Name: quantifying fish swimming behavior in response to acute exposure of aqueous copper using computer assisted video and digital image analysis
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Description: Watch this Scientific Journal Video about Quantifying Fish Swimming Behavior in Response to Acute Exposure of Aqueous Copper Using Computer Assisted Video and Digital Image Analysis at JoVE.com

We thank the staff in the Ecology Branch, Toxicology Branch and Environmental Chemistry Branch of the U.S. Geological Survey, Columbia Environmental Research Center for technical and analytical assistance. Funding provided in part by Teck American Incorporated through an agreement with the US Environmental Protection Agency (USEPA) Region 10 with funds provided by USEPA to US Geological Survey (USGS) through the Department of Interior Central Hazmat Fund.

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汚染物質への暴露に行動の変化は、多くの場合、サブ致死毒性のエンドポイントとして使用されるが、測定することが困難な場合があります。一般的に、行動応答は、目視観察し、処理するために多くの時間を必要とする手動によるデータ分析により測定されています。技術を進めるとともにしかし、水泳の活動を定量化するための方法は、ビデオ撮影18および運動解析や処理および分析時間を短縮し、デジタルトラッキングソフトウェアを使用することに焦点を当てています。ビデオの分析の間ので、チョウザメの遊泳行動を分析するためのより効果的かつ効率的な方法を提供し、映像データの記録や魚のトラッキングソフトウェアの使用を消費手動で非常に時間があったであろう水泳変数を定量化、データを捕獲しました。手順は、魚の遊泳行動を強調したが、このような両生類や水生無脊椎動物のような他の生物のためにカスタマイズすることは簡単な変更を必要とするであろう。どのような行動endpoinに応じて、TSは、実験的なデザインとカメラシステムは、ちょうど約あらゆる市販の追跡ソフトウェアパッケージで使用するために開発することができ、アドレス指定されています。 この方法は、溶解した銅を用いて実証するが、他の水性汚染物質又は温度又は酸素含有量等の特性に適用可能です。本論文で開発し、提示されたプロトコルは、記録装置のような単純なデジタルビデオカメラを利用しました。デジタルファイルは、容易にコンピュータに転送し、運動解析ソフトウェアにアップロードされます。方法は絶えず変更され、定量化プロセスを合理化するために洗練されています。ビデオ品質を追跡するために、各個々の魚を識別するために解析ソフトウェアために高解像度であることが不可欠です。データファイルを処理しようとすると魚とのコントラストしない任意の背景には、問題が発生します。 2次元ビデオトラッキングと別の一般的な問題は、個人を識別しています水泳のパスが渡ります。これは、パスの横断中に各魚を識別し、ソフトウェア内経路セグメントを連結することによって、手動で補正することができます。あるいは、総活性は、グループの平均として各反復室から決定することができます。 1魚をそれぞれ含むものといくつかの個々のチャンバは、個々の魚の動きを計算するために、同じ視野で撮影することができます。 現在、我々は、高精細デジタルビデオ記録装置（HD-DVR）にリンクされている露光室以上のオーバーヘッドの監視カメラのシリーズを使用してアップグレードしました。しかし、高解像度のMPEG-4ビデオを録画することができます任意のカメラシステムの使用が動作します。 HD-DVRは、指定した時刻に記録するように設定し、7日間までプログラムすることができます。この手オフの自動化のアプローチは、魚の行動を損なう可能性外乱を最小限に抑えながら、一貫性を維持するために、同時に複数のビデオのキャプチャを可能にします。 HD-DVRのsysteMSは、そのようにファイルの転送は比較的簡単である内部ネットワークに接続されています。自動化されたカメラシステムは、遊泳行動を定量化するためのはるかに改善された技術ですが、まだ毒性試験中に行動障害を文書化するためなどの追加支援情報を提供するために視覚的な観察を行うことが有益です。 1960年代初頭19,20,21にさかのぼる金属への暴露に起因する文献の文書化変更された魚の行動の長い歴史があります。銅はブルーギル22でこのような活動低下などの活動レベルの変化（ ブルーギル属macrochirus rafinesque）とカワマス23（ イワナ fontinalis）の運動と摂食活動の変化を引き起こすことが示されています。少なくともいくつかの稚魚の捕食者を検出し、回避するために、匂いの感覚に頼る、および銅誘導性の化学感覚の剥奪は、アラーム化学物質24,25,26の検出に関連した行動に影響を与える可能性があります</sup&gt;。嗅上皮は、それによって、見当識障害につながる可能性があり感覚のメカニズム、行動の回避、低減し供給および嗅覚27に案内され、他の行動に影響を与えることにより、銅暴露に破損しています。これらの変更された行動は、エクスポージャーの間に観察されたものと一致しました。 白チョウザメの遊泳行動が大幅に銅水溶液濃度に致死量以下の露光時に影響を受けました。これらの結果は、動作が銅の亜致死濃度で影響を受け、毒性ストレスの指標として使用することができる方法を示しています。ビデオベースの分析では、遊泳行動を定量化するのに有効証明し、また銅にさらさチョウザメに深刻な影響の定性的な視覚的なドキュメントを務めていました。解析ソフトウェアは、他の様々な行動のエンドポイントを定量化することが可能です。リストについては、 表1を参照してください。露光システムは、各エンドポイントに対処するために変更することができますリアルタイムモードと関心のさまざまな汚染物質への曝露に関連した動作の違いを定量化するために使用することができます。 水生毒性学的研究における行動のエンドポイントの使用がますます採用されており、適応行動機能は、環境傷害9の決意に重要であるため、汚染物質の影響を評価する際に考慮すべきです。魚の行動への環境汚染物質の影響は、標準的な方法の不在下で亜致死エンドポイントを扱う場合は特に、多くの場合、主観的かつ挑戦的です.. 厳密に監視することができ、これらの方法を使用して定量化活性を水泳、生物に最小限のストレスで非破壊的であり、繰り返すことができます。水泳の動作は、標準的な毒性試験5の感度を拡大するテストプロトコルに組み込まれるべきである亜致死毒性の有効かつ一貫性の指標です。

汚染物質の曝露は行動にどのように影響するかを理解することは時間に非常に困難かつ主観的です。行動は、一般的に、中枢神経系を介して動作と、生き残る成長し、再現するために生物を有効に明白な、観察可能な、全身の一連の活動として定義されています。生存率1と比較した場合に起因する毒物への暴露に行動の変化は、環境ストレス、多くの場合、10から100の間の倍の感度の最も敏感な指標の一つです。これらの研究の大半は、スイミングアクティビティ、換気、および魚の2,3,4の採餌行動を調べました。水泳の活動は、毒性試験5中の汚染物質に反応して行動変化を決定する上で最も頻繁に使用される亜致死のエンドポイントです。水泳変数は、周波数や旅の移動、速度と距離の期間、頻度およびターンの角度、位置水柱中や水泳のパターンを含みます。水泳活性があります遊泳行動の有効な手段水生毒性の本の基礎にランド6により、第9章で提案された基準に基づいて、毒性物質に対する感受性を評価します。 本稿では、早期開発の様々な段階での早期のライフステージ白チョウザメへの銅の毒性（ シロチョウザメ ）水のみの遊泳行動を評価したチョウザメする相対的なエクスポージャー方法の例として、毒性学的研究を提示し、水泳を定量する方法を示しています動作。 以前の研究では、有害な行動反応は銅に急性および慢性暴露の最初の数日の間に早期発症と明らかにしたと暴露時間と濃度の7,8,9の上に次第により厳しいなりました。これらの行動反応の発症の大きさとタイミングが長期生存を制限する可能性が十分であり、したがってrecruiに影響与え懸念されますtment障害10。正確に金属露出方法および手順に、この感度の意義を解釈するために銅濃度を基準に行動の減損の時間経過と程度を定量化するために開発されました。 行動機能と開発のための試験プロトコルは、露光チャンバ内でチョウザメのオーバーヘッドビデオサンプルを分析することによって確立しました。ビデオサンプルは、暴露期間を通して銅処理間発達と機能の定性的な評価を提供します。行動と避難所を追究活動を行うようにと協調、平衡、および呼吸を泳いで、避難所のシーク、無気力に影響を与える減損の時系列を特徴づけるために時間を決定するために、水性銅濃度に対するエクスポージャーの間に評価しました。また、ビデオ録画を定量的に文書化する自発的な水泳ACTIの目的のために複製それぞれからサブサンプリングされたチョウザメで作られていましたVITY。これらのエンドポイントは、持続時間の測定が含まれるか、または時間が移動費やし、遊泳速度及び距離は、商業的に利用可能なデジタル画像分析ソフトウェアパッケージを用いて5,11を旅しました。このソフトウェアは、視野内の各画像の輪郭を定義し、このことから、各画像の重心を定義します。ソフトウェアは、運動の経路を決定するために、フレームの連続によってフレーム内の各重心の位置を追跡することができます。 動物福祉法の規定（9 CFR）の最終規則の該当するすべてのセクションで、文化と実験中の試験生物の人道的な扱いのためのすべての施設のガイドラインを遵守し、この研究。研究の終了時に、すべての魚は、米国地質調査所のための制度的動物実験委員会、コロンビア環境研究センターによって承認されたように、適切なガイドラインに従って安楽死させました。

<table><tbody><tr><td>copper II sulfate pentahydrate</td><td>Sigma-Aldrich</td><td></td><td>contaminant of concern</td></tr><tr><td>syringe dispenser</td><td>Hamilton MicroLab 600 Series</td><td></td><td>apparatus to spike chemical</td></tr><tr><td>2 L volumetric flask</td><td></td><td></td><td>container for holding stock solution</td></tr><tr><td>24-1.5 L glass jars</td><td></td><td></td><td>test chamber for 2 dph sturgeon</td></tr><tr><td>video camera</td><td>Sony Handycam HDR-CX550V</td><td></td><td></td></tr><tr><td>digital tracking software</td><td>Noldus Ethovision</td><td></td><td></td></tr><tr><td>3-17&quot; flat screen monitors</td><td></td><td></td><td></td></tr><tr><td>24 surveillance cameras</td><td>Model CL101</td><td></td><td></td></tr><tr><td>3-16 channel digital recording devices</td><td></td><td></td><td></td></tr><tr><td>DO meter</td><td>YSI</td><td></td><td></td></tr><tr><td>pH meter</td><td>Orion 940</td><td></td><td></td></tr><tr><td>ph probe</td><td>Orion&amp;nbsp;</td><td></td><td></td></tr><tr><td>ammonia meter</td><td></td><td></td><td></td></tr><tr><td>ammonia probe</td><td>Orion</td><td></td><td></td></tr><tr><td>chiller unit</td><td></td><td></td><td></td></tr><tr><td>recirculating water pump</td><td></td><td></td><td></td></tr></tbody></table>

quantifying fish swimming behavior in response to acute exposure of aqueous copper using computer assisted video and digital image analysis

Behavioral responses of aquatic organisms to environmental contaminants can be precursors of other effects such as survival, growth, or reproduction. However, these responses may be subtle, and measurement can be challenging. Using juvenile white sturgeon (Acipenser transmontanus) with copper exposures, this paper illustrates techniques used for quantifying behavioral responses using computer assisted video and digital image analysis. In previous studies severe impairments in swimming behavior were observed among early life stage white sturgeon during acute and chronic exposures to copper. Sturgeon behavior was rapidly impaired and to the extent that survival in the field would be jeopardized, as fish would be swept downstream, or readily captured by predators. The objectives of this investigation were to illustrate protocols to quantify swimming activity during a series of acute copper exposures to determine time to effect during early lifestage development, and to understand the significance of these responses relative to survival of these vulnerable early lifestage fish. With mortality being on a time continuum, determining when copper first affects swimming ability helps us to understand the implications for population level effects. The techniques used are readily adaptable to experimental designs with other organisms and stressors.

手動で処理する視覚観測データは、2日後に孵化（DPH）チョウザメ（ 図2）で開始曝露のわずか72時間後に増加銅濃度とともに増加パーセントの異常を示しました。ビデオサンプルは、チョウザメの遊泳行動（動画2）上の銅暴露の極端な影響を文書化し、その結果としての行動障害を定義する際に支援します。別の例では、30 DPHでのチョウザメは、死亡率のみに基づいて40.3μg/ Lを96時間半数致死影響濃度（LC50）と銅の露出に敏感であることが判明しました。しかし、平衡および固定化の損失の亜致死行動エンドポイントは、死亡率と一緒に含まれている場合2.4〜5.0μgの/ Lの範囲の推定96時間中央値は影響濃度（EC50）での感度が高くなります。記録された行動異常のビデオドキュメントこれらの亜致死影響を捕獲し、さらに検証人間観察露光中。水泳活動を分析する場合、デジタルトラッキングソフトウェアの使用は、後処理時間を減少させました。魚の遊泳速度は、時間が動いて過ごした、との距離はすべて大幅に増加銅濃度で（ 図3）減少し 、トリップアドバイザー。水泳経路も増加銅濃度（ 図4）で還元しました。 <img alt="図1" src="/files/ftp_upload/53477/53477fig1.jpg" /> 図1：試験原液濃度目標濃度に基づいて、スプレッドシートのテンプレートを使用して決定した希釈毒性試験のための化学の株式の準備を決定するために使用されるスプレッドシートのテンプレートのコンピュータのスクリーンショット <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53477/53477fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 図2：希釈のセットアップと実験デザイン白チョウザメのさまざまなライフステージが銅に曝露しました。露光のために使用される希釈のサイズは、魚の大きさに基づいて選択しました。 （a）は初期ライフステージのチョウザメは、大きな希釈のセットアップを使用して露光した小希釈のセットアップおよび（b）は 、古いライフステージのチョウザメを用いて露光しました。 <img alt="図3" src="/files/ftp_upload/53477/53477fig3.jpg" /> 図3：初期生活段階の白チョウザメ（ シロチョウザメ ）72時間の銅暴露（[DPH]魚30日の孵化後から始まる ） から 、 行動結果をスイミング 。白チョウザメDPH 30の間での移動の水泳活動のエンドポイントは、（a）の期間; （b）は、水泳のスピード。そして（c </移動strong&gt;の）距離は96時間さらさ白チョウザメの中銅濃度の増加に伴って減少しました。アスタリスクは対照からの有意差を示し、エラーバーは標準偏差を表します。 <img alt="図4" src="/files/ftp_upload/53477/53477fig4.jpg" /> 図4：初期生活段階の白チョウザメ（シロチョウザメ）の結果96時間の銅暴露（2日目の孵化後[DPH]魚で始まる）の72時間後の平衡および固定化の損失を示す2 DPHで白チョウザメを存続。銅濃度の増加に伴って96時間暴露。アスタリスクは対照からの有意差を示し、エラーバーは標準偏差を表します。 <img alt="図5" src="/files/ftp_upload/53477/53477fig5.jpg" /> 図5：初期生活段階の白チョウザメ（チョウザメ属transmoから例水泳パス結果ntanus）96時間銅暴露（30日の孵化後[DPH]魚）と凝視。チョウザメの水泳経路が（N = 5魚）と（b）高い治療から（50（a）の制御の反復からのデジタルトラッキングソフトウェアを使用してデジタル化96時間曝露後μgの/ L）の反復（n = 3の魚）。不活性であったため、いくつかの魚の遊泳経路の数は、チャンバ内の魚の存在の数を表すものではありません。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53477/53477fig5large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <table fo:keep-together.within-page="1"><tbody><tr><td> 定量化可能なパラメータ </td><td> 観測パラメータ </td></tr><tr><td>速度</td><td>無気力/多動</td></tr><tr><td>距離移動</td><td>均衡の喪失</td></tr><tr><td>ゾーンで過ごした時間の長さ</td><td>痙攣/振戦/逆さまダウン</td></tr><tr><td>選択したゾーン間の時間生物の移動のゾーン遷移数</td><td>水柱内の位置</td></tr><tr><td>見出し興味のある点に向かって、動物のパスの偏差をポイント計算</td><td>呼吸（速い/遅いです） </td></tr><tr><td>見出しは、選択されたボディポイントの見出しを算出し、 </td><td>着色</td></tr><tr><td> 2サンプル間の見出しに角度差を回し</td><td>隠蔽</td></tr><tr><td>角速度計算サンプル間隔によって回転角度を分割することにより</td><td></td></tr><tr><td>移動距離によって旋回角度を分割することにより蛇行計算しました。異なる速度で移動する動物で回し比較するために使用されます</td><td></td></tr><tr><td>時間が動いて過ごしました</td><td></td></tr><tr><td>モビリティでもセントた場合、動物のように検出された完全なエリアが変更されている期間を状態は、計算しますER点は同じまま</td><td></td></tr><tr><td>選択されたボディポイントが360°の累積回転角を有するとき、回転一回転が終了します。無視される閾値未満の反対方向に回転。 </td><td></td></tr><tr><td>移動度は、中心点が同じであっても、検出された動物の完全な領域の移動度の割合を連続計算します。 </td><td></td></tr><tr><td>間の距離全ての関係者と選択した受信機との間の距離を被験者は、計算します</td><td></td></tr><tr><td>俳優であるか、または受信機に近接していないする期間を近接は、計算します</td><td></td></tr><tr><td>相対運動</td><td></td></tr><tr><td>ネット加重移動-（ポジティブ）に俳優の動きとそれらの間の距離によって重み付け（負の）受信機、から</td><td></td></tr><tr><td>加重移動から、moveme<html>それらの間の距離によって重み付け受信機から俳優のntは、。 </td><td></td></tr><tr><td>それらの間の距離によって重み付け受信機に俳優の動き対加重移動、 </td><td></td></tr><tr><td> 2試用コントロール要素のイベント、または1つの要素内の間の試用制御state-期間</td><td></td></tr><tr><td>あなたが裁判コントロールに定義されている要素内のイベントが発生した裁判制御イベントモーメント。 </td><td></td></tr></tbody></table> 表1：デジタル追跡ソフトウェアを介して定量化行動のエンドポイントは、これらのエンドポイントは、個人またはグループで使用され、また、露光時の行動障害を視覚的に観察チェックのリストとして機能することができます。 <img alt="ビデオ1" src="/files/ftp_upload/53477/53477movie1.jpg" /> /jove.unicartagenaproxy.elogim.com/files/ftp_upload/53477/53477video1.avi &quot;&gt; ビデオ1：影響を受けた白チョウザメが示す異常行動のビジュアル定義（右クリックしてダウンロード）毎日観察チェックリストは、異常を文書化するために使用されたザ・。。平衡および固定化の損失は、曝露の間に観察された最も一般的な異常であった。Calfee らから変更された。7 <img alt="動画2" src="/files/ftp_upload/53477/53477movie2.jpg" /> <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53477/53477video2.avi">ビデオ2：白チョウザメの遊泳行動の例を強調ビジュアルドキュメント。 （右クリックしてダウンロード）</a>白チョウザメ水泳活性が大幅に増加銅濃度への暴露で還元しました。このビデオに描かチョウザメは、コントロール、中低、および96時間銅水溶液の終わりに高い治療からのものです暴露。 白チョウザメはまだ生きていたが、魚が大幅にコントロールと比較して治療が損なわれた明白です。 Calfee らから変更。7

Watch this Scientific Journal Video about Quantifying Fish Swimming Behavior in Response to Acute Exposure of Aqueous Copper Using Computer Assisted Video and Digital Image Analysis at JoVE.com

Quantifying Fish Swimming Behavior in Response to Acute Exposure of Aqueous Copper Using Computer Assisted Video and Digital Image Analysis

コンピュータ支援ビデオとデジタル画像解析を用いた銅水溶液の急性暴露に応答して、魚の遊泳行動を定量化

魚の行動に環境汚染物質の影響を測定することは、亜致死エンドポイントを扱う場合は特に、多くの場合、主観的かつ挑戦的です。我々は、中および銅の種々の濃度に対する96時間の急性曝露後初期生活段階白チョウザメ（ シロチョウザメ ）の遊泳行動を定量化するためのビデオ技術を含む方法が記載されています。

Behavioral responses of aquatic organisms to environmental contaminants can be precursors of other effects such as survival, growth, or reproduction.  ...

quantifying-fish-swimming-behavior-response-to-acute-exposure-aqueous

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

11.1K ビュー.  US Geological Survey. 魚の行動に環境汚染物質の影響を測定することは、亜致死エンドポイントを扱う場合は特に、多くの場合、主観的かつ挑戦的です。我々は、中および銅の種々の濃度に対する96時間の急性曝露後初期生活段階白チョウザメ（ シロチョウザメ ）の遊泳行動を定量化するためのビデオ技術を含む方法が記載されています。 

ビデオ: Quantifying Fish Swimming Behavior in Response to Acute Exposure of Aqueous Copper Using Computer Assisted Video and Digital Image Analysis

Using Touch-evoked Response and Locomotion Assays to Assess Muscle Performance and Function in Zebrafish

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many myopathies affecting skeletal muscle function can be quickly and easily assessed in zebrafish over the first few days of embryogenesis. By 24 hr post-fertilization (hpf), wildtype zebrafish spontaneously contract their tail muscles and by 48 hpf, zebrafish exhibit controlled swimming behaviors. Reduction in the frequency of, or other alterations in, these movements may indicate a skeletal muscle dysfunction. To analyze swimming behavior and assess muscle performance in early zebrafish development, we utilize both touch-evoked escape response and locomotion assays.
Touch-evoked escape response assays can be used to assess muscle performance during short burst movements resulting from contraction of fast-twitch muscle fibers. In response to an external stimulus, which in this case is a tap on the head, wildtype zebrafish at 2 days post-fertilization (dpf) typically exhibit a powerful burst swim, accompanied by sharp turns. Our method quantifies skeletal muscle function by measuring the maximum acceleration during a burst swimming motion, the acceleration being directly proportional to the force produced by muscle contraction.
In contrast, locomotion assays during early zebrafish larval development are used to assess muscle performance during sustained periods of muscle activity. Using a tracking system to monitor swimming behavior, we obtain an automated calculation of the frequency of activity and distance in 6-day old zebrafish, reflective of their skeletal muscle function. Measurements of swimming performance are valuable for phenotypic assessment of disease models and high-throughput screening of mutations or chemical treatments affecting skeletal muscle function.

Zebrafish are an excellent model to study muscle function and disease. During early embryogenesis zebrafish begin regular muscle contractions producing rhythmic swimming behavior, which is altered when the muscle is disrupted. Here we describe a touch-evoked response and locomotion assay to examine swimming performance as a measure of muscle function.

ゼブラフィッシュで筋肉のパフォーマンスと機能を評価するためにタッチ誘発応答と歩行アッセイを使用して

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many ...

発生生物学

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity testing has been demonstrated. Overall, the sensitivity of the species to toxic compounds is in the range with, or higher than, that of other model species.
This work describes protocols for acute, chronic, and multigenerational bioassays of single and combined stressor effects on N. furzeri. Due to its short maturation time and life-cycle, this vertebrate model allows the study of endpoints such as maturation time and fecundity within four months. Transgenerational full life-cycle exposure trials can be performed in as little as 8 months. Since this species produces eggs that are drought-resistant and remain viable for years, the on-site culture of the species is not needed but individuals can be recruited when required. The protocols are designed to measure life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

In this work, we describe an acute, chronic and multigenerational bioassay to study the effects of single and combined stressors on the Turquoise killifish Nothobranchius furzeri. This protocol is designed to study life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

急性および慢性毒性試験ターコイズ メダカによる furzeriのためのプロトコル

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity ...

環境学

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish&#160;were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

稚魚の行動応答プロファイルを調べるための実験プロトコル: 神経興奮剤カフェインへの応用

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a &lt;1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes.
In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts.
In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.

ゼブラフィッシュで自動化されたハイコンテンツ炎症アッセイ：創薬を促進する

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and ...

免疫・感染学

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the visual scene. However, the OKR requires that an animal detect moving stripes and it is possible that fish that fail to exhibit an OKR may not be completely blind. One zebrafish mutant, the no optokinetic response c (nrc) has no OKR under any light conditions tested and was reported to be completely blind. Previously, we have shown that OFF-ganglion cell activity can be recorded in these mutants. To determine whether mutant fish with no OKR such as the nrc mutant can detect simple light increments and decrements we developed the visual motor behavioral assay (VMR). In this assay, single zebrafish larvae are placed in each well of a 96-well plate allowing the simultaneous monitoring of larvae using an automated video-tracking system. The locomotor responses of each larva to 30 minutes light ON and 30 minutes light OFF were recorded and quantified. WT fish have a brief spike of motor activity upon lights ON, known as the startle response, followed by return to lower-than baseline activity, called a freeze. WT fish also sharply increase their locomotor activity immediately following lights OFF and only gradually (over several minutes) return to baseline locomotor activity. The nrc mutants respond similarly to light OFF as WT fish, but exhibit a slight reduction in their average activity as compared to WT fish. Motor activity in response to light ON in nrc mutants is delayed and sluggish. There is a slow rise time of the nrc mutant response to light ON as compared to WT light ON response. The results indicate that nrc fish are not completely blind. Because teleosts can detect light through non-retinal tissues, we confirmed that the immediate behavioral responses to light-intensity changes require intact eyes by using the chokh (chk) mutants, which completely lack eyes from the earliest stages of development. In our VMR assay, the chk mutants exhibit no startle response to either light ON or OFF, showing that the lateral eyes mediate this behavior. The VMR assay described here complements the well-established OKR assay, which does not test the ability of zebrafish larvae to respond to changes in light intensities. Additionally, the automation of the VMR assay lends itself to high-throughput screening for defects in light-intensity driven visual responses.

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

We developed the Visual-Motor Response to quantitate the motor output of larval zebrafish in response to light increments and decrements. We also examined zebrafish vision mutants, including the no optokinetic response (nrc) mutants, which were thought to be completely blind when tested by another vision assay, the optokinetic reflex.

光強度の変化にゼブラフィッシュの応答性を測定する行動アッセイ

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the ...

生物学

Behavioral Tracking and Neuromast Imaging of Mexican Cavefish

Cave-dwelling animals have evolved a series of morphological and behavioral traits to adapt to their perpetually dark and food-sparse environments. Among these traits, foraging behavior is one of the useful windows into functional advantages of behavioral trait evolution. Presented herein are updated methods for analyzing vibration attraction behavior (VAB: an adaptive foraging behavior) and imaging of associated mechanosensors of cave-adapted tetra, Astyanax mexicanus. In addition, methods are presented for high-throughput tracking of a series of additional cavefish behaviors including hyperactivity and sleep-loss. Cavefish also show asociality, repetitive behavior and higher anxiety. Therefore, cavefish serve as an animal model for evolved behaviors. These methods use free-software and custom-made scripts that can be applied to other types of behavior. These methods provide practical and cost-effective alternatives to commercially available tracking software.

Here, we present methods for high-throughput study of a series of the Mexican cavefish behaviors and vital staining of a mechanosensory system. These methods use free-software and custom-made scripts, providing a practical and cost-effective method for the studies of behaviors.

行動追跡とメキシコの血脈の Neuromast イメージング

Cave-dwelling animals have evolved a series of morphological and behavioral traits to adapt to their perpetually dark and food-sparse environments. Among ...

行動学

Assessing the Influence of Personality on Sensitivity to Magnetic Fields in Zebrafish

To orient themselves in their environment, animals integrate a wide array of external cues, which interact with several internal factors, such as personality. Here, we describe a behavioral protocol designed for the study of the influence of zebrafish personality on their orientation response to multiple external environmental cues, specifically water currents and magnetic fields. This protocol aims to understand whether proactive or reactive zebrafish display different rheotactic thresholds (i.e., the flow speed at which the fish start swimming upstream) when the surrounding magnetic field changes its direction. To identify zebrafish with the same personality, fish are introduced in the dark half of a tank connected with a narrow opening to a bright half. Only proactive fish explore the novel, bright environment. Reactive fish do not exit the dark half of the tank. A swimming tunnel with low flow rates is used to determine the rheotactic threshold. We describe two setups to control the magnetic field in the tunnel, in the range of the earth&#8217;s magnetic field intensity: one that controls the magnetic field along the flow direction (one dimension) and one that allows a three-axial control of the magnetic field. Fish are filmed while experiencing a stepwise increase of the flow speed in the tunnel under different magnetic fields. Data on the orientation behavior are collected through a video-tracking procedure and applied to a logistic model to allow the determination of the rheotactic threshold. We report representative results collected from shoaling zebrafish. Specifically, these demonstrate that only reactive, prudent fish show variations of the rheotactic threshold when the magnetic field varies in its direction, while proactive fish do not respond to magnetic field changes. This methodology can be applied to the study of magnetic sensitivity and rheotactic behavior of many aquatic species, both displaying solitary or shoaling swimming strategies.

We describe a behavioral protocol designed to assess how zebrafish&#8217;s personalities influence their response to water currents and weak magnetic fields. Fishes with the same personalities are separated based on their explorative behavior. Then, their rheotactic orientation behavior in a swimming tunnel with a low flow rate and under different magnetic conditions is observed.

ゼブラフィッシュにおける磁界感度に人格の影響を評価します。

To orient themselves in their environment, animals integrate a wide array of external cues, which interact with several internal factors, such as ...

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and social interactions can be best studied by observing unrestrained, freely behaving animals. Weakly electric fish (WEF) display readily observable exploratory and social behaviors by emitting electric organ discharge (EOD). Here, we describe three effective techniques to synchronously measure the EOD, body position, and posture of a free-swimming WEF for an extended period of time. First, we describe the construction of an experimental tank inside of an isolation chamber designed to block external sources of sensory stimuli such as light, sound, and vibration. The aquarium was partitioned to accommodate four test specimens, and automated gates remotely control the animals&#39; access to the central arena. Second, we describe a precise and reliable real-time EOD timing measurement method from freely swimming WEF. Signal distortions caused by the animal&#39;s body movements are corrected by spatial averaging and temporal processing stages. Third, we describe an underwater near-infrared imaging setup to observe unperturbed nocturnal animal behaviors. Infrared light pulses were used to synchronize the timing between the video and the physiological signal over a long recording duration. Our automated tracking software measures the animal&#39;s body position and posture reliably in an aquatic scene. In combination, these techniques enable long term observation of spontaneous behavior of freely swimming weakly electric fish in a reliable and precise manner. We believe our method can be similarly applied to the study of other aquatic animals by relating their physiological signals with exploratory or social behaviors.

We describe a set of techniques for studying spontaneous behavior of freely swimming weakly electric fish over an extended period of time, by synchronously measuring the animal&#39;s electric organ discharge timing, body position and posture both accurately and reliably in a specially designed aquarium tank inside a sensory isolation chamber.

自由水泳弱電気魚の長期的な行動の追跡

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and ...

神経科学

Swimming Performance Assessment in Fishes

Swimming performance tests of fish have been integral to studies of muscle energetics, swimming mechanics, gas exchange, cardiac physiology, disease, pollution, hypoxia and temperature. This paper describes a flexible protocol to assess fish swimming performance using equipment in which water velocity can be controlled. The protocol involves one to several stepped increases in flow speed that are intended to cause fish to fatigue. Step speeds and their duration can be set to capture swimming abilities of different physiological and ecological relevance. Most frequently step size is set to determine critical swimming velocity (Ucrit), which is intended to capture maximum sustained swimming ability. Traditionally this test has consisted of approximately ten steps each of 20 min duration. However, steps of shorter duration (e.g. 1 min) are increasingly being utilized to capture acceleration ability or burst swimming performance. Regardless of step size, swimming tests can be repeated over time to gauge individual variation and recovery ability. Endpoints related to swimming such as measures of metabolic rate, fin use, ventilation rate, and of behavior, such as the distance between schooling fish, are often included before, during and after swimming tests. Given the diversity of fish species, the number of unexplored research questions, and the importance of many species to global ecology and economic health, studies of fish swimming performance will remain popular and invaluable for the foreseeable future.

The lives of the majority of fish are predicated on swimming. This protocol describes techniques for capturing a range of swimming modes available to individual and schooling fish, and includes metrics associated with swimming physiology and behaviour.

魚類の遊泳性能評価

Swimming performance tests of fish have been integral to studies of muscle energetics, swimming mechanics, gas exchange, cardiac physiology, disease, ...

Assessment of Swim Endurance and Swim Behavior in Adult Zebrafish

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. Following complete transection of their spinal cord, zebrafish extend glial and axonal bridges across severed tissue, regenerate neurons proximal to the lesion, and regain their swim capacities within 8 weeks of injury. Recovery of swim function is thus a central readout for functional spinal cord repair. Here, we describe a set of behavioral assays to quantify zebrafish motor capacity inside an enclosed swim tunnel. The goal of these methods is to provide quantifiable measurements of swim endurance and swim behavior in adult zebrafish. For swim endurance, zebrafish are subjected to a constantly increasing water current velocity until exhaustion, and time at exhaustion is reported. For swim behavior assessment, zebrafish are subjected to low current velocities and swim videos are captured with a dorsal view of the fish. Percent activity, burst frequency, and time spent against the water current provide quantifiable readouts of swim behavior. We quantified swim endurance and swim behavior in wild-type zebrafish before injury and after spinal cord transection. We found that zebrafish lose swim function after spinal cord transection and gradually regain that capacity between 2 and 6 weeks post-injury. The methods described in this study could be applied to neurobehavioral, musculoskeletal, skeletal muscle regeneration, and neural regeneration studies in adult zebrafish.

Capable of functional recovery after spinal cord injury, adult zebrafish is a premier model system to elucidate innate mechanisms of neural regeneration. Here, we describe swim endurance and swim behavior assays as functional readouts of spinal cord regeneration.

成体ゼブラフィッシュの水泳持久力と水泳行動の評価

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. ...

コンピュータ支援ビデオとデジタル画像解析を用いた銅水溶液の急性暴露に応答して、魚の遊泳行動を定量化

要約

さらに動画を探す

この動画の章

ゼブラフィッシュで筋肉のパフォーマンスと機能を評価するためにタッチ誘発応答と歩行アッセイを使用して

急性および慢性毒性試験ターコイズメダカによる furzeriのためのプロトコル

稚魚の行動応答プロファイルを調べるための実験プロトコル: 神経興奮剤カフェインへの応用

ゼブラフィッシュで自動化されたハイコンテンツ炎症アッセイ：創薬を促進する

光強度の変化にゼブラフィッシュの応答性を測定する行動アッセイ

行動追跡とメキシコの血脈の Neuromast イメージング

ゼブラフィッシュにおける磁界感度に人格の影響を評価します。

自由水泳弱電気魚の長期的な行動の追跡

魚類の遊泳性能評価

成体ゼブラフィッシュの水泳持久力と水泳行動の評価

コンピュータ支援ビデオとデジタル画像解析を用いた銅水溶液の急性暴露に応答して、魚の遊泳行動を定量化

要約

さらに動画を探す

この動画の章

ゼブラフィッシュで筋肉のパフォーマンスと機能を評価するためにタッチ誘発応答と歩行アッセイを使用して

急性および慢性毒性試験ターコイズ メダカによる furzeriのためのプロトコル

稚魚の行動応答プロファイルを調べるための実験プロトコル: 神経興奮剤カフェインへの応用

ゼブラフィッシュで自動化されたハイコンテンツ炎症アッセイ：創薬を促進する

光強度の変化にゼブラフィッシュの応答性を測定する行動アッセイ

行動追跡とメキシコの血脈の Neuromast イメージング

ゼブラフィッシュにおける磁界感度に人格の影響を評価します。

自由水泳弱電気魚の長期的な行動の追跡

魚類の遊泳性能評価

成体ゼブラフィッシュの水泳持久力と水泳行動の評価

急性および慢性毒性試験ターコイズメダカによる furzeriのためのプロトコル