R01AA016837（JCB）とP20AA017828（AGDとJCB）：これらの研究は、国立衛生研究所、国立アルコール依存症とアルコール乱用研究所からの補助金によってサポートされていました。

アッセイの前日に実行するための手順1. <ol><li>新鮮な線虫の増殖培地へのL4段階のワームをピック（NGM）プレートはOP50 E.の芝生を播種20 CO / Nにおける大腸菌 、文化、それらを。各アッセイ条件は、10ワームが必要です。ワームのO / N損失を可能にするためにワームの過剰を選ぶ。 <ol><li>最初の日の成人のみアッセイ動物;多くの変異体は野生型よりも遅い速度で成長する。試験した全ての動物は最初の日の成人になるように発達の遅れを持っている株でピッキングのタイミングを調整します。 </li></ol></li></ol> 2.手順アッセイの日に実行する方法<ol><li>アッセイのための準備： <ol><li>標準播種していない60×15ミリメートルNGMプレート上でアッセイを行う。蓋をオフにして、2時間37℃で使用する全てのプレートを乾燥させる。それぞれの実験試験のために、4乾燥したNGMプレートを使用します。これらは、0 MMと400 mMのエタノールアッセイプレートとそれに付随する順化プレートになります。 注意：NGMプレートの使用はここに重要です。プレートの組成の違いは、特にそれらのオスモル濃度は、強く行動にエタノールの用量応答に影響を与えることができる、これは、少なくとも部分的には、動物20によって蓄積されたエタノール量の変化に起因する。 NGM寒天は160ミリオスモルである。 </li><li>乾燥させた後、アッセイに使用される非シードNGMプレートの各計量および体重を記録します。空のプレートの重量に基づいて、プレート内でのメディアの音量を決定します。ボリュームの寒天の重量の変換を近似するために、メディアは等量の水と同じ重さと仮定する。 注：私達の最も一貫した結果は、元の10ml容量が8.3〜8.9ミリリットルの間の乾燥後の体積を有することを十分に脱水したNGM培地で見出されている。 2時間の乾燥時間の代替は、プレートがインキュベータの違いを考慮して、この容量範囲に達するまで乾燥させることである。 </li><li> 4銅リング（1の内径を溶かす。6センチメートル）炎で鉗子でworms.Grasp各リングの異なる遺伝子型または治療群のための囲い、熱として作用するプレートの表面に（ブンゼンバーナーからの強い炎がうまく機能）約3秒。まだ「スキップ」からそれを防ぐために、鉗子でリングを保持している間すぐにプレートにリングを置きます。 <ol><li>リングはリング上のいくつかの点で鉗子で軽く押し下げて、寒天の表面に対して平らに載ることを確認してください。リングを配置する場合、追加の3つのリングのための余地を残すように注意してください。 </li><li>すべての4つのカメラの視野内になるように互いにできるだけ近くにそれらを配置するように注意しながら、プレートの表面に3つの追加の銅リングを溶かす。 注：寒天で良好なシールを作るには、アッセイの間のリングでワームを保つために不可欠である。板の上に周りのスキップリングが寒天で正しいシールを作る可能性は低いとワームは穴を掘ることを可能にするとワームを可視化を妨げる寒天を、傷跡があります。 </li><li>各アッセイプレート乾燥し、播種していないと何エタノールを受信しませんする必要があります添付「順化」のプレートを準備。これらのプレート上の4つの銅リングを置きます。 </li></ol></li><li>リング自体の視野内に書き込まないように注意しながら、次のリングで使用するワーム株とそれぞれのリングに、プレートの下にラベルを付けます。それに付随する順化プレートとアッセイプレート上のラベルと一致。 </li><li>最終濃度が0mMのまたは400mMのエタノール（体積に対する重量）となるように各アッセイプレートに追加する100％エタノールの量を計算する。各実験（n = 1 の場合 ）、1 0mMの一つの400mMエタノール板が存在する。順化プレートはエタノールを受信しません。プレートの表面のまわりでそれをピペットで、エタノールを追加します。プレートをシールするために実験用フィルムを使用し、それが2時間ベンチ上に平衡化させる。 </li><li> 1.5時間が経過すると、アッセイの順応ステップ、ステップ2.2を開始する。 </li></ol></li><li>アッセイプレート：歩行運動アッセイを実施<ol><li>慎重に順化板上の銅リングの中心に各実験群の10ワームを転送する。ワームのピックで軽くこすることによって、この時点で寒天上に表示されている任意の食品を削除します。同株は、臨床試験で同じ順序でプレート上に置かれないように、実験群は、実験的試験を横切るプレート上に置かれる順序を変化させる。 注：目標は、順応プレート上の食品がアッセイプレートに移された場合、ワームは、食品の周りに集約され、運動に対する薬物の効果が隠されるため、食品の最小限の量でプレートに動物を転送することである。 </li><li>これはワームが穴を掘ることができますし、アッセイを妨害するように、ピックと寒天の表面を壊さないように注意してください。室温で30分間ワームをインキュベートする。 </li><li>各プレートのイニシエーションの間に適切な間隔があることを確認します。経験豊富な実験者は、&lt;1.5分で4輪用の40の動物、10 /リングを移動させることができますが、2分までの任意の間隔が許容される。標準的なアッセイは、10〜12分であり、0 MMおよび400 mMのエタノールの両方の曝露の30-32分で記録映画を持っています。 </li><li>第二のムービーが開始しなければならない前に、最初のムービーファイルの保存を可能にするために、約2分30秒離れて400 mMの露光用の0 mMの曝露と順化板に順化プレートを開始します。 </li><li> 30分間の順応期間の後、アッセイプレートに順応プレートからワームを転送する。それらは各プレートの補完の間のタイミングを追跡する、順応プレートに添加したのと同じ順序でアッセイプレートにワーム（0 mM以下の400mMエタノール）を転送する。気化エタノールの損失を最小限にするために実験用フィルムでプレートを密封する。 </li><li> U薄い刃の平坦化ワームにすくい運動は、平坦化されたピックの上にワームを収集するために選ぶSE。それはピックにワームを接着するのに役立ちますように、一般的にワームを搬送する際に用いられる細菌、を使用せずにアッセイプレートに播種していない順化プレートからワームの転送を実行します。 注意：プレートの最初のウォームをプレートに加え、最後のワームよりも長いエタノールにさらさ​​れるので、動物がこの段階で転送される速度が非常に重要であり、以前の時点でいくつかの時間依存効果を示し得る。これは株は最初の実験の反復全体のプレート上に配置されて回転させるための主な理由である。 </li></ol></li><li>歩行アッセイを実行します。ビデオ撮影<ol><li>そのような2048x2048 7.4と0.5倍の顕微鏡対物レンズ、0.8倍の倍率とカメラとして、視野（約42x42 mm 2の正方形）のすべての4輪の同時イメージングを可能にし、顕微鏡/カメラの組み合わせを使用して、81、m個の画素CCD。 </li><li>強度閾値ステップで物体認識を助ける視野を横切っての照明にも使用する（下記参照）。 3「X3」バックライトはうまく動作します。 </li><li>イメージメディア側を上に配置プレート上のワームの光源を送信した伝統的な顕微鏡と比較してバックライトを使用する際に失われたコントラストを生成し（蓋ダウン）、。 </li><li>移動するワームのタイムラプスムービーをキャプチャする画像解析ソフトウェアを準備します。 2分（1​​20フレーム）ムービーとして、1秒毎に12ビットグレースケール画像をキャプチャするためのソフトウェアを設定します。まだ十分な画像解像度を維持しつつ、出力ファイルのサイズを低減するために、1024×1024ピクセルの画像をキャプチャする2×2ビニングモードを使用する。 </li><li>映画を記録します。最後のワームがそのプレート上に配置した10分後に最初のプレート（0 mMのエタノール）の120フレームの動画撮影を開始します。ムービーファイルを保存します。 </li><li>第二のプレート（400 mMのエタノール）を記録します。このプロセスを繰り返します最後にワーム30分後に始まる両プレートは、各露光のために30〜32分の時間点を捕捉する第一プレート（0 mMのエタノール）上に置いた。 注：分析すべき次のプレートの記録を開始する前に、各キャプチャセッション後の画像ファイルを保存するのに十分な時間を確保してください。 </li><li>アーカイブされた映画の未来プルーフ用の8ビット256グレースケールのTIFFファイルに各ムービーのコピーを変換し、これらの映画が開かれ、そのようなImageJの28など、他のパブリックドメインのオープンソースイメージングソフトウェアプログラムによって分析されることを可能にする。 注：ここで説明する画像解析ソフトウェアは、独自のファイル形式を使用しています。 </li></ol></li><li>イメージプロ（ImagePro）ソフトウェアを使用して映画の分析。 <ol><li>キャプチャされたら、画像-Proソフトウェアまたはその同等Plusを使用して映画を分析する。 注：他のオブジェクトトラッキングソフトウェアの様々な利用可能で正常に複数C.を追跡するために使用されたこと1時間29などで虫オブジェクト識別ステップは同様の原理に基づいているソフトウェアは、合理的に、ここで説明したものに代替できた。記載されている方法は、画像プロプラスソフトウェアのバージョン6.0から6.3と7.0であって、イメージ·プロプラスソフトウェアの新しいバージョンでは、マイナーな違いがある場合がございます。 </li><li> 2分（1​​20フレーム）のセグメントに映画を分析します。まず、背景を平坦とウォームオブジェクトのコントラストを向上させるために画像にフィルタを適用する。次のようにフィルタを選択：メニュー&gt;プロセス&gt;フィルタ&gt;強化&gt;フラット化：パラメータ：背景=ダーク。 WIDTH = 20 Pixのを備えています。 </li><li>フィルタリングステップの後に映画を再保存が、常にそのフィルタリングされていない形でオリジナルのムービーを保持します。 <ol><li>配置され、銅リングとオーバーラップするような大きさの関心（ROI）の円形領域とは別に、各環における動物の運動を分析します。コマンド...メニュー&gt;メジャー&gt;トラックオブジェクトとワームを特定し、追跡する。これはトラッキングDが表示されますATA表ウィンドウ。追跡オプションボタンは、特定のトラックが除外されるように、任意の実験的なアーティファクトを制限することができます。 </li></ol></li><li>自動追尾]タブで、次のパラメータを使用します。トラックパラメータ：速度制限（検索半径）= 400ミクロン/フレーム、加速限界=オート、最小の総トラック長を=400μmで、優勢なモーションタイプ=混沌。トラックパラメータ内のオブジェクトは：部分トラック= YES、最小長= 21フレーム、トラッキング予測深さ= 1フレームを許可します。 </li><li>追跡プロセスを開始するには、自動的にすべてのトラックを探すカウント/サイズオプション]ダイアログボックスと追跡ダイアログボックスを開き、ボタンをクリックして機能する。分析用のワームオブジェクトを強調するために、軽量化背景ピクセルを無視するワームピクセルのグレースケール強度を使用して重要なしきい値ステップを提供してカウント/サイズ]ダイアログボックス、内の強度範囲の選択のためのマニュアルのオプションを選択します。 <ol><li>強度閾値SLIを調整しますDERをすべて暗いオブジェクトを強調包括的な範囲を作成するには（下限値を設定する上限、1を設定するには、1）。 0-4,095の規模0-1,500の範囲は、より細かいチューニングのための良い出発点である。 </li><li>そのような銅リング、プレート上の任意の破片のような単一のワームオブジェクトより大きく、小さいオブジェクトを除外するためにサイズのフィルタを適用します。カウント/サイズオプション]ダイアログボックスの2尺度との姿勢の様々な個々のワームのためにサイズをカバーこれを行うには、サイズのパラメータ選択&gt;計測メニュー項目を設定します。 （エリア範囲：28,000-120,000μm2で、境界範囲：600-2,500ミクロン）。 </li><li>変異株を分析するプレート上の他の動物よりも有意に小さい、または大きい場合、設定を変更する必要がないように、最初の動物を追跡​​する前に、株の異なるサイズに対応するために、物体認識のためのフィルタ設定の範囲を広げる半ば分析。 </li></ol> &lt;/ LI&gt; <li>追跡ダイアログボックスで[続行]をクリックしてトラッキング処理を完了します。視覚自動フィルタの設定に基づいて、それを排除する正当な理由がない限り、すべてのワームが表現されていることを確実にするために映画の中の各ワームの進行に出力トラックを比較する。手動でサイズのフィルタ基準を満たして確認された非ワームのオブジェクトの存在によって生産されたトラックを削除します。 </li><li>各フレーム間の各ワームの速度を計算するためのソフトウェアを使用します（距離はフレーム間の1秒あたりのオブジェクトの重心のために旅し）とにおけるワームの人口のためにすべてのトラック全体で各トラックの平均速度と平均速度を表示銅リング。この最終的な平均は実験的な臨床試験の面ではn = 1であると考えています。統計分析とデータアーカイブのためのスプレッドシートプログラムにデータをエクスポートします。 </li><li>データは、プレート上の第一のリングのために記録された後、次のリングにROIを移動させパラメータを変更することなく、追尾処理を繰り返す。 </li><li> ：を通して移動の相対速度を算出する</li><li>相対速度（％）=未処理（400 mM）の速度/（0 mM）の速度×100。 注：別の遺伝子操作は、多くの場合、動物の基礎歩行速度を変える。動物の歩行速度に対するエタノールの効果を決定するために、異なる遺伝子型または条件にわたってこれらの効果を比較することができるようにするために、相対速度を算出する。 </li></ol></li></ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+role+of+unc-79+in+ethanol+responses+in+lightweight+mutant+mice.">Conserved role of unc-79 in ethanol responses in lightweight mutant mice.</a> PLoS Genet. 6, e1001057 (2010).</li><li>Thiele, T. E., Badia-Elder, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+neuropeptide+Y+in+alcohol+intake+control:+evidence+from+human+and+animal+research.">A role for neuropeptide Y in alcohol intake control: evidence from human and animal research.</a> Physiol. Behav. 79, 95-101 (2003).</li><li>Treistman, S. N., Martin, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BK+Channels:+mediators+and+models+for+alcohol+tolerance.">BK Channels: mediators and models for alcohol tolerance.</a> Trends Neurosci. 32, 629-637 (2009).</li><li>Han, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+GWASs+and+human+protein+interaction+networks+identifies+a+gene+subnetwork+underlying+alcohol+dependence.">Integrating GWASs and human protein interaction networks identifies a gene subnetwork underlying alcohol dependence.</a> Am. J. Hum. Genet. 93, 1027-1034 (2013).</li><li>Kendler, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomewide+association+analysis+of+symptoms+of+alcohol+dependence+in+the+molecular+genetics+of+schizophrenia+(MGS2)+control+sample.">Genomewide association analysis of symptoms of alcohol dependence in the molecular genetics of schizophrenia (MGS2) control sample.</a> Alcohol. Clin. Exp. Res. 35, 963-975 (2011).</li><li>Schuckit, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autosomal+linkage+analysis+for+the+level+of+response+to+alcohol.">Autosomal linkage analysis for the level of response to alcohol.</a> Alcohol. Clin. Exp. Res. 29, 1976-1982 (2005).</li><li>Alaimo, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+metabolism+and+osmolarity+modify+behavioral+responses+to+ethanol+in+C.+elegans.">Ethanol metabolism and osmolarity modify behavioral responses to ethanol in C. elegans.</a> Alcohol. Clin. Exp. Res. 36, 1840-1850 (2012).</li><li>Morgan, P. G., Sedensky, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+affecting+sensitivity+to+ethanol+in+the+nematode,+Caenorhabditis+elegans.">Mutations affecting sensitivity to ethanol in the nematode, Caenorhabditis elegans.</a> Alcohol. Clin. Exp. Res. 19, 1423-1429 (1995).</li><li>Mitchell, P. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+concentration-dependent+effects+of+ethanol+on+Caenorhabditis+elegans+behaviour.">The concentration-dependent effects of ethanol on Caenorhabditis elegans behaviour.</a> Pharmacogenomics J. 7, 411-417 (2007).</li><li>Vidal-Gadea, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+selects+distinct+crawling+and+swimming+gaits+via+dopamine+and+serotonin.">Caenorhabditis elegans selects distinct crawling and swimming gaits via dopamine and serotonin.</a> Proc. Natl. Acad. Sci. U S A. 108, 17504-17509 (2011).</li><li>Bettinger, J. C., Leung, K., Bolling, M. H., Goldsmith, A. D., Davies, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+environment+modulates+the+development+of+acute+tolerance+to+ethanol+in+Caenorhabditis+elegans.">Lipid environment modulates the development of acute tolerance to ethanol in Caenorhabditis elegans.</a> PLoS ONE. 7, e35129 (2012).</li><li>Davies, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+genes+influence+toluene-+and+ethanol-induced+locomotor+impairment+in+C.+elegans.">Different genes influence toluene- and ethanol-induced locomotor impairment in C. elegans.</a> Drug Alcohol Depend. 122, 47-54 (2012).</li><li>Newlin, D., Thomson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alcohol+challenge+with+sons+of+alcoholics:+a+critical+review+and+analysis.">Alcohol challenge with sons of alcoholics: a critical review and analysis.</a> Psychol. Bull. 108, 383-402 (1990).</li><li>Ponomarev, I., Crabbe, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+to+assess+initial+sensitivity+and+acute+functional+tolerance+to+hypnotic+effects+of+ethanol.">A novel method to assess initial sensitivity and acute functional tolerance to hypnotic effects of ethanol.</a> J. Pharmacol. Exp. Ther. 302, 257-263 (2002).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9, 671-675 (2012).</li><li>Husson, S. J., Costa, W. S., Schmitt, C., Gottschalk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+track+of+worm+trackers.">Keeping track of worm trackers.</a> WormBook. , (2012).</li></ol>

The authors have nothing to disclose.

C.で利用可能なシンプルな神経生物学と遺伝的ツール虫は、ワームの行動に対するエタノールの影響の分子基盤を研究するために優れたモデルにする。ここでは、エタノール8,10,20,24,25に対する急性行動応答のいくつかの分子および環境メディエーターを同定するために使用されたアッセイを記載する。この方法は、二つの異なるエタノール応答挙動の表現型、初期感度?...

Alcohol use disorders (AUD) are widespread and produce serious health, social, and economic problems. In humans, the susceptibility to developing an AUD is heavily influenced by both genetics and the environment1,2. A strong physiological predictor of abuse liability is the initial level of response (LR) to alcohol (ethanol) that is exhibited by na&#239;ve drinkers3-5. This LR phenotype is influenced by genetics and non-genetic components6. Determining the molecular mechanisms that influence the LR to ethanol is an important goal of the study of ethanol response behaviors.
The nematode, Caenorhabditis elegans, has been increasingly used as a model for studying the effects of ethanol on behavior7-9. There is strong molecular conservation in the machinery of nervous system function between worms and mammals, and several genes that have been shown to influence the LR to ethanol in worms have been shown to influence LR to ethanol in mammals10-16, and have been implicated in abuse liability in humans17-19. 
Ethanol intoxicates worms, which is reflected in a decrease in their locomotion speed. Several different laboratories have developed behavioral assays that differ in several ways, for example, the locomotion behavior that they study (crawling versus swimming11,12,14,20,21) or in the composition of the solutions in which the assays are performed (nematode growth medium versus Dent&#8217;s saline20,22). Interestingly, these diverse assays have yielded somewhat different dose response profiles for the effects of ethanol. These results have pointed to important differences in the underlying behaviors of crawling and swimming9,23, as well as a role for the environmental variable osmolarity in ethanol responses20, and have highlighted the importance of describing experimental detail of the various assays.
An assay to measure the acute effects of ethanol on crawling behavior is presented here. This assay has been used extensively to study the genetic and environmental influences on the LR to ethanol8,10,20,24,25. The mammalian LR phenotype is a composite of at least two components, initial sensitivity to ethanol and acute functional tolerance to ethanol26,27. In worms, the LR phenotype has been shown to be separable into these two components through the use of this behavioral assay. The influences of genetic and environmental manipulations on both phenotypes can be examined using this single assay. Importantly, these two phenotypes are genetically separable.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>C. elegans strains</td>
			<td>Caenorhabditis Genetics Center</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>60 x15 mm Petri plates, triple vented</td>
			<td>Greiner Bio-One</td>
			<td>628161</td>
			<td>Other plate brands will suffice.</td>
		</tr>
		<tr>
			<td>NGM agar</td>
			<td>Various</td>
			<td></td>
			<td>NaCl (3g/L), agar (17g/L), peptone (2.5g/L), 1 mL cholesterol (5mg/mL in ethanol), 1 mL (1M) MgSO4, 1 mL (1M) CaCl2, 25 mL (1M) KPO4, pH=6, 975 mL H2O</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Various</td>
			<td></td>
			<td>e.g. Fisher Scientific #10300</td>
		</tr>
		<tr>
			<td>37&#176;C Incubator</td>
			<td>Various</td>
			<td></td>
			<td>For drying agar</td>
		</tr>
		<tr>
			<td>Digital balance</td>
			<td>Various</td>
			<td></td>
			<td>For determining plate weights and agar volume</td>
		</tr>
		<tr>
			<td>Copper rings</td>
			<td>Plumbmaster</td>
			<td>STK#35583 (48 cap thread gasket)</td>
			<td>1.6 cm inner diameter, 1.8 cm outer diameter copper rings</td>
		</tr>
		<tr>
			<td>100% ethanol</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Bemis</td>
			<td>PM996</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>QImaging</td>
			<td>RET-4000R-F-M-12</td>
			<td>This camera has a large field of view.</td>
		</tr>
		<tr>
			<td>Stereomicroscope with C-mount and 0.5X objective</td>
			<td>Leica</td>
			<td>MZ6</td>
			<td>Discontinued model, M60 is current equivalent.</td>
		</tr>
		<tr>
			<td>Light source</td>
			<td>Schott</td>
			<td>A08923</td>
			<td>3&#8221;x3&#8221;&#160; backlight for even illumination across the field of view</td>
		</tr>
		<tr>
			<td>Imaging and tracking software</td>
			<td>Media Cybernetics</td>
			<td>ImagePro-Plus v6.0-6.3</td>
			<td>Newer versions of the software have tracking functions.</td>
		</tr>
	</tbody>
</table>

an assay for measuring the effects of ethanol on the locomotion speed of caenorhabditis elegans

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

いくつかの異なる遺伝子型とその対になったコントロールからの代表的なデータ（ 図1）は、8,24 を提示されている。データは、具体的にアッセイした動物において、そのハイライトの違いを選択した。曝露の10分での効果の程度は、 図1B-Gの左軸に示されている株の初期感度と考えられている。 10分での対照よりも大きな相対速度を有する変異株は、...

Watch this Scientific Journal Video about An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans at JoVE.com

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

の歩行速度に対するエタノールの効果を測定するためのアッセイ<em&gt;線虫（Caenorhabditis elegans）</em

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

an-assay-for-measuring-effects-ethanol-on-locomotion-speed

Research

JoVE Journal

Neuroscience

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

8.5K ビュー.  Virginia Commonwealth University. C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay. 

ビデオ: An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

ガスクロマトグラフィーによるオペラントエタノール自己投与とエタノール判別中エタノールのマイクロダイアリシス

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

神経科学

An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form during the earliest stages of brain development and are maintained throughout the animal's life. The brain ventricular system is found in vertebrates, and the ventricles develop after neural tube formation, when the central lumen fills with cerebrospinal fluid (CSF) 1,2. CSF is a protein rich fluid that is essential for normal brain development and function3-6.
In zebrafish, brain ventricle inflation begins at approximately 18 hr post fertilization (hpf), after the neural tube is closed. Multiple processes are associated with brain ventricle formation, including formation of a neuroepithelium, tight junction formation that regulates permeability and CSF production. We showed that the Na,K-ATPase is required for brain ventricle inflation, impacting all these processes 7,8, while claudin 5a is necessary for tight junction formation 9. Additionally, we showed that "relaxation" of the embryonic neuroepithelium, via inhibition of myosin, is associated with brain ventricle inflation.
To investigate the regulation of permeability during zebrafish brain ventricle inflation, we developed a ventricular dye retention assay. This method uses brain ventricle injection in a living zebrafish embryo, a technique previously developed in our lab10, to fluorescently label the cerebrospinal fluid. Embryos are then imaged over time as the fluorescent dye moves through the brain ventricles and neuroepithelium. The distance the dye front moves away from the basal (non-luminal) side of the neuroepithelium over time is quantified and is a measure of neuroepithelial permeability (Figure 1). We observe that dyes 70 kDa and smaller will move through the neuroepithelium and can be detected outside the embryonic zebrafish brain at 24 hpf (Figure 2).
This dye retention assay can be used to analyze neuroepithelial permeability in a variety of different genetic backgrounds, at different times during development, and after environmental perturbations. It may also be useful in examining pathological accumulation of CSF. Overall, this technique allows investigators to analyze the role and regulation of permeability during development and disease.

We describe a live whole animal quantitative measurement for permeability of the embryonic zebrafish brain. The technique analyzes the ability to retain cerebrospinal fluid and molecules of different molecular weights within the neural tube lumen and quantifies their movement out of the ventricles. This method is useful for determining differences in epithelial permeability and maturation during development and disease.

ゼブラフィッシュ胚性神経上皮の透水性に関するアッセイ

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form ...

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

A<em&gt;線虫（Caenorhabditis elegans）</emAmylopathyの研究のための&gt;モデルシステム

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping1,2. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than APDs.

抗精神病薬の作用のメカニズムを研究するための方法<em&gt;線虫（Caenorhabditis elegans）</em

Caenorhabditis elegans is a simple genetic organism amenable to  large-scale forward and reverse genetic screens and chemical genetic screens. The C.  ...

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in their etiology. The fruit fly, Drosophila melanogaster, is a powerful model for exploring molecular-genetic mechanisms underlying alcohol-related behaviors and therefore holds great promise for identifying and understanding the function of genes that influence AUD. The use of the Drosophila model for these types of studies depends on the availability of assays that reliably measure behavioral responses to ethanol. This report describes an assay suitable for assessing ethanol sensitivity and rapid tolerance in flies. Ethanol sensitivity measured in this assay is influenced by the volume and concentration of ethanol used, a variety of previously reported genetic manipulations, and also the length of time the flies are housed without food immediately prior to testing. In contrast, ethanol sensitivity measured in this assay is not affected by the vigor of fly handling, sex of the flies, and supplementation of growth medium with antibiotics or live yeast. Three different methods for quantitating ethanol sensitivity are described, all leading to essentially indistinguishable ethanol sensitivity results. The scalable nature of this assay, combined with its overall simplicity to set-up and relatively low expense, make it suitable for small and large scale genetic analysis of ethanol sensitivity and rapid tolerance in Drosophila.

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Straightforward assays for measuring ethanol sensitivity and rapid tolerance in Drosophila facilitate the use of this model organism for investigating these important ethanol-related behaviors. Here, a relatively simple, scalable assay for measuring ethanol sensitivity and rapid tolerance in flies is described.

エタノール鎮静感度との急速な寛容を測定するための安価な、スケーラブルな行動アッセイ<em&gt;ショウジョウバエ</em

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

の長期カルシウムイメージングのためのアガロースマイクロチャンバー<em&gt;線虫（Caenorhabditis elegans）</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. Embryonic elongation of the nematode Caenorhabditis elegans is a late developmental stage consisting of the elongation of the embryo along its longitudinal axis. This developmental stage is controlled by intercellular communication between hypodermal cells and underlying body-wall muscles. These signaling mechanisms control the morphology of hypodermal cells by remodeling the cytoskeleton and the cell-cell junctions. Measurement of embryonic lethality and developmental arrest at larval stages as well as alteration of cytoskeleton and cell-cell adhesion structures in hypodermal and muscle cells are classical phenotypes that have been used for more than 25 years to dissect these signaling pathways. Recent studies required the development of novel metrics specifically targeting either early or late elongation and characterizing morphogenic defects along the antero-posterior axis of the embryo. Here, we provide detailed protocols enabling the accurate measurement of the length and the width of the elongating embryos as well as the length of synchronized larvae. These methods constitute useful tools to identify genes controlling elongation, to assess whether these genes control both early and late phases of this stage and are required evenly along the antero-posterior axis of the embryo.

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo.

新規メトリックは、線虫の胚伸びを特徴づけるために、<em&gt;線虫（Caenorhabditis elegans）</em

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

の記録細胞内の電圧応答のための電気生理学的手法<em&gt;ショウジョウバエ</em&gt;光刺激に感光体と介在ニューロン<em&gt;インビボ</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their locomotory patterns upon detection of diverse environmental stimuli and can modulate their suite of behavioral responses in response to starvation conditions. Nematodes typically exhibit a decreased aversive response when removed from a food source for over 30 min. Observation of behavioral changes in response to a changing nutritional status can provide insight into the mechanisms that regulate the transition from a well-fed to starved state.
We have developed an assay that measures a nematode&#39;s ability to cross an aversive barrier (i.e. copper) then reach a food source over a prolonged period of time. This protocol builds upon previous work by integrating multiple variables in a manner that allows for continued data collection as the organisms shift towards an increasingly starved condition. Moreover, this assay permits an increased sample size so that larger populations of nematodes can be simultaneously evaluated.
Organisms defective for the ability to detect or respond to copper immediately cross the chemical barrier, while wild type nematodes are initially repelled. As wild type worms are increasingly starved, they begin to cross the barrier and reach the food source. We designed this assay to evaluate a mutant that is incapable of responding to diverse environmental cues, including food sensation or detection of aversive chemicals. When evaluated via this protocol, the defective organisms immediately crossed the barrier, but were also incapable of detecting a food source. Hence, these mutants repeatedly cross the chemical barrier despite temporarily reaching a food source. This assay can straightforwardly test populations of worms to evaluate potential pathway defects related to aversion and starvation.

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

Here, we present a Caenorhabditis elegans-specific assay designed to evaluate changes in copper aversion behavior and the ability to locate a common food source, as the organism progresses from a well-fed to starved nutritional state.

A<em&gt; Caenorhabditis elegans</em&gt;栄養状態ベースの銅回避アッセイ

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

線虫の行動で個々 のニューロンのレシオ メトリック カルシウム イメージング

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...