Name: a novel method to model chronic traumatic encephalopathy in drosophila
Uploaded: 2017-07-04T13:00:00.000+00:00
Duration: 10 min 15 s
Description: Watch this Scientific Journal Video about A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila at JoVE.com

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ストライク装置の組み立て（ 図1 ） <ol><li>プランジャーを1mLツベルクリンシリンジから取り出します。 1 mLのマークでバレルをカットします。 </li><li> 200μLのピペットチップからエアロゾルバリヤ（高さ3mm×直径4mm）を取り外し、インパクタとして使用する。インパクターをシリンジバレルの内側に置きます。ゆっくりとバレルをタップしてインパクターを先端に移動させ、平らな面がノズルの開口部を覆うようにします。 </li><li> ショウジョウバエ麻酔ステーションの二酸化炭素（CO 2 ）フローレギュレーターに接続されているプラ​​スチックチューブにバレルの先端を取り付けます。 </li><li>バレルを垂直に持ち、インパクタがバレルの底にとどまるように標準のクランプホルダースタンドにクランプします。 </li><li> 200μlのピペットチップをフライホルダーに変更します。 <ol><li>先端から4 mmをカットして直径0.8 mmの開口部を作り、フライヘッドのみを露出させます。 注：thoレックスとフライボディーの他の部分はピペットチップの内側に留まります。 </li></ol></li><li> 1000μLピペットチップと1 mLシリンジ針キャップを変更してコネクターを作成します。 <ol><li>先端の開口部から44mmの位置で切断します。 1ミリリットルシリンジ針キャップの長さを6 mmにして、1,000μLピペットチップの残りの部分にしっかりと押し込みます。 </li></ol></li></ol> 2.ストライク装置の動作<ol><li>フライパッドにCO 2を使用して、 2日齢の成人女性フライを麻酔する。 </li><li>細かい筆を使って軽くフライホルダーに移してください。フライヘッドが先端の外側に見えるように、ホルダーを静かにタップします。フライ・プローブがチップの外側に露出している場合は、鈍い1mLシリンジ・ニードルでチップの内側に静かに戻します。 注：フライポイコスをホルダーの内側に入れてください。さもなければ、ハエは吸う胸部の傷害によって死ぬかもしれない。 </li><li>フライホーを締めなさいフライヘッドが下向きになるように、コネクターでシリンジバレルに接続してください。 </li><li>ガス圧を100 kPaに設定します。実験計画に従って流量を調整する。 </li><li>インパクタがフライヘッドを一度叩くように、フローレギュレータのトグルスイッチを素早くオン/オフします。 </li><li>フライホルダーを持ち上げ、フライパッドの上に移動します。フライホルダーを逆さまにして軽く飛ばして、フライを出す。空のバイアルにフライを入れて回復させる。 </li></ol> 3.ビデオ支援移動追跡<ol><li>トラッキングアリーナを作るために、直径6cmのペトリ皿に透明なシリコンエラストマーを充填する。シリコンとフライパンの間に3mmのスペースを空けて、ハエが自由に歩いて飛行することができます。 </li><li>毎回擬似群または治療群のいずれかから4匹のハエを麻酔し、それらをアリーナに入れる。フライを22℃で1時間放置する。 </li><li>アリーナの上に電荷結合素子（CCD）カメラを置き、5分間記録する。</li><li> Ctraxソフトウェア（Caltechから自由に入手可能）を使用して、記録された移動軌跡を分析します。トラッキングされたデータをプログラミング言語（Matlabなど）互換の形式でエクスポートし、フレームあたりの移動距離に基づいてデータを分析します。各フライの平均歩行距離を計算し、それを他のすべての記録されたフライ/グループと組み合わせて、同じグループ内のファイルの集団が移動した平均累積距離を得る。 </li></ol>

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この研究は、ジョンズ・ホプキンス大学医学部の教員スタートアップファンドであるLC

神経生理学的変化、神経病理学的特徴、および神経行動障害を含むCTEの特徴を忠実にモデル化する動物モデルは、疾患メカニズムの解明および診断および治療標的の開発に不可欠である。臨床的に関連するすべてのエンドポイントを模倣するのに、ヒト疾患の動物モデルは完全ではないことは理解できる。しかし、堅牢なCTEモデルは、次の3つの要件を満たす必要があると考えています。（1?...

慢性外傷性脳症（CTE）は、最近、アルツハイマー病などの他のタウオパシーとは別個の別個の神経変性障害として認識されている1 。アルツハイマー病や他の一般的なタウオパシーとは異なり、CTEはその名の通り、脳の外傷の病歴と密接に関連していると言えます。接触したスポーツアスリートでは、ボクサーやフットボールの選手、軍用退役軍人のようなもの2,3,4,5。それは頭に繰り返し震えと副交渉の打撃によって開始されると考えられています。患者は、アルツハイマー病、前頭側頭と重複する認知機能障害、気分および行動変化、および運動障害などの症状および徴候を示すことがある認知症、レヴィー小体認知症、およびパーキンソン病6 。対照的に、脳組織の死後検査は、皮質溝の深さにある小血管周囲の過剰リン酸化タウ蓄積の明確なパターンを示し、他の変性状態では見られない病理学的特徴である7 。しかしながら、これまでのところ、疾患発現につながる病因についてはほとんど知られていない。これは大部分が忠実な動物モデルが欠如していることに起因しており、最近ではげっ歯類モデルが作成されたばかりです5,8。これらのモデル生物は、費用重視のケアおよび比較的長い寿命の欠点を有し、神経変性疾患研究にはあまり適していない。 哺乳動物のそれと比較して、 ショウジョウバエなどの無脊椎動物は費用対効果の高いメンテナンスを備えた優れた代替品であり、遺伝的決定因子を解剖するための広範なツール、および比較的短い寿命9 。注目すべきことに、ハエとヒトの脳は、進化的に保存された分子および細胞経路、ならびに解剖学的類似点を共有している10,11,12。外傷性脳損傷を研究するための2つの巧妙なショウジョウバエモデルが以前に報告されている13,14 。 Katzenbergerらによって設計された最初の &quot;High Impact Trauma&quot;（HIT）装置は、金属ばね13,15の自由端に結合されたプラスチックバイアル内に自由に動く飛行機を含んでいた。プラスチック製のバイアルを直立させて離したときに、ポリウレタン製のパッドに当たって、バイアルの壁に跳ね返り、跳ね返りながら飛行機に外傷を与えました。対照的に、Barekatらは、異なる送達方法を設計したOmni Bead Ruptor-24ホモジナイザープラットフォーム14を用いて行った 。ハエはCO 2で不能化され、ホモジナイザーに固定された2mLスクリューキャップ管に入れられ、予めプログラムされた振盪条件に供された。組織ホモジナイザーシステムを使用することの1つの利点は、実験者が傷害の強度、傷害の持続時間、および傷害の回数を調節することができることである。しかし、両方のレジームにも同じ欠点があります。頭部への一次損傷は、インパクトの場所と強度に関してランダムに影響を受けます。さらに、両方の方法は、身体および内臓の他の部分への必然的な副次的損傷によって引き起こされる、かなりの死亡をもたらした。ここでは、ミバエのmTBIを誘導する新規な方法を説明する。私たちの装置は、ガス推進弾道衝撃子で構成されています。既存のショウジョウバエのモデル14,15と比較すると、本発明者らの方法は、meas自由に動くフライヘッドのみに向けることができるので、衝撃の重大性、衝撃の時間間隔、持続する衝撃の総数などの様々な要因を正確に制御することができます。

<table><tbody><tr><td>Aerosol Barrier</td><td>USA Scientific</td><td>1120-8810</td><td>Used as an impactor</td></tr><tr><td>200 &amp;#956;L Pipette Tip</td><td>USA Scientific</td><td>1111-0706</td><td>Used as a fly head holder</td></tr><tr><td>1000 &amp;#956;L Pipette Tip</td><td>USA Scientific</td><td>1122-1830</td><td>Used as a connector</td></tr><tr><td>1 mL Tuberculin Syringe</td><td>Becton Dickinson</td><td>309625</td><td></td></tr><tr><td>60 mm Petri Dishes</td><td>Fisher Scientific</td><td>FB0875713A</td><td>Used as a tracking arenas</td></tr><tr><td>Flow Regulator</td><td>Genesee Scientific</td><td>59-122WC</td><td></td></tr><tr><td>Standard Clamp Holder/stand</td><td>EISCO Scientific</td><td>CH0688</td><td></td></tr><tr><td>Fine Brush</td><td>Genesee Scientific</td><td>59-204</td><td></td></tr><tr><td>Flypad</td><td>Genesee Scientific</td><td>59-114</td><td></td></tr><tr><td>Sylgard Silicone Elastomer</td><td>Dow Corning</td><td>4019862</td><td></td></tr><tr><td>CCD Camera</td><td>Microsoft&amp;nbsp;</td><td>HD-5000</td><td></td></tr><tr><td>Ctrax Walking Fly Tracker</td><td>Caltech</td><td>Ctrax 0.2.11</td><td></td></tr><tr><td>MATLAB Image Processing Toolbox</td><td>MATLAB</td><td>R2015b</td><td></td></tr></tbody></table>

a novel method to model chronic traumatic encephalopathy in drosophila

慢性外傷性脳症（CTE）は、繰り返し軽度の外傷性脳傷害（mTBI）への暴露と密接に関連する確立された神経変性疾患である。神経病理学的基準を定義するための最近のコンセンサスにもかかわらず、その複雑な病理学的変化の原因となっている機構は、依然として分かりにくいままである。ここでは、 ショウジョウバエメラノガスター （ ショウジョウバエ ）におけるCTEのモデルを開発して、特徴的な過剰リン酸化タウの蓄積および脳における神経細胞の死につながる重要な遺伝子および経路を同定するための新しい方法を説明する。軽度の閉鎖傷害を与えるための調整可能な衝撃は、ヘッドを急速な加減速にさらし、フライヘッドに直接供給されます。我々の方法は、他のショウジョウバエの mTBIモデルに内在する潜在的な問題を排除する（ 例えば、動物死は、身体の他の部分または内部器官）。労力とコストを重視する動物の手入れが少なく、寿命が短く、遺伝的ツールが豊富で、CTEの病因を研究し、ゲノム規模の大規模な遺伝的および薬理学的スクリーニングを行うことができます。我々は、モデルの進行中の特徴付けが、疾患の予防および治療アプローチに関する重要な機構的な洞察を生成すると予想する。

成体ショウジョウバエを使用してCTEモデルを確立するために、私たちは単一の閉鎖頭傷害を与える際の装置の有効性を判断した。遺伝子型、性別、年齢に関するバリエーションを排除するために、我々は実験で2日齢のCanton-S WT雌ハエを使用した。 100kPaの一定のガス圧でCO 2の流量を調節することによって、インパクターの強度を容易に制御することがで...

Watch this Scientific Journal Video about A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila at JoVE.com

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

ここでは、 ショウジョウバエのメラノガスター（melanogaster）における閉鎖頭部外傷性脳傷害を引き起こす新しいアプローチについて説明する。私たちの方法は、調整可能な強さで反復的な衝撃を直接ヘッドに伝えるという利点があります。無脊椎系のさらなる探索は、慢性外傷性脳症の病因を明らかにするのに役立つだろう。

慢性外傷性脳症モデルの新しい方法<em&gt;ショウジョウバエ</em

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic ...

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Neuroscience

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

8.2K ビュー.  Johns Hopkins University School of Medicine. ここでは、 ショウジョウバエのメラノガスター（melanogaster）における閉鎖頭部外傷性脳傷害を引き起こす新しいアプローチについて説明する。私たちの方法は、調整可能な強さで反復的な衝撃を直接ヘッドに伝えるという利点があります。無脊椎系のさらなる探索は、慢性外傷性脳症の病因を明らかにするのに役立つだろう。 

ビデオ: A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

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

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

郭清ときのこ体の免疫蛍光染色と大人のショウジョウバエ脳光受容器ニューロン

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

発生生物学

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 ...

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

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and thus current therapeutic treatment strategies are not curative. Through the use of whole exome sequencing, an increasing number of genes have been identified to play a role in human locomotion. Despite identifying these genes, it is not known how these genes are crucial to normal locomotive functioning. Therefore, a reliable assay, which utilizes model organisms to elucidate the role of these genes in order to identify novel targets of therapeutic interest, is needed more than ever. We have designed a sensitized version of the negative geotaxis assay that allows for the detection of milder defects earlier and has the ability to evaluate these defects over time. The assay is performed in a glass graduated cylinder, which is sealed with a wax barrier film. By increasing the threshold distance to be climbed to 17.5 cm and increasing the experiment duration to 2 min&#160;we have observed a greater sensitivity in detecting mild mobility dysfunctions. The assay is cost effective and does not require extensive training to obtain highly reproducible results. This makes it an excellent technique for screening candidate drugs in Drosophila mutants with locomotion defects.

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 ...

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies using Drosophila have contributed important breakthroughs in understanding neurological processes. Thus, with the goal of understanding the cellular and molecular basis of TBI pathologies in humans, we developed the High Impact Trauma (HIT) device to inflict closed head TBI in flies. Flies subjected to the HIT device display phenotypes consistent with human TBI such as temporary incapacitation and progressive neurodegeneration. The HIT device uses a spring-based mechanism to propel flies against the wall of a vial, causing mechanical damage to the fly brain. The device is inexpensive and easy to construct, its operation is simple and rapid, and it produces reproducible results. Consequently, the HIT device can be combined with existing experimental tools and techniques for flies to address fundamental questions about TBI that can lead to the development of diagnostics and treatments for TBI. In particular, the HIT device can be used to perform large-scale genetic screens to understand the genetic basis of TBI pathologies.

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Here we describe a method to inflict closed head traumatic brain injury (TBI) in Drosophila. This method provides a gateway to investigate the cellular and molecular mechanisms that underlie TBI pathologies using the vast array of experimental tools and techniques available for flies.

で閉鎖性頭部外傷性脳損傷を負わせる方法<em&gt;ショウジョウバエ</em

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

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 ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

転写産物および代謝物からの精製<em&gt;ショウジョウバエ</em&gt;ヘッド

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

生物学

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our understanding of the molecular mechanisms underlying axon degeneration remains limited. Injury-induced axon degeneration serves as a simple model to study how severed axons execute their own disassembly (axon death). Over recent years, an evolutionarily conserved axon death signaling cascade has been identified from flies to mammals, which is required for the separated axon to degenerate after injury. Conversely, attenuated axon death signaling results in morphological and functional preservation of severed axons and their synapses. Here, we present three simple and recently developed protocols that allow for the observation of axonal morphology, or axonal and synaptic function of severed axons that have been cut-off from the neuronal cell body, in the fruit fly Drosophila. Morphology can be observed in the wing, where a partial injury results in axon death side-by-side of uninjured control axons within the same nerve bundle. Alternatively, axonal morphology can also be observed in the brain, where the whole nerve bundle undergoes axon death triggered by antennal ablation. Functional preservation of severed axons and their synapses can be assessed by a simple optogenetic approach coupled with a post-synaptic grooming behavior. We present examples using a highwire loss-of-function mutation and by over-expressing dnmnat, both capable of delaying axon death for weeks to months. Importantly, these protocols can be used beyond injury; they facilitate the characterization of neuronal maintenance factors, axonal transport, and axonal mitochondria.

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Here, we provide protocols to perform three simple injury-induced axon degeneration (axon death) assays in Drosophila melanogaster to evaluate the morphological and functional preservation of severed axons and their synapses.

ショウジョウバエメラノガスターにおける軸索死における軸索とそのシナプスの形態学的および機能的評価

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our ...

Tracking Freely Walking Flies: A Method to Assess Locomotion in Drosophila

Source: Sun, M. and Chen, L. L. <a href="https://www.jove.com/video/55602/" target="_blank">A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila</a>. J. Vis. Exp. (2017).
This video describes how to track freely walking flies to assess their general locomotion ability. The featured protocol demonstrates the assay&#39;s setup and data analysis using video tracking software.

自由に歩くハエの追跡:ショウジョウバエの移動を評価する方法

Source: Sun, M. and Chen, L. L. A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila. J. Vis. Exp. (2017).
This video describes how to ...

JoVE Encyclopedia of Experiments

Drosophila melanogaster（ショウジョウバエ）

成虫

A Technique to Inflict Closed-Head Traumatic Brain Injury in Drosophila Flies

Source: Sun, M., et al. <a href="https://app.jove.com/v/55602">A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila.</a> J. Vis. Exp. (2017).
This video demonstrates a procedure to inflict a closed-head traumatic injury in anesthetized Drosophila flies using a strike device with a gas-propelled impactor. The injury affects neuronal function and impairs walking ability.

ショウジョウバエに閉鎖性外傷性脳損傷を負わせる技術

1. Assembly of the Strike Device (Figure 1)

	Remove the plunger from a 1 mL tuberculin syringe. Cut the barrel at the 1 mL mark.
	Remove an aerosol ...

慢性外傷性脳症モデルの新しい方法

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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

郭清ときのこ体の免疫蛍光染色と大人のショウジョウバエ脳光受容器ニューロン

神経変性表現型を評価する

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

で閉鎖性頭部外傷性脳損傷を負わせる方法

に神経変性を定量化するために、マス組織学

転写産物および代謝物からの精製

ショウジョウバエメラノガスターにおける軸索死における軸索とそのシナプスの形態学的および機能的評価

自由に歩くハエの追跡:ショウジョウバエの移動を評価する方法

ショウジョウバエに閉鎖性外傷性脳損傷を負わせる技術