Name: a simple technique to assay locomotor activity in drosophila
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この研究は、東呉大学と中国国家科学財団(NSFC)(82171414)からの特別打ち上げ基金によって支援されました。Chunfeng Liu教授の研究室メンバーの議論とコメントに感謝します。

W1118 成虫のハエと3齢幼虫を本研究に用いた。
1. 実験準備
注: ショウジョウバエ の移動追跡のためのオープンフィールドアリーナは、無色無臭のシリカゲルで作られています。
<ol><li>試薬Aと試薬Bを、シリカキットの製造元の説明書に従って1:10の比率で混合します( 材料表を参照)。色が白に変わるまで...

<ol>
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M., 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=Automated+real-time+quantification+of+group+locomotor+activity+in+Drosophila+melanogaster.">Automated real-time quantification of group locomotor activity in Drosophila melanogaster.</a> Scientific Reports. 9 (1), 4427 (2019).</li><li>Werkhoven, Z., Rohrsen, C., Qin, C., Brembs, B., de Bivort, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MARGO+(Massively+Automated+Real-time+GUI+for+Object-tracking),+a+platform+for+high-throughput+ethology.">MARGO (Massively Automated Real-time GUI for Object-tracking), a platform for high-throughput ethology.</a> PLoS One. 14 (11), e0224243 (2019).</li><li>Gargano, J. W., Martin, I., Bhandari, P., Grotewiel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+iterative+negative+geotaxis+(RING):+a+new+method+for+assessing+age-related+locomotor+decline+in+Drosophila.">Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila.</a> Experimental Gerontology. 40 (5), 386-395 (2005).</li><li>Cichewicz, K., Hirsh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ShinyR-DAM:+a+program+analyzing+Drosophila+activity,+sleep+and+circadian+rhythms.">ShinyR-DAM: a program analyzing Drosophila activity, sleep and circadian rhythms.</a> Communications Biology. 1, 25 (2018).</li><li>McParland, A. L., Follansbee, T. L., Ganter, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+larval+activity+in+the+Drosophila+activity+monitor.">Measurement of larval activity in the Drosophila activity monitor.</a> Journal of Visualized Experiments. 98, e52684 (2015).</li><li>Walter, T., Couzin, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRex,+a+fast+multi-animal+tracking+system+with+markerless+identification,+and+2D+estimation+of+posture+and+visual+fields.">TRex, a fast multi-animal tracking system with markerless identification, and 2D estimation of posture and visual fields.</a> eLife. 10, (2021).</li><li>Maia Chagas, A., Prieto-Godino, L. L., Arrenberg, A. B., Baden, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+&#8364;100+lab:+A+3D-printable+open-source+platform+for+fluorescence+microscopy,+optogenetics,+and+accurate+temperature+control+during+behaviour+of+zebrafish,+Drosophila,+and+Caenorhabditis+elegans.">The &#8364;100 lab: A 3D-printable open-source platform for fluorescence microscopy, optogenetics, and accurate temperature control during behaviour of zebrafish, Drosophila, and Caenorhabditis elegans.</a> PLoS Biology. 15 (7), e2002702 (2017).</li><li>Nichols, C. D., Becnel, J., Pandey, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+assay+Drosophila+behavior.">Methods to assay Drosophila behavior.</a> Journal of Visualized Experiments. (61), (2012).</li><li>Xiao, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+assay+the+behavior+of+Drosophila+melanogaster+for+toxicity+study.">Methods to assay the behavior of Drosophila melanogaster for toxicity study.</a> Methods in Molecular Biology. 2326, 47-54 (2021).</li><li>Schindelin, 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Johnson, M. E., Bobrovskaya, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+update+on+the+rotenone+models+of+Parkinson's+disease:+their+ability+to+reproduce+the+features+of+clinical+disease+and+model+gene-environment+interactions.">An update on the rotenone models of Parkinson's disease: their ability to reproduce the features of clinical disease and model gene-environment interactions.</a> Neurotoxicology. 46, 101-116 (2015).</li><li>Coulom, H., Birman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+exposure+to+rotenone+models+sporadic+Parkinson's+disease+in+Drosophila+melanogaster.">Chronic exposure to rotenone models sporadic Parkinson's disease in Drosophila melanogaster.</a> The Journal of Neuroscience. 24 (48), 10993-10998 (2004).</li><li>Kumar, P. P., Bawani, S. S., Anandhi, D. U., Prashanth, K. V. 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フィジーの画像処理プログラムと互換性のあるオープンソースの AnimalTracker APIに基づいて、研究者が成虫と個々の幼虫の両方のハエを追跡することにより、自発運動活動を体系的に評価できるようにする方法を設計しました。 AnimalTracke はJavaで書かれたツールであり、既存のデータベースや他のツールに簡単に統合して、アプリケーションで設計された動物の追跡行動の分析を?...

ショウジョウバエメラノガスターは、遺伝子改変1、薬物治療2、老化3によって作成された神経疾患モデルにおける細胞および分子機能を調べるための優れたモデル生物を提供します。ヒトとショウジョウバエの間の生物学的経路、物理的特性、および疾患関連ホモログ遺伝子の高い保存により、ショウジョウバエは分子レベルから行動レベル4までの理想的な模倣になります。多くの疾患モデルにおいて、行動不全は重要な指標であり、さまざまなヒトニューロパシーに有用なモデルを提供します5,6。ショウジョウバエは現在、複数のヒト疾患、神経発達、およびパーキンソン病や筋萎縮性側索硬化症などの神経変性疾患の研究に使用されています7,8。疾患モデルの運動能力を検出することは、病原性の進行を理解するために重要であり、疾患プロセスの根底にある分子メカニズムとの表現型の相関関係を提供する可能性があります。
最近、市販のソフトウェアツールと費用対効果の高いプログラムが、グループ化されたハエ9,10でのハイスループットテストやリアルタイムでの移動の測定11,12など、ショウジョウバエの自発運動検出戦略のために開発されました。そのような従来のアプローチの1つは、クライミングアッセイとも呼ばれる迅速なインタラクティブネガティブジオタキシス(RING)であり、同じ性別と年齢の大規模なハエの個体群を封じ込めることを可能にする複数のチャネルを含み、データ収集中の変動を減らします9,13。自発運動挙動を分析するための別の事前試験方法は、TriKinetics ショウジョウバエ活動モニタ(DAM)であり、これは、薄いガラス管14内のハエ活動運動を検出するために複数のビームを使用する装置である。この装置は、位置を連続的に記録し、これは、より長い期間にわたるハエの活動および概日リズムを研究するためにビーム交差を計算することによって自動移動を表す15。これらの方法は、ショウジョウバエの行動欠陥を分析して行動移動の変化を決定する際に広く使用されてきましたが、常に特別な試験装置または複雑な分析プロセスが必要であり、限られた単純な装置で一部のモデルでは適用が制限されています。FlyGrAM11やショウジョウバエアイランドアッセイ10など、成虫のショウジョウバエをテストするための動物追跡グループベースの戦略は、事前定義された領域で社会的募集と個々の追跡を実装します。それにもかかわらず、反抗された地域での社会的個人的制限は、ハエの衝突または重なりによって引き起こされる画像内の識別に悪影響を与える可能性があります。TRex16、MARGO 12、FlyPi17などのオープンソースのマテリアルベースの方法には緊急事態がありますが、行動テストで柔軟に使用することで、ハエを迅速に追跡できます。これらのテストアプローチは、精巧な実験装置の設置、特別なソフトウェア要件、またはプロのコンピューター言語に関連しています。幼虫の場合、単位時間18あたりのグリッド境界線の数を横切って移動した総距離を測定するか、または個人の体壁収縮を手動で大まかに数えること19は、自発運動能力を評価するための主要な方法です。機器や装置、分析方法の精度が不足しているため、幼虫の行動移動が検出を逃れ、行動運動、特に微細な動きを正確に評価することが困難になる可能性があります15。
本開発手法は,画像処理プログラム「フィジー(ImageJ)」と互換性のあるアプリケーションプログラミングインターフェース(API) AnimalTracker を利用し,高精細(HD)映像からハエの追跡行動を解析し,成虫と仔魚の運動活動を系統的に評価する手法である.フィジーはオープンソースソフトウェアです ImageJディストリビューション 堅牢なソフトウェアライブラリと多数のスクリプト言語を組み合わせることができるため、画像処理アルゴリズムのラピッドプロトタイピングが可能になり、画像分析機能で生物学者の間で人気があります20。現在のアプローチでは、フィジーの AnimalTracker APIへの統合を利用して、パーソナライズされたアルゴリズム挿入を備えた独自の ショウジョウバエ 行動アッセイを開発し、自発運動行動の堅牢な分析機能をサポートするための詳細なドキュメントとチュートリアルの有用なステップを提供します(図1)。ハエの衝突または重なりによって引き起こされる画像内の客観的識別の複雑さを回避するために、各アリーナは1つのハエのみをホストするように制限されています。アプローチの追跡精度を評価した上で、パーキンソン病の動物モデルに一般的に使用されている毒性薬物ロテノンを投与された ショウジョウバエ の運動運動を追跡および定量化するために実施され、最終的に薬物治療における移動障害を発見した21。オープンソースでフリーなソフトウェアを採用したこの方法論は、高コストの計測を必要とせず、 ショウジョウバエ の行動運動を正確かつ再現性よく解析することができます。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Animal tracker</td>
			<td>Hungarian Brain Research Program</td>
			<td>version: 1.7</td>
			<td>pfficial website: http://animaltracker.elte.hu/main/downloads</td>
		</tr>
		<tr>
			<td>Camera software</td>
			<td>Microsoft</td>
			<td>version: 2021.105.10.0</td>
			<td>built-in windows 10 system</td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>DELL</td>
			<td>Vostro-14-5480</td>
			<td>a comupter running win 10 system is available</td>
		</tr>
		<tr>
			<td>Drosophila carbon dioxide anesthesia workstation</td>
			<td>Wu han Yihong technology</td>
			<td>#YHDFPCO2-018</td>
			<td>official website: http://www.yhkjwh.com/</td>
		</tr>
		<tr>
			<td>Fiji software</td>
			<td>Fiji team</td>
			<td>version: 1.53v</td>
			<td>official website: https://fiji.sc/</td>
		</tr>
		<tr>
			<td>Format factory software</td>
			<td>Pcfreetime</td>
			<td>version: X64 5.4.5</td>
			<td>official website: http://www.pcfreetime.com/formatfactory/CN/index.html</td>
		</tr>
		<tr>
			<td>Graph pad prism</td>
			<td>GraphPad Software</td>
			<td>version: 8.0.2</td>
			<td>official website: https://www.graphpad-prism.cn</td>
		</tr>
		<tr>
			<td>Hight definition camera</td>
			<td>TTQ</td>
			<td>Jingwang2 (HD1080P F1.6 6-60mm)</td>
			<td>official website: http://www.ttq100.com/product_show.php?id=35</td>
		</tr>
		<tr>
			<td>Office software</td>
			<td>Microsoft</td>
			<td>version: office 2019</td>
			<td>official website: https://www.microsoftstore.com.cn/software/office</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Bkman</td>
			<td>110301003</td>
			<td>size: 60 mm</td>
		</tr>
		<tr>
			<td>Silica gel</td>
			<td>DOW</td>
			<td>SYLGARD 184 Silicone Elastomer Kit</td>
			<td>Mix well according to the instructions</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Macklin</td>
			<td>#144-55-8</td>
			<td>Mix well with silica gel</td>
		</tr>
	</tbody>
</table>

a simple technique to assay locomotor activity in drosophila

ショウジョウバエは 、高度な遺伝子操作技術が豊富で、多様な行動特徴があるため、さまざまな疾患を研究するための理想的なモデル生物です。動物モデルにおける行動不全の特定は、例えば、患者が運動機能の障害を経験することが多い神経変性疾患において、疾患の重症度の重要な尺度である。しかし、薬物治療やトランスジェニック個体など、ハエモデルの運動障害を追跡および評価するためのさまざまなシステムが利用できるため、複数の角度から正確に評価するための経済的でユーザーフレンドリーなシステムはまだ不足しています。ここでは、フィジーの画像処理プログラムと互換性のある AnimalTracker アプリケーションプログラミングインターフェース(API)に基づく手法を開発し、記録されたビデオから成虫と幼虫の両方の個体の移動活動を体系的に評価し、追跡行動の分析を可能にします。この方法は、行動を記録および分析するために高解像度カメラとコンピューター周辺機器の統合のみを必要とするため、トランスジェニックまたは環境行動障害のあるハエモデルをスクリーニングするための手頃な価格で効果的なアプローチになります。薬理学的に処理されたハエを用いた行動試験の例を挙げて、成虫のハエと幼虫の両方の行動変化を再現性の高い方法で検出する方法を示します。

本研究では、ロテノンで処理された成虫のハエと3齢幼虫の自発運動障害を調べ、薬物溶媒ジメチルスルホキシド(DMSO)を与えられた対照ハエの運動活動と比較しました。ショウジョウバエにおけるロテノンによる治療は、脳22においてドーパミン作動性ニューロン喪失を引き起こし、重大な自発運動障害23を引き起こすことが示されている。図<strong cla...

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A Simple Technique to Assay Locomotor Activity in Drosophila

本プロトコルは、オープンソースソフトウェアフィジーを使用して手作りのアリーナでのハエの動きを追跡および分析することにより、ショ ウジョウバエ の自発運動を評価し、高解像度ビデオ録画に基づいて各フレームのピクセルをセグメント化し、速度、距離などのパラメータを計算するプラグインと互換性があります。

ショウジョウバエの自発運動活性をアッセイする簡単な技術

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and ...

この方法は、オープンソースソフトウェアに基づいており、記録されたビデオから成虫と幼虫のハエの追跡行動について局所的な運動活動を体系的に評価します。この方法は、行動を記録および分析するための低コストのデバイス統合を必要とするため、ハエの移動をスクリーニングするための手頃な価格で効果的なアプローチになります。この技術は、局所運動障害のあるヒト疾患のハエモデルにおける行動変容の同定に用いられているが、疾患、障害、困難の治療には適していない。分析プロセス中に関連するビデオウィンドウ、つまり元のビデオウィンドウと処理中のビデオウィンドウをクリアすることが不可欠です。まず、製造元の指示に従って、シリカキットの試薬Aと試薬Bを1対10の比率で混合し、ショウジョウバエの移動追跡用のオープンフィールドアリーナを準備します。次に、HDカメラを三脚にセットし、カメラのレンズがシリカアリーナの表面に垂直になるように調整します。次に、フィジーでビデオを開きます。進行状況バーを最初のフレームにドラッグし、暗黙のうちに承認します。フリーハンド選択ツールを使用してフライの全身を選択します。画像、調整、明るさとコントラストをクリックして、選択した領域のグレー値が広い背景に近づくまでホワイトバランスを調整します。麻酔をかけたフライをオープンフィールドアリーナに移します。ハエが麻酔から回復したら、ハエと一緒にアリーナディッシュをカメラの下に置き、左右にすばやく振って、記録の開始時にハエが動いていることを確認します。カメラアプリケーションの録画ボタンを押して、ビデオ録画を開始します。録画が完了したら、停止ボタンを押してビデオ録画を終了します。録画したビデオをMJ異教徒コーディングでAVI形式に変換して、フィジーを使用して開いて分析できるようにします。また、ビデオのフレーム/秒を、成虫のハエの場合は15 fps、幼虫の場合は12 fpsに設定します。フィジーでビデオを開くには、ポップアップウィンドウで2つのオプションを選択し、仮想スタックを使用してグレースケールに変換します。動物トラッカープラグインのアクティブな画像の設定ツールを使用して処理ウィンドウを取得し、楕円形ツールを使用して元のビデオウィンドウのアリーナを一周する追跡領域を作成します。処理ウィンドウの最初の空白フレームのフィルターと 2 つのフィルターのパラメーターを設定します。次に、元のビデオウィンドウの次のフレームを選択し、処理ウィンドウのフィルタリングされたサーフェスを選択します。フィルタリングされた処理ウィンドウを選択したら、[しきい値の設定]ツールを使用して、処理ウィンドウで覆われた赤いプロファイルでトラクトフライを回転させます。次に、set blob 検出器を使用して、処理ウィンドウで覆われた赤いプロファイルでハエをコンピューターに認識させます。テキスト原稿に記載されているように、成虫のハエまたは幼虫の最後のフレームを設定します。BLOB の表示ツールを使用して、元のビデオ ウィンドウに追跡用の四角形を表示します。追跡を開始し、監視が完了したら追跡ファイルをエクスポートします。動物トラッカーと追跡アナライザープラグインを使用して、追跡ファイルとゾーンファイルをロードします。ゾーン設定を使用して目的のインデックスを選択し、パラメーター設定を変更します。フレームレートを使用してフレーム間隔の時間を計算します。トラッキングアナライザーで分析した後、スプレッドシートソフトウェアとグラフパッドプリズムを使用して定量分析チャートを作成します。トラックファイルを開きます。すべての座標を Microsoft Office Excel にコピーし、スペース キーを使用してセルを分割します。フレーム間隔あたりの不動時間を計算するには、すべての計算結果を選択し、縦棒グラフを挿入して、縦棒グラフ全体の余白で不動時間を視覚的に表示します。方向角度の変化を示すには、すべての計算結果を選択し、散布図を挿入します。ロテノンで処理された成虫のハエと3齢幼虫は、溶媒DMSOを与えられた対照のハエと比較して有意な自発運動障害を持っていました。成虫のロテノン処理の定量分析では、移動距離と平均速度の有意な減少が示されました。そして、不動時間の大幅な増加。代表的なグラフは、対照のものと比較して、ロテノンを給餌したハエのフレーム間隔あたりの移動速度のピークが少ないことを示し、自発運動活動障害の重症度を示しています。フレームあたりの移動ピクセルの制度的不動列も、対照ハエと比較して、ロテノン給餌ハエの1分以内の動きが有意に少ないことを示しました。ロテノン給餌動物および対照動物における移動方向角変化を示す代表的なグラフも、ハエが選択した方向の変化を明らかにした。同様の結果は、3齢幼虫についても観察されました。その結果、ロテノンを給餌した追跡幼虫の行動運動は、対照と比較して著しく損なわれていることが明らかになりました。分析プロセスの最も重要なステップは、ソフトウェアが効果的にチェックできるかどうかを証明するため、最初の空白フレームを形成することです。この技術により、研究者は神経や筋肉の損傷などの複数の欠陥の根底にあるメカニズムをより深く掘り下げることができます。

a-simple-technique-to-assay-locomotor-activity-in-drosophila

Research

JoVE Journal

Behavior

A Simple Technique to Assay Locomotor Activity in Drosophila

Mouse Short- and Long-term Locomotor Activity Analyzed by Video Tracking Software

Locomotor activity (LMA) is a simple and easily performed measurement of behavior in mice and other rodents. Improvements in video tracking software (VTS) have allowed it to be coupled to LMA testing, dramatically improving specificity and sensitivity when compared to the line crossings method with manual scoring. In addition, VTS enables high-throughput experimentation. While similar to automated video tracking used for the open field test (OFT), LMA testing is unique in that it allows mice to remain in their home cage and does not utilize the anxiogenic stimulus of bright lighting during the active phase of the light-dark cycle. Traditionally, LMA has been used for short periods of time (mins), while longer movement studies (hrs-days) have often used implanted transmitters and biotelemetry. With the option of real-time tracking, long-, like short-term LMA testing, can now be conducted using videography. Long-term LMA testing requires a specialized, but easily constructed, cage so that food and water (which is usually positioned on the cage top) does not obstruct videography. Importantly, videography and VTS allows for the quantification of parameters, such as path of mouse movement, that are difficult or unfeasible to measure with line crossing and/or biotelemetry. In sum, LMA testing coupled to VTS affords a more complete description of mouse movement and the ability to examine locomotion over an extended period of time.

Locomotor activity (LMA) is a simple and easily performed measurement of behavior in mice. Coupling of video tracking software (VTS) and LMA allows for the improvement of specificity and sensitivity, especially when compared with the manual, line crossing method of LMA analysis. Additionally VTS allows long-term tracking of mouse LMA.

ビデオトラッキングソフトウェアによって分析されるマウスの短期および長期の自発運動

Locomotor  activity (LMA) is a simple and easily performed measurement of behavior in mice  and other rodents. Improvements in video tracking software ...

行動学

Behavioral and Locomotor Measurements Using an Open Field Activity Monitoring System for Skeletal Muscle Diseases

The open field activity monitoring system comprehensively assesses locomotor and behavioral activity levels of mice. It is a useful tool for assessing locomotive impairment in animal models of neuromuscular disease and efficacy of therapeutic drugs that may improve locomotion and/or muscle function. The open field activity measurement provides a different measure than muscle strength, which is commonly assessed by grip strength measurements. It can also show how drugs may affect other body systems as well when used with additional outcome measures. In addition, measures such as total distance traveled mirror the 6 min walk test, a clinical trial outcome measure. However, open field activity monitoring is also associated with significant challenges: Open field activity measurements vary according to animal strain, age, sex, and circadian rhythm. In addition, room temperature, humidity, lighting, noise, and even odor can affect assessment outcomes. Overall, this manuscript provides a well-tested and standardized open field activity SOP for preclinical trials in animal models of neuromuscular diseases. We provide a discussion of important considerations, typical results, data analysis, and detail the strengths and weaknesses of open field testing. In addition, we provide recommendations for optimal study design when using open field activity in a preclinical trial.

Open field activity levels are used to assess locomotive and behavioral activity levels. This protocol provides a well-designed, standardized protocol to use in preclinical trials for neuromuscular disorders.

骨格筋の病気のためにオープンフィールドアクティビティ·モニタリングシステムを用いた行動と自発運動の測定

The open field activity monitoring system comprehensively assesses locomotor and behavioral activity levels of mice. It is a useful tool for assessing ...

Use of the Open Field Maze to Measure Locomotor and Anxiety-like Behavior in Mice

Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of the most commonly used platforms to measure behaviors in animal models. It is a fast and relatively easy test that provides a variety of behavioral information ranging from general ambulatory ability to data regarding the emotionality of the subject animal. As it relates to rodent models, the procedure allows the study of different strains of mice or rats both laboratory bred and wild-captured. The technique also readily lends itself to the investigation of different pharmacological compounds for anxiolytic or anxiogenic effects. Here, a protocol for use of the open field maze to describe mouse behaviors is detailed and a simple analysis of general locomotor ability and anxiety-related emotional behaviors between two strains of C57BL/6 mice is performed. Briefly, using the described protocol we show Wild Type mice exhibited significantly less anxiety related behaviors than did age-matched Knock Out mice while both strains exhibited similar ambulatory ability.

A protocol is provided to use an Open Field Maze to access general locomotor activity, anxiety and emotionality in a laboratory mouse model.

マウスにおける自発運動と不安様行動を測定するために、オープンフィールド迷路の利用

Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of ...

The Treadmill Fatigue Test: A Simple, High-throughput Assay of Fatigue-like Behavior for the Mouse

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of current interventions to adequately treat fatigue in all patients leaves a need for new treatment options. Despite the therapeutic need and importance of preclinical research in helping identify promising novel treatments, few preclinical assays of fatigue are available. Moreover, the most common preclinical assay used to assess fatigue-like behavior, voluntary wheel running, is not suitable for use with some strains of mice, may not be sensitive to drugs that reduce fatigue, and has relatively low throughput. The current protocol describes a novel, non-voluntary preclinical assay of fatigue-like behavior, the treadmill fatigue test, and provides evidence of its efficacy in detecting fatigue-like behavior in mice treated with a chemotherapy drug known to cause fatigue in humans and fatigue-like behavior in animals. This assay may be a beneficial alternative to wheel running, as fatigue-like behavior and potential interventions can be assessed in a greater number of mice over a shorter time frame, thus permitting faster discovery of new therapeutic options.

Fatigue is a common, undertreated and frequently poorly-understood symptom in many diseases and disorders. New preclinical assays of fatigue may help to improve current understanding and future treatment of fatigue. To that end, the current protocol provides a novel means of measuring fatigue-like behavior in the mouse.

トレッドミル疲労試験：マウスの​​疲労のような挙動のシンプルな、ハイスループットアッセイ

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of ...

Using a Split-belt Treadmill to Evaluate Generalization of Human Locomotor Adaptation

Understanding the mechanisms underlying locomotor learning helps researchers and clinicians optimize gait retraining as part of motor rehabilitation. However, studying human locomotor learning can be challenging. During infancy and childhood, the neuromuscular system is quite immature, and it is unlikely that locomotor learning during early stages of development is governed by the same mechanisms as in adulthood. By the time humans reach maturity, they are so proficient at walking that it is difficult to come up with a sufficiently novel task to study de novo locomotor learning. The split-belt treadmill, which has two belts that can drive each leg at a different speed, enables the study of both short- (i.e., immediate) and long-term (i.e., over minutes-days; a form of motor learning) gait modifications in response to a novel change in the walking environment. Individuals can easily be screened for previous exposure to the split-belt treadmill, thus ensuring that all experimental participants have no (or equivalent) prior experience. This paper describes a typical split-belt treadmill adaptation protocol that incorporates testing methods to quantify locomotor learning and generalization of this learning to other walking contexts. A discussion of important considerations for designing split-belt treadmill experiments follows, including factors like treadmill belt speeds, rest breaks, and distractors. Additionally, potential but understudied confounding variables (e.g., arm movements, prior experience) are considered in the discussion.

We describe a protocol for investigating human locomotor adaptation using the split-belt treadmill, which has two belts that can drive each leg at a different speed. We specifically focus on a paradigm designed to test the generalization of adapted locomotor patterns to different walking contexts (e.g., gait speeds, walking environments).

分割トレッドミルを使用した運動適応の一般化の評価

Understanding the mechanisms underlying locomotor learning helps researchers and clinicians optimize gait retraining as part of motor rehabilitation. ...

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors remains poorly understood. In Drosophila melanogaster, environmental cues, such as food availability, affect mating activity offering a tractable system to investigate the mechanisms modulating sexual behavior. In D. melanogaster, environmental cues are often sensed via the chemosensory gustatory and olfactory systems. Here, we present a method to test the effect of environmental chemical cues on mating behavior. The assay consists of a small mating arena containing food medium and a mating couple. The mating frequency for each couple is continuously monitored for 24 h. Here we present the applicability of this assay to test environmental compounds from an external source through a pressurized air system as well as manipulation of the environmental components directly in the mating arena. The use of a pressurized air system is especially useful to test the effect of very volatile compounds, while manipulating components directly in the mating arena can be of value to ascertain a compound's presence. This assay can be adapted to answer questions about the influence of genetic and environmental cues on mating behavior and fecundity as well as other male and female reproductive behaviors.

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

We demonstrate an assay to analyze the environmental and genetic cues that influence mating behavior in the fruit fly Drosophila melanogaster.

環境内の交信が交配行動に及ぼす影響を調べる方法<em&gt;ショウジョウバエmelanogaster</em

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors ...

A Computational Method to Quantify Fly Circadian Activity

In most animals and plants, circadian clocks orchestrate behavioral and molecular processes and synchronize them to the daily light-dark cycle. Fundamental mechanisms that underlie this temporal control are widely studied using the fruit fly Drosophila melanogaster as a model organism. In flies, the clock is typically studied by analyzing multiday locomotor recording. Such a recording shows a complex bimodal pattern with two peaks of activity: a morning peak that happens around dawn, and an evening peak that happens around dusk. These two peaks together form a waveform that is very different from sinusoidal oscillations observed in clock genes, suggesting that mechanisms in addition to the clock have profound effects in producing the observed patterns in behavioral data. Here we provide instructions on using a recently developed computational method that mathematically describes temporal patterns in fly activity. The method fits activity data with a model waveform that consists of four exponential terms and nine independent parameters that fully describe the shape and size of the morning and evening peaks of activity. The extracted parameters can help elucidate the kinetic mechanisms of substrates that underlie the commonly observed bimodal activity patterns in fly locomotor rhythms.

A method to quantify the main temporal features seen in fly circadian locomotor rhythms is presented. The quantification is achieved by fitting fly activity with a multi-parametric model waveform. The model parameters describe the shape and size of the morning and evening peaks of daily activity.

フライの日周期活動を定量化する手法

In most animals and plants, circadian clocks orchestrate behavioral and molecular processes and synchronize them to the daily light-dark cycle. ...

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

Cells display different morphologies and complex anatomical relationships. How do cells interact with their neighbors? Do the interactions differ between cell types or even within a given type? What kinds of spatial rules do they follow? The answers to such fundamental questions in vivo have been hampered so far by a lack of tools for high resolution single cell labeling. Here, a detailed protocol to target single cells with a MultiColor FlpOut (MCFO) technique is provided. This method relies on three differently tagged reporters (HA, FLAG and V5) under UAS control that are kept silent by a transcriptional terminator flanked by two FRT sites (FRT-stop-FRT). A heat shock pulse induces the expression of a heat shock-induced Flp recombinase, which randomly removes the FRT-stop-FRT cassettes in individual cells: expression occurs only in cells that also express a GAL4 driver. This leads to an array of differently colored cells of a given cell type that allows the visualization of individual cell morphologies at high resolution. As an example, the MCFO technique can be combined with specific glial GAL4 drivers to visualize the morphologies of the different glial subtypes in the adult Drosophila brain.

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

Cells display different morphologies and establish a variety of interactions with their neighbors. This protocol describes how to reveal the morphology of single cells and to investigate cell-cell interaction by using the well-established Gal4/UAS expression system.

多色 FlpOut 技術の高分解能単一細胞の形態とショウジョウバエにおけるグリア細胞の細胞間相互作用の研究への応用

Cells display different morphologies and complex anatomical relationships. How do cells interact with their neighbors? Do the interactions differ between ...

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration

Neurodegenerative diseases are frequently associated with a progressive loss of movement ability, reduced life span, and age-dependent neurodegeneration. To understand the mechanism of these cellular events, and their causal relationships with each other, Drosophila melanogaster, with its sophisticated genetic tools and diverse behavioral features, are used as disease models for assessing neurodegenerative phenotypes. Here we describe a high-throughput method to analyze Drosophila adult negative geotaxis behavior, as an indication for possible motor defects associated with neurodegeneration. An automated machine is designed and developed to drive fly synchronization using an initial electric impulse, later allowing the recording of negative geotaxis behavior over a course of secs to mins. Images from the digitally recorded video are then processed with the self-designed RflyDetection software for statistical data manipulation. Different from the manually controlled negative geotaxis assay based on single fly, this precise, fast, and high-throughput protocol allows data acquisition from more than hundreds of flies simultaneously, providing an efficient approach to advance our understanding in the underlying mechanism of locomotor deficits associated with neurodegeneration.

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration

This step-by-step protocol analyzes Drosophila negative geotaxis behavior using an automated multi-cylinder system that hosts hundreds of flies and synchronizes their action by an electric motor. Upon synchronization, fly negative geotaxis behavior is assayed, digitally recorded, and analyzed using the self-designed RflyDetection software.

神経変性のショウジョウバエモデル アダルト登山行動を分析するための自動化された迅速な反復負走アッセイ

Neurodegenerative diseases are frequently associated with a progressive loss of movement ability, reduced life span, and age-dependent neurodegeneration. ...

A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation in mouse models is a non-invasive and reproducible activity assay that is directly analogous to measures of patient ambulation such as the 6-minute walk test and related mobility scores. Many common methods for testing mouse ambulation speed and distance are based on the open field test, where an animal&#39;s free movement within an arena is measured over time. One major downside to this approach is that commercial software and equipment for high-resolution motion tracking is expensive and may require transferring mice to specialized facilities for testing. Here, we describe a low-cost, video-based system for measuring mouse ambulation that utilizes free and open-source software. Using this protocol, we demonstrate that voluntary ambulation in the dystrophin-null mdx mouse model for Duchenne muscular dystrophy (DMD) is decreased relative to wild-type mouse activity. In mdx mice expressing the utrophin transgene, these activity deficits are not observed and the total distance traveled is indistinguishable from wild-type mice. This method is effective for measuring changes in voluntary ambulation associated with dystrophic pathology, and provides a versatile platform that can be readily adapted to diverse research settings.

This protocol describes a flexible, low-cost system for measuring mouse ambulation in an open field activity assay. We show that a 6-minute ambulation assay based on this system detects a decrease in voluntary movement in mdx mice, and accurately distinguishes improvement in a muscle-specific rescue of these animals.

筋ジストロフィーのマウス ・ モデルで歩行を測定するためのシンプルで低コスト分析

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation ...