位置するペースメーカーニューロンが脳内で6,7クロック内在青色光センサーとして機能し、光に敏感なタンパク質クリプト（クライ）を発現することが知られている。このアッセイでテストするときに興味深いことに、野生型動物のphotophobicityは、昼と夜のコースの中の異なる時点でのサーカディアンコンポーネントを示しています。明暗好み7のためにテストされたときに採餌L3幼虫の光への応答は夕暮れ夜明け強くphotophobicityと下位photophobicityを示した。 RH6-PRのは必須では間に興味深いだけRH5-PRのは、光の回避のために必要とされる。 、RH5-PRのとRH6-PRの両方が光8で分子時計をリセットすることに関与している。クライ経路は内の適切な行動の出力を調整するために、他の光センシング経路と調整する必要がありますその日のコースです。 PRの中でアセチルコリンは光回避行動と同様、分子時計の同調に不可欠な役割を果たしている。のPRから概日ペースメーカーニューロンにアセチルコリン神経伝達をブロックすると、光闇の好みアッセイ8羞明の応答を低減します。同じアッセイを採用し、ニューロンの2つの対称のペアは最近ショウジョウバエ 9の三齢幼虫の光優先を切り替えることが同定されている。動物はおそらく適切なpupariationのサイトを見つけるために食べ物を残したときにニューロンのこれら二つのペアは、後期幼生の段階で機能していることがあります。しかし、視覚的な経路が相互作用し、概日的に幼虫の視覚的な動作を制御する方法の問題は、主に未回答のままです。光優先アッセイは異なる光質下概日時点、フライラインとサーカディアン状態間の比較を可能にします。アッセイは、容易に調製と安価であり、私は以前に役立っているnの幼虫の光派生動作を記述し、勉強するには、いくつかのラボ。

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Agar</td>
 <td>Sigma-Aldrich</td>
 <td>A5093-500G</td>
 <td>2.5%; Sigma-Aldrich, 9471 Buchs, Switzerland </td>
 </tr>
 <tr>
 <td>Petri dishes</td>
 <td>Greiner Bio-One GmbH</td>
 <td>633180</td>
 <td>90-mm diameter; Greiner Bio-One GmbH, 4550 Kremsmeinster, Austria</td>
 </tr>
 <tr>
 <td>LEDs Lamp</td>
 <td>OSARAM </td>
 <td>80012 White</td>
 <td>LED lamp, 80012 White</td>
 </tr>
 <tr>
 <td>Environment Meter</td>
 <td>PCE</td>
 <td>PCE EM882</td>
 <td>Lux, Temp, RH%</td>
 </tr>
 <tr>
 <td>Thermostatic cabinet</td>
 <td>Aqua Lytic (Liebherr)</td>
 <td>ET636-6</td>
 <td></td>
 </tr>
 <tr>
 <td>Light timer</td>
 <td>Timer T</td>
 <td>6185.104</td>
 <td>230V/50HZ (check specifications for your country)</td>
 </tr>
 <tr>
 <td>Universal thermostat</td>
 <td>Conrad</td>
 <td>UT200</td>
 <td></td>
 </tr>
 <tr>
 <td>Humidifier</td>
 <td>Boneco</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Balck tape</td>
 <td>Tesa</td>
 <td></td>
 <td>5 cm </td>
 </tr>
 <tr>
 <td>Glue</td>
 <td>Uhu</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>lncubator lamp</td>
 <td>Phillips</td>
 <td>Softtone</td>
 <td>5W</td>
 </tr>
 <tr>
 <td>Timer clock</td>
 <td>Ziliss</td>
 <td></td>
 <td>Ziliss, Switzerland</td>
 </tr>
 <tr>
 <td>Excel Software</td>
 <td>Microsoft</td>
 <td></td>
 <td>Excel</td>
 </tr>
 <tr>
 <td>Origin Software 8.5</td>
 <td>OriginLab</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Backer Yeast</td>
 <td>Migros Switzerland</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Iron support stand 17X28CM</td>
 <td>Fisher Scientific</td>
 <td>S47808</td>
 <td></td>
 </tr>
 <tr>
 <td>Acetic acid</td>
 <td>Sigma Aldrich</td>
 <td>A6283-100ML</td>
 <td>20% acetic acid dilluted in H2O</td>
 </tr>
 <tr>
 <td>Red light lamp</td>
 <td>Phillips</td>
 <td>PFE712E*8C</td>
 <td></td>
 </tr>
 <tr>
 <td>Spatula </td>
 <td>Fisher Scientific</td>
 <td>14-373-25A</td>
 <td></td>
 </tr>
 <tr>
 <td>Power supply</td>
 <td>EA</td>
 <td>EA PS 2042-06B</td>
 <td>Optional</td>
 </tr>
 <tr>
 <td>Aluminium foil</td>
 <td>Prix Coop</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Heater</td>
 <td>GOON</td>
 <td>NSB200C</td>
 <td></td>
 </tr>
 <tr>
 <td>Microwave Oven</td>
 <td>Intertronic</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Standard corn meal fly food</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Destilled water </td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

light preference assay to study innate and circadian regulated photobehavior in drosophila larvae

光は様々なレベルで動物の行動を制御するための環境シグナルとして機能します。 ショウジョウバエの幼虫の神経系は、光情報が処理され、迅速かつ概日行動の間で共有される方法についての基本的な質問に答えるためにユニークなモデルとして使用されます。光にさらされたときに、ショウジョウバエの幼虫はステレオタイプ回避行動を表示します。比較的単純な明暗嗜好試験を適用することができる光依存性挙動を調査する。脊椎動物と節足動物では部分的に視覚入力を感知すると、処理に関わる神経経路は、光の概日情報を処理したものと重複する。光センシングシステムと概日システムが行動の出力が調整さ保つためにどのように相互作用するかの魅惑的な質問は、主に未踏のままです。 ショウジョウバエは脳内の神経細胞の数が少ないために、これらの質問に接近する影響を与える生物学的モデル、および遺伝的なツールの可用性です神経manipul用ATION。提示明暗選好アッセイは、走光性の概日制御を含む視覚的な行動の範囲の調査を可能にします。

上記プロトコルに続いて、我々は、野生型カントン-Sの初期三幼虫スタジアムで明暗好みをテストした二つの異なる概日時間にCT0とCT12を飛ぶ。大人は、12時間の光-12-時間暗を飼育し、12時間のために卵を産むために残っていた。幼虫は同じ明暗政権下で最初の二日間育つ。我々は一定の条件（概日時計のフリーランニング）の下で概日変調をテストしたかったので、テストが（ 図3A）</s...

Watch this Scientific Journal Video about Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae at JoVE.com

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

ここでは、ショウジョウバエの幼虫のために明暗選好テストを記述します。このアッセイは、光センシングと処理photobehaviorの自然免疫と概日規制に関する情報を提供します。

に自然免疫とサーカディアン規制Photobehaviorを勉強する光プアッセイ<em&gt;ショウジョウバエ</em&gt;幼虫

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer ...

light-preference-assay-to-study-innate-circadian-regulated

Research

JoVE Journal

Neuroscience

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

12.8K ビュー.  University of Fribourg. ここでは、ショウジョウバエの幼虫のために明暗選好テストを記述します。このアッセイは、光センシングと処理photobehaviorの自然免疫と概日規制に関する情報を提供します。

ビデオ: Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

のレーザーキャプチャーマイクロダイセクションショウジョウバエ末梢ニューロン

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

神経科学

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal's endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila manifesting altered circadian or sleep properties.

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

We describe procedures for recording daily locomotor activity rhythms of Drosophila and subsequent data analysis. Locomotor activity rhythms are a reliable behavioral output of animal circadian clocks and are used as the standard readout of clock function when studying circadian mutants or examining how the environment regulates the circadian system.

に概日リズムと睡眠のパラメータを研究するために運動活性を測定するショウジョウバエ</em

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) ...

Measuring Circadian and Acute Light Responses in Mice using Wheel Running Activity

Circadian rhythms are physiological functions that cycle over a period of approximately 24 hours (circadian- circa: approximate and diem: day)1, 2. They are responsible for timing our sleep/wake cycles and hormone secretion. Since this timing is not precisely 24-hours, it is synchronized to the solar day by light input. This is accomplished via photic input from the retina to the suprachiasmatic nucleus (SCN) which serves as the master pacemaker synchronizing peripheral clocks in other regions of the brain and peripheral tissues to the environmental light dark cycle3-7. The alignment of rhythms to this environmental light dark cycle organizes particular physiological events to the correct temporal niche, which is crucial for survival8. For example, mice sleep during the day and are active at night. This ability to consolidate activity to either the light or dark portion of the day is referred to as circadian photoentrainment and requires light input to the circadian clock9. Activity of mice at night is robust particularly in the presence of a running wheel. Measuring this behavior is a minimally invasive method that can be used to evaluate the functionality of the circadian system as well as light input to this system. Methods that will covered here are used to examine the circadian clock, light input to this system, as well as the direct influence of light on wheel running behavior.

This article will review methods that can be used to determine circadian function and light responsiveness in mice.

ホイール実行中のアクティビティを使用して、マウスの概日リズムと急性ライト応答を測定する

Circadian rhythms are physiological functions that cycle over a period of approximately 24 hours (circadian- circa: approximate and diem: day)1, 2.  They ...

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 ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

でProprioceptorsの可視化<em&gt;ショウジョウバエ</em&gt;幼虫および蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

で食欲連想嗅覚学習<em&gt;ショウジョウバエ</em&gt;幼虫

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

げっ歯類におけるランニングホイール活動の概日リズムの記録および分析

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

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 ...

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 ...