This work is supported by a Canadian Health Research Institute (CIHR) Team Grant to Dr. Bolduc (co-PI) and Dr. Guy Rouleau (PI). We would like to thank Dr. Oksana Suchowersky, Dr. Kathryn Todd, the members of the University of Alberta fly club and Dr. Clayton Dickson for help in development of the method and statistical analysis.

キイロショウジョウバエの研究は、アルバータ州の研究指針の大学を遵守していました。 1.フライコレクション<ol><li>食品を含む25ミリメートルX 95ミリメートルの回収バイアル中のCO 2（g）の麻酔と場所を使用して、20ハエを収集します。 </li><li>水平にハエを含むストアバイアルは、バイアル底に蓄積する可能性のある液体でハエをトラップ避けるために。 </li><li>約15時間、インキュベーター中で45％の湿度で22℃で少なくとも21時間、ハエをインキュベートします。暗サイクル：12時間の明とインキュベーターを設定します。 </li></ol> 2.クライミングアッセイ<ol><li>翌朝は、メスシリンダー250mlのガラスに単一バイアルから20ハエを転送します。それを一定の日常を維持するために、シリンダの位置をマークします。遺伝子型間の交差汚染を防止するために、遺伝子型ごとにガラスシリンダーを使用してください。各実験及びROの最後に洗います遺伝子型との間でそれらをテート。 <ol><li>温度22℃、湿度40％であった（視覚の潜在的な欠陥がある場合、または赤色光）は、周囲の光の中で実験を行います。概日リズムの交絡を回避するために、常に一日の同じ時間に実験を行います。 </li></ol></li><li>任意のハエ（ 図2）の漏れを防止するためのバリア膜（ワックスフィルム）とシリンダーの上部をシール。 </li><li>三脚にビデオカメラを設定します。 250ミリリットルの190ミリリットルライン上の焦点カメラは、シリンダ（17.5センチメートル）を卒業しました。 </li><li>シリンダーの底部に、食品バイアルに死んだハエの数をカウントします。死亡率としてこの番号を記録します。 </li><li>非常に軽く内側底面にハエを変位させるように十分な力で繰り返しクローズドセルフォームパッドに対してシリンダーをタップします。カメラの記録を押すようにもう一方の手を使用しながら10回 - 5をタップします。 </li><li>カメラの「録音」ボタンを押してください。 </li><li> VIDを開始EOカメラ記録とは別個の非リズミカルなパターンでシリンダーを6回タップします。 </li><li>ハエが最後のタップされた時点から2分間各試験を実施し、選択した各時点で17.5センチメートル（190ミリリットル）（10秒ごとに定量化）の高さを横切るハエの数を記録します。注：シリンダーのマーキングミリリットルを直径に応じて、1気筒モデルから別のものに変化します。エラーを回避するには、使用される各気筒の高さを測定します。 </li><li>裁判が終わった後、95％エタノールにハエを処分。 </li><li>すべての複製が新鮮なハエとするたびにテストされるまで繰り返して、2.9に2.1を繰り返します。 注：5の複製は、変異が運動に強い影響を持つ十分であるかもしれないが、20ハエ（200ハエ）の10の生物学的反復は、より小さな差を検出することをお勧めします。 </li><li>実験の完了後、再使用する実験用食器洗い機、乾燥O / Nでのシリンダを洗浄します。 </li></ol> 3.分析<ol><li> ANALY各フライ試験のZE動画。各10秒は、ターゲットラインを通過したハエの総数を記録します。 <ol><li>フライバックダウン登っまたは低下した場合、-1のように飛ぶし、レコードはバックダウン登ったり下ったハエと同じ数として目標ラインと交差するように、次のフライを数えます。 15 番目のフライが目標ラインを下回った場合、（16 番目が飛ぶ）ラインを通過する次のハエは、15 番目、16番目を飛行していないと考えられています。 </li></ol></li><li>裁判に残るハエの数を得るために、ハエ（20）の合計数から死亡を引きます。各時点で、ターゲット·ラインの上のハエの画分を得ました。 </li><li>各時点での各割合をプロットする（ 図3参照 ）。 </li><li> 120秒のデータポイントでパフォーマンスを分析し、2基が存在する場合、またはANOVAとボンフェローニ修正と多重比較のための事後テストは、（計画および計画外の共同のためのテューキーのためのスチューデントt検定を行いますmparisons）。コルモゴロフ-スミルノフ17はまた、正規と同等の分散を確認するために行われるだけでなく、コントロールに変異群の分布を比較するためにテストします。 </li><li> 、老化を介してデータを現在進行赤字（ 図4）があるかどうかを確認するために、異なる年齢のハエで120秒に（2日、1週間、2週間）を登るハエの割合をプロットします。 </li></ol>

<ol>
	<li>Auluck, P. K., Bonini, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+prevention+of+Parkinson+disease+in+Drosophila.">Pharmacological prevention of Parkinson disease in Drosophila.</a> Nature medicine. 8, 1185-1186 (2002).</li><li>Bonini, N. M., Fortini, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neurodegenerative+disease+modeling+using+Drosophila.">Human neurodegenerative disease modeling using Drosophila.</a> Annual review of neuroscience. 26, 627-656 (2003).</li><li>Bolduc, F. V., Bell, K., Cox, H., Broadie, K. S., Tully, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excess+protein+synthesis+in+Drosophila+fragile+X+mutants+impairs+long-term+memory.">Excess protein synthesis in Drosophila fragile X mutants impairs long-term memory.</a> Nature neuroscience. 11, 1143-1145 (2008).</li><li>McBride, S. 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=Pharmacological+rescue+of+synaptic+plasticity,+courtship+behavior,+and+mushroom+body+defects+in+a+Drosophila+model+of+fragile+X+syndrome.">Pharmacological rescue of synaptic plasticity, courtship behavior, and mushroom body defects in a Drosophila model of fragile X syndrome.</a> Neuron. 45, 753-764 (2005).</li><li>Parker, L., Padilla, M., Du, Y., Dong, K., Tanouye, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+a+model+for+epilepsy:+bss+is+a+gain-of-function+mutation+in+the+para+sodium+channel+gene+that+leads+to+seizures.">Drosophila as a model for epilepsy: bss is a gain-of-function mutation in the para sodium channel gene that leads to seizures.</a> Genetics. 187, 523-534 (2011).</li><li>Marley, R., Baines, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+persistent+Na++current+contributes+to+seizure+in+the+slamdance+bang-sensitive+Drosophila+mutant.">Increased persistent Na+ current contributes to seizure in the slamdance bang-sensitive Drosophila mutant.</a> Journal of neurophysiology. 106, 18-29 (2011).</li><li>Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+Mutants+Of+Drosophila+Isolated+By+Countercurrent+Distribution.">Behavioral Mutants Of Drosophila Isolated By Countercurrent Distribution.</a> Proceedings of the National Academy of Sciences of the United States of America. 58, 1112-1119 (1967).</li><li>Ganetzky, B., Flanagan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+relationship+between+senescence+and+age-related+changes+in+two+wild-type+strains+of+Drosophila+melanogaster.">On the relationship between senescence and age-related changes in two wild-type strains of Drosophila melanogaster.</a> Experimental gerontology. 13, 189-196 (1978).</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 : JoVE. , (2012).</li><li>Toma, D. P., White, K. P., Hirsch, J., Greenspan, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+genes+involved+in+Drosophila+melanogaster+geotaxis,+a+complex+behavioral+trait.">Identification of genes involved in Drosophila melanogaster geotaxis, a complex behavioral trait.</a> Nature genetics. 31, 349-353 (2002).</li><li>Inagaki, H. K., Kamikouchi, A., Ito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+quantifying+simple+gravity+sensing+in+Drosophila+melanogaster.">Methods for quantifying simple gravity sensing in Drosophila melanogaster.</a> Nature protocols. 5, 20-25 (2010).</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, 386-395 (2005).</li><li>Botella, J. 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=The+Drosophila+carbonyl+reductase+sniffer+prevents+oxidative+stress-induced+neurodegeneration.">The Drosophila carbonyl reductase sniffer prevents oxidative stress-induced neurodegeneration.</a> Current biology : CB. 14, 782-786 (2004).</li><li>Feany, M. B., Bender, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+model+of+Parkinson's+disease.">A Drosophila model of Parkinson's disease.</a> Nature. 404, 394-398 (2000).</li><li>Chakraborty, R., 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=Characterization+of+a+Drosophila+Alzheimer's+disease+model:+pharmacological+rescue+of+cognitive+defects.">Characterization of a Drosophila Alzheimer's disease model: pharmacological rescue of cognitive defects.</a> PLoS One. 6, e20799 (2011).</li><li>Orso, 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=Disease-related+phenotypes+in+a+Drosophila+model+of+hereditary+spastic+paraplegia+are+ameliorated+by+treatment+with+vinblastine.">Disease-related phenotypes in a Drosophila model of hereditary spastic paraplegia are ameliorated by treatment with vinblastine.</a> J Clin Invest. 115, 3026-3034 (2005).</li><li>Lehmann, E. L., D'Abrera, H. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nonparametrics : statistical methods based on ranks. , (2006).</li><li>Sherwood, N. T., Sun, Q., Xue, M., Zhang, B., Zinn, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+spastin+regulates+synaptic+microtubule+networks+and+is+required+for+normal+motor+function.">Drosophila spastin regulates synaptic microtubule networks and is required for normal motor function.</a> PLoS biology. 2, e429 (2004).</li><li>Inlow, J. K., Restifo, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+comparative+genetics+of+mental+retardation.">Molecular and comparative genetics of mental retardation.</a> Genetics. 166, 835-881 (2004).</li><li>Palladino, M. J., Hadley, T. J., Ganetzky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-sensitive+paralytic+mutants+are+enriched+for+those+causing+neurodegeneration+in+Drosophila.">Temperature-sensitive paralytic mutants are enriched for those causing neurodegeneration in Drosophila.</a> Genetics. 161, 1197-1208 (2002).</li></ol>

The authors have nothing to disclose.

ショウジョウバエはすでにパーキンソン病14および他の神経変性条件1,2に優れたモデルであることが証明されました。 ショウジョウバエで利用可能な遺伝子ツールに加えて、そのゲノムは非常に神経疾患19に関与する遺伝子のために保存されています。 （全体exomeシーケンシングを含む）ゲノムワイド遺伝子スクリーニング法の出現は、人間の運動障害?...

パーキンソン病、筋萎縮性側索硬化症、および遺伝性痙性対麻痺のような壊滅的な神経変性疾患は、ますます認識されています。残念ながら、これらの神経変性疾患のほとんどは、治療せずに残っ​​ています。このような全体exomeシーケンシングなどのゲノムワイドな、公平な遺伝子検査の広範な臨床使用は、ヒト機関車の障害に関与している遺伝子の数の増加につながっています。この進歩にもかかわらず、初期後期への病理学的進行は、これらの疾患でとらえどころのないままになります。 ショウジョウバエは、制御された空間及び時間的に遺伝子の要件を研究するための遺伝的ツールとのうちの1つを提供します。また、 ショウジョウバエは、パーキンソン1、アルツハイマー2、知的障害3,4およびとりわけてんかん5,6などの神経学的状態のための薬剤のスクリーニングに有用であることが証明されました。私たちの目的は、効果的なコストを開発することでしたまだモータ性能の小さな変化を検出するのに十分な感度であろう、高スループット分析を可能にする信頼性の高いアッセイ。 ショウジョウバエクライミング挙動に関する遺伝子変異及び/又は環境条件の影響を定量化するために使用されるいくつかのアッセイがあります。アッセイのほとんどは負の走地性、またはクライミングアッセイとして知られて登るハエの自然な傾向を生かします。 Benzer 7は走光性の研究のために使用されるカウンター現在の装置はまた、重力走性を研究するために使用することができることを1967年に提案しました。それ以来、Ganetsky 8および他の多くの9 -12は最初のアッセイ上に構築されてきました。原理は、バイアルにハエの既知の数を配置し、ハエがバイアルの底に落下させ、硬い表面に対して強くバイアルをタップすることです。それは生得的行動であるように、ハエは、重力に反対し、バイアルの上部に登るしようとします。このアッセイは、定量的およびmeasuです割り当てられた時間期間の間に、バイアル上のマーカーを越えて上昇しているどのように多くのハエのres。代わりにハエクライミングの総数の速度の測定は、信頼性の高いパラメータとなり、ハエの基準の数は13に有意ではなかった場合の欠陥を示しています。 クライミングアッセイは、パーキンソン病14を含む多くの神経変性疾患の研究に有用であることが証明されました。しかし、我々は、機関車の欠陥は、神経変性が既に病理学的研究14に見られる時に検出されない場合があることに留意しました。したがって、従来のアッセイの使用は、疾患の発症の初期段階を研究するための能力を制限することができます。病理学の後の段階の間に機関車の欠陥の外観は、その進行の完全な救済のためのあまりに高度である疾患を反映することができます。 これは、従来のクライミングアッセイの感度を持つ潜在的な問題を提起します。 tradiの潜在的なことができないこと軽度の機関車の欠陥を検出するため的なクライミングアッセイはハエを登るために必要とされた高さに帰することができます。正常に10〜20秒で2〜5センチメートルの高さを乗り越えるために、従来のアッセイ15,16対策ハエの数。

<table><tbody><tr><td>Drosophila stocks</td><td></td><td></td><td>The stocks are selected depending on the experiments. The temperature and humidity in the room and in the incubator must be controled and consistent to avoid flies being too staticky or too wet.</td></tr><tr><td>Video camera</td><td></td><td></td><td>Any digital camcorder will do. Make sure they can focus on close object.</td></tr><tr><td>Graduated cylinder</td><td>Kimble</td><td>20028W</td><td>Different models of graduated cylinder may have different diameter. It is therefore imporant to measure the height.</td></tr><tr><td>Computer</td><td></td><td></td><td>Any model will do. We used the computer to monitor the climbing of the flies and record the number of flies at each time point.</td></tr></tbody></table>

quantitative analysis of climbing defects in a drosophila model of neurodegenerative disorders

神経変性疾患に起因する機関車の欠陥は、無症候性変性の年後に、疾患の遅発性の症状であることができ、したがって、現在の治療の治療戦略は治癒的ではありません。全体exomeシーケンスを使用することにより、遺伝子の増加は、ヒトの移動において役割を果たすことが同定されています。これらの遺伝子の同定にもかかわらず、これらの遺伝子は、正常な機関車の機能に重要であるかは知られていません。そのため、治療目的の新規標的を同定するために、これらの遺伝子の役割を解明するためのモデル生物を利用し信頼性の高いアッセイは、これまで以上に必要とされています。我々は以前より穏やかな欠陥の検出を可能にし、時間をかけてこれらの欠陥を評価する能力を有する負の走地性アッセイの感作されたバージョンを設計しました。ガラス中で行われるアッセイは、ワックスバリアフィルムで封止されているシリンダー、メス。閾値距離を増加させることによって、17.5℃に上昇させます私たちは穏やかなモビリティ機能障害を検出するのに高い感度を観察している2分の実験期間をMと増加。アッセイは、費用対効果がされており、再現性の高い結果を得るために広範囲の訓練を必要としません。これは、運動欠陥のショウジョウバエの変異体では、候補薬剤をスクリーニングするための優れた手法になります。

登山は強く、再現可能な動作です。 （ - 30秒25）実際、1日齢の野生型ハエが急速に目標距離クライミング性能に達します。変異体ハエがターゲットに登ることができないことを完了するために、穏やかな（または遅延）からパフォーマンスの範囲を提示します。我々は2つ​​の異なる突然変異対立遺伝子とここにこれを説明します。最初のものはspastin遺伝子（温泉5.75）18の完全な?...

Watch this Scientific Journal Video about Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders at JoVE.com

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets.

神経変性疾患のショウジョウバエモデルにおけるクライミング欠陥の定量分析

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

quantitative-analysis-climbing-defects-drosophila-model

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

18.5K ビュー.  University of Alberta. We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets. 

ビデオ: Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination2. Here we use behavioral assays, including the negative geotaxis assay3 and the aversive phototaxic suppression assay (APS assay)4,5, to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.

でアッセイする運動、学習、記憶障害ショウジョウバエ</em神経変性の>モデル

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

神経科学

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic screening using chemical mutagens is a useful strategy for mapping mutant phenotypes to genes among Drosophila and other model organisms that share conserved cellular pathways with humans. If the mutated gene of interest is not lethal in early developmental stages of flies, a climbing assay can be conducted to screen for phenotypic indicators of decreased brain functioning, such as low climbing rates. Subsequently, secondary histological analysis of brain tissue can be performed in order to verify the neuroprotective function of the gene by scoring neurodegeneration phenotypes. Gene mapping strategies include meiotic and deficiency mapping that rely on these same assays can be followed by DNA sequencing to identify possible nucleotide changes in the gene of interest.

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

We present a protocol using a forward genetic approach to screen for mutants exhibiting neurodegeneration in Drosophila melanogaster. It incorporates a climbing assay, histology analysis, gene mapping and DNA sequencing to ultimately identify novel genes related to the process of neuroprotection.

ショウジョウバエメラノガスターの新しい神経保護遺伝子を同定する生体内前方遺伝スクリーン

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic ...

遺伝学

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. ...

行動学

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's 5.
Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human &alpha;-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function 2. Accordingly, this model has been successfully used to identify potential therapeutic targets in PD 8.
Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line 3, 10.

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

神経変性表現型を評価する<em&gt;ショウジョウバエ</em&gt;クライミングアッセイによりドーパミン作動性ニューロンと脳全体の免疫染色

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the ...

A Simple Technique to Assay Locomotor Activity in Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and diverse behavioral features. Identifying behavioral deficiency in animal models is a crucial measure of disease severity, for example, in neurodegenerative diseases where patients often experience impairments in motor function. However, with the availability of various systems to track and assess motor deficits in fly models, such as drug-treated or transgenic individuals, an economical and user-friendly system for precise evaluation from multiple angles is still lacking. A method based on the AnimalTracker application programming interface (API) is developed here, which is compatible with the Fiji image processing program, to systematically evaluate the movement activities of both adult and larval individuals from recorded video, thus allowing for the analysis of their tracking behavior. This method requires only a high-definition camera and a computer peripheral hardware integration to record and analyze behavior, making it an affordable and effective approach for screening fly models with transgenic or environmental behavioral deficiencies. Examples of behavioral tests using pharmacologically treated flies are given to show how the techniques can detect behavioral changes in both adult flies and larvae in a highly repeatable manner.

A Simple Technique to Assay Locomotor Activity in Drosophila

The present protocol assesses the locomotor activity of Drosophila by tracking and analyzing the movement of flies in a hand-made arena using open-source software Fiji, compatible with plugins to segment pixels of each frame based on high-definition video recording to calculate parameters of speed, distance, etc.

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 ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

なぜ定量事項：での表現型のキャラクタリゼーション<em&gt;ショウジョウバエ</em&gt;幼虫の神経筋接合部

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Mass Histology to Quantify Neurodegeneration in Drosophila

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health worldwide. Although mammalian models have provided important insights into the underlying mechanisms of pathogenicity, the complexity of mammalian systems together with their high costs are limiting their use. Therefore, the simple but well-established Drosophila model-system&#160;provides an alternative for investigating the molecular pathways that are affected in these diseases. Besides behavioral deficits, neurodegenerative diseases are characterized by histological phenotypes such as neuronal death and axonopathy. To quantify neuronal degeneration and to determine how it is affected by genetic and environmental factors, we use a histological approach that is based on measuring the vacuoles in adult fly brains. To minimize the effects of systematic error and to directly compare sections from control and experimental flies in one preparation, we use the &#39;collar&#39; method for paraffin sections. Neurodegeneration is then assessed by measuring the size and/or number of vacuoles that have developed in the fly brain. This can either be done by focusing on a specific region of interest or by analyzing the entire brain by obtaining serial sections that span the complete head. Therefore, this method allows one to measure not only severe degeneration but also relatively mild phenotypes that are only detectable in a few sections, as occurs during normal aging.

Mass Histology to Quantify Neurodegeneration in Drosophila

Drosophila is widely used as a model system to study neurodegeneration. This protocol describes a method by which degeneration, as determined by vacuole formation in the brain, can be quantified. It also minimizes effects due to the experimental procedure by processing and sectioning control and experimental flies as one sample.

に神経変性を定量化するために、マス組織学<em&gt;ショウジョウバエ</em

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health ...

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. Drosophila larval neuronal dendritic arborization (da) is an ideal model for studying morphogenesis of neural dendrites and gene function in the development of nervous system. There are four classes of da neurons. Class IV is the most complex with a branching pattern that covers almost the entire area of the larval body wall. We have previously characterized the effect of silencing the Drosophila ortholog of SOX5 on class IV neuronal dendritic arborization complexity (NDAC) using four parameters: the length of dendrites, the surface area of dendrite coverage, the total number of branches, and the branching structure. This protocol presents the workflow of NDAC quantitative analysis, consisting of larval dissection, confocal microscopy, and image analysis procedures using ImageJ software. Further insight into da neuronal development and its underlying mechanisms will improve the understanding of neuronal function and provide clues about the fundamental causes of neurological and neurodevelopmental disorders.

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

This protocol focuses on quantitative analysis of neuronal dendritic arborization complexity (NDAC) in Drosophila, which can be used for studies of dendritic morphogenesis.

ショウジョウバエのニューロン樹状分枝複雑さの定量解析

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

ImageJ を用いた蛍光タグ付きタンパク質の公平な分析によるショウジョウバエの神経変性の定量細胞生物学

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

Visualizing Synaptic Degeneration in Adult Drosophila in Association with Neurodegeneration

Drosophila serves as a useful model for assessing synaptic structure and function associated with neurodegenerative diseases. While much work has focused on neuromuscular junctions (NMJs) in Drosophila larvae, assessing synaptic integrity in adult Drosophila has received much less attention. Here we provide a straightforward method for dissection of the dorsal longitudinal muscles (DLMs), which are required for flight ability. In addition to flight as a behavioral readout, this dissection allows for the both DLM synapses and muscle tissue to be amenable to structural analysis using fluorescently labeled antibodies for synaptic markers or proteins of interest. This protocol allows for the evaluation of the structural integrity of synapses in adult Drosophila during aging to model the progressive, age-dependent nature of most neurodegenerative diseases.

Visualizing Synaptic Degeneration in Adult Drosophila in Association with Neurodegeneration

The goal of this procedure is to dissect the dorsal longitudinal muscle (DLM) tissue to assess the structural integrity of DLM neuromuscular junctions (NMJs) in neurodegenerative disease models using Drosophila melanogaster.

神経変性と関連した成人 ショウジョウバエ におけるシナプス変性の可視化

Drosophila serves as a useful model for assessing synaptic structure and function associated with neurodegenerative diseases. While much work has focused ...

神経変性疾患のショウジョウバエモデルにおけるクライミング欠陥の定量分析

要約

さらに動画を探す

この動画の章

でアッセイする運動、学習、記憶障害ショウジョウバエモデル

ショウジョウバエメラノガスターの新しい神経保護遺伝子を同定する生体内前方遺伝スクリーン

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

神経変性表現型を評価する

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

なぜ定量事項：での表現型のキャラクタリゼーション

に神経変性を定量化するために、マス組織学

ショウジョウバエのニューロン樹状分枝複雑さの定量解析

ImageJ を用いた蛍光タグ付きタンパク質の公平な分析によるショウジョウバエの神経変性の定量細胞生物学

神経変性と関連した成人ショウジョウバエにおけるシナプス変性の可視化

神経変性疾患のショウジョウバエモデルにおけるクライミング欠陥の定量分析

要約

さらに動画を探す

この動画の章

でアッセイする運動、学習、記憶障害ショウジョウバエモデル

ショウジョウバエメラノガスターの新しい神経保護遺伝子を同定する生体内前方遺伝スクリーン

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

神経変性表現型を評価する

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

なぜ定量事項：での表現型のキャラクタリゼーション

に神経変性を定量化するために、マス組織学

ショウジョウバエのニューロン樹状分枝複雑さの定量解析

ImageJ を用いた蛍光タグ付きタンパク質の公平な分析によるショウジョウバエの神経変性の定量細胞生物学

神経変性と関連した成人 ショウジョウバエ におけるシナプス変性の可視化

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

神経変性と関連した成人ショウジョウバエにおけるシナプス変性の可視化