The authors have nothing to disclose.

すべての手順および行動試験は、実験動物の管理と使用のための動物ケアのガイドラインについては、カナダ人評議会に準拠しているプロトコル番号06-11-12、下マキュワン大学の動物実験倫理委員会によって承認された。 1.投与タンク、ソリューション、および投与スケジュールを準備<ol><li>動物は視覚刺激に時間やバイアスのいずれ交絡を避けるために、同じ環境で、一日の同じ時間の間に投与されるような投与スケジュールを準備します。 </li><li>グループサイズの数に対して必要な限り多くの同一、1.5 L、明確なポリプロピレン産卵タンクを入手します。魚の2つのグループを可能にし、タンクあたり8魚の使用グループは手順で後述日あたりテストされる（ ステップ3を参照）。 1保持タンクやグループごとに1つの投与タンク（グループの2倍合計数）を使用します。 <ol><li>すべての保持タンクに400μmの産卵インサートを置きます。 F病気生息水でタンクまたは温度魚と一致している（ゼブラフィッシュのため、25〜28℃）、正しい温度で逆浸透水は、通常に収容されている。 注：一部の薬剤および緩衝生息水の化学成分間の望ましくない化学的相互作用が存在し得る。この状況では、逆浸透薬物投与のための最小または全く水槽塩で緩衝水、ならびに対照群のために使用する。 </li><li>タンクは、投薬時に外部視覚刺激にコンディショニングの魚を避けるために、中立的な環境であることを確認します。 </li></ol></li><li>薬液を準備します。産卵タンクで生息地の水と適切な量の薬剤を混ぜる。水1497ミリリットルでハイグレードのエタノール（95％非変性エタノール）の3ミリリットルを組み合わせることにより、0.2％エタノール溶液を調製します。水1479ミリリットルとエタノール21ミリリットルを組み合わせることにより、1.4％エタノール溶液を調製します。 </li></ol> 2.ネッティング魚、そしてEthanol管理手順<ol><li>慎重にネット生息タンクから魚や産卵挿入物を含む適切な保持タンクに移す。理想的には、産卵中の家の魚は完全にネッティングを排除するために挿入します。 </li><li>それぞれの保持タンク内のすべての魚と、優しく保持タンクの外に挿入し、適切な薬液タンク（ 図1A）にそれを置いて産卵を持ち上げる。 <ol><li>必要に応じて、投与時間を記録します。ここで説明する手順のためのエタノール溶液に30分を使用してください。 </li><li>可能な場合、アシスタントは、正確な投薬時間を確保すると同時に薬液を全ての実験群の転送を支援している。代替的に、一度に1つのグループを転送し、個々のグループの投薬回数（ 図1A）を追跡する。 </li></ol></li><li>必要な投与期間の終わりに、慎重に産卵インサートを持ち上げることにより、エタノール溶液から魚を外す薬液のうち、背面保持タンクに優しく置く。 </li><li>優しくネット保持タンク内の魚や次回のスケジュール、投与時まで戻って自分の生息地にそれらを配置するか、産卵を配置ネッティングを排除するために戻って保持タンクに挿入します。 </li><li>動物の住宅設備機器、住宅貯蔵タンクとして使用されているものと同じタンクと産卵インサートにおける動物のパラメータの範囲内で以前に、可能な場合に述べたように。これは投与手順の間に完全にネッティングがなくなります。 </li></ol> 3.行動試験<ol><li>長い55センチメートルと白の防水床（ 図1B）との深い9.5センチメートル9.5センチ幅の明/暗アリーナを取得します。黒で覆われた白と半分に覆われたアリーナの半分で、マジックテープを使用して、アリーナの内壁に白と黒の防水無反射紙を貼る。 25-28℃の温度で、生息地の水で5cmの深さにアリーナを埋める。通販テスト全体でこの温度をAINの。 </li><li>アリーナ内に位置されるためには、白三面囲いを構築することにより、外部視覚刺激をMinimalize。テストエリアが水面上に反射を起こさない頭上の照明を拡散持って、まだ十分に運動追跡ソフトウェア、またはポストのために明るいあることを確認してくださいビデオ画像から-hoc手動定量。 </li><li>エンクロージャにアリーナを置き、行動追跡ソフトウェアの記録と運動解析パラメータを設定します。研究課題に応じて、5〜15分までの試用期間を設定します。 注：ここでは、5分を使用していました。 </li><li>魚の群が生息タンク内の研究領域にテスト​​される輸送し、アリーナエンクロージャの外に配置します。 10分間の魚を順応さ。 </li><li>明/暗アリーナの中央に該当するグループと場所から静かにネット魚は、AVOするアリーナの長軸に平行に配置されたとき、魚を解放することを確認され光や暗いゾーンに魚をバイアスするID。 </li><li>動物が解放された直後に記録動作を開始します。魚を追跡または魚のジャンプや凍結のための任意のソフトウェアの問題を監視します。被験者の半数に起因アリーナの端が筐体の開口端に向かって配向されているから生じる偏見にどんな交絡を防ぐためにテストされた後に、アリーナを180°回転させます。 </li><li>裁判の後、終了優しくネットと保持タンクや生息地のタンクにアリーナから魚を削除しています。 </li></ol> 4.分析<ol><li>ダークゾーンに対する光の中で費やされた時間を調べます。グループごとに、それぞれの魚、光と闇のゾーンで過ごした相対的な時間を得て、1 標本t -test使用して分析する（またはウィルコクソンをノンパラメトリックデータのための順位検定を締結し、総試行時間の（半分）150秒との違い）グループは大幅に他の上の1つの領域を好むかどうかを判断する。 </li><li>環境設定を比較するには、prefeを計算暗いゾーンで過ごした時間からの光帯に過ごし、群間の差を比較する時間を減算することによりrenceインデックス。 のt-試験は、2つのグループを比較するために使用され得る。 TukeyのHSDポストホックテスト、必要な場合（ノンパラメトリックデータ用またはクラスカル·ワリス検定Dunnの多重比較ポストホック付きテスト）を利用した一元配置分散分析で複数のグループを比較してください。 </li><li>速度、ゾーン遷移の数、蛇行、およびグループ間で不動の比較。 TukeyのHSDポストホック検定を用いた一元配置分散分析を使用して、必要に応じて（Dunnの多重比較、ノンパラメトリックデータのための事後検定又はクラスカル·ワリス検定）。 </li></ol>

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著者らは、図1に使用される彼の写真撮影装置の使用のためにジョシュアギャラップを認める。この作品は、（TJHに）自然科学と工学研究評議会（NSERC）カナダディスカバリーグラントによってサポートされていました。

ゼブラフィッシュにおける薬剤投与を伴う以前の研究では、単純に薬物溶液12,16に自宅タンクからそれらを輸送するために魚をネッティングに頼ってきた。ネッティングは常に一貫していないとしばしばかなりの個体差があるゼブラフィッシュの逃避応答に予想よりも時間がかかります。伝統的な転送方法は、有用ではあるが、魚が水の外に費やす合計時間を減少させるだけでなく、...

ゼブラフィッシュ（ ゼブラフィッシュ）は 、行動1および医学研究2,3ための有用なモデル生物であるインド発の小さな硬骨魚種である。ゼブラフィッシュは、一般に行動への影響を特徴付けるために、様々な薬理学的物質の試験において使用されている。薬物投与の様々な投与量およびスケジュールは、覚せい剤4、抗不安5およびエタノール6-8のような化合物の投与後のゼブラフィッシュの挙動を調査するために使用されてきた。 私たちの研究室はまた、一般scototaxicアッセイと呼ばれる、よく検証ダーク/ライトアッセイ9、20にゼブラフィッシュ不安や歩行にエタノール投与の異なるスケジュールの影響を調査しました。エタノール投与の新しい方法は、6時間の長い期間（21日間）にわたって反復、毎日の投与のための効率を高めるために開発された</s&gt;まで。以前に使用された方法は実用的であったが、我々は、その関連する時間コストは、ネッティングを低減する方法を開発しようと、魚の大量の目的とする薬物の同時、正確に時限投与を可能にした。エタノールを用いた従来の研究では、ゼブラフィッシュは、1つのタンクからエタノールと水10~12の適切な混合物を含む別に相殺され、転送される。この方法は広く受け入れられているが、ゼブラフィッシュを相殺すること導入し、薬物溶液から魚を除去するために要する時間のばらつきを増大させることができる。そのため、目的の化合物への正確な露出は反復投与を伴う実験の過程にわたって変化し得る。輸送時間の変動に起因する誤差の発生源を減少させる方法がが望ましい。私たちの方法では、我々はそれぞれの魚で同一の投与時間で、その結果、同時にすべての魚を移動することができます。 （ここで説明）エタノール曝露後、ゼブラフィッシュは、任意のnumbeにおいて試験することができる不安を評価するものも含めて行動アッセイ、のrを。新しいメソッドを使用して、魚のグループを投与すると、正確に被験者間や魚のグループ間での投与を複製し、標準化する能力を超え、実用用途があります。一度に複数の魚の追跡を可能にする新しいソフトウェアの出現は、研究者が実験に複製能と精度を確保するために、私たちの方法を利用することがあります。行動神経科学のためのモデル生物としてゼブラフィッシュの普及を考えると、この方法では、将来の薬理学的研究における効率性と実用性が向上します。 現在のパラダイムでは、反復投与スケジュールは、およそ人間の飲用スケジュールを反映していることを採用した。コントロール、毎日の中等度、または毎週-どんちゃん騒ぎ：魚を無作為に3つの群に割り当てた。投与スケジュールは、それが大幅に過去の研究7に露光時間を超えたため選ばれ、期間は21日であった。対照魚はゼロALCOを受けHOL、毎日の中等度の魚は1日1回、0.2％アルコールを受け、毎週-どんちゃん騒ぎの魚は、1週間に1回1.4％アルコールを受け取った。明/暗タスクが離脱の2日後に不安を評価した。これは、一方の側の壁が白色であり、他の側に9暗いする矩形アリーナを使用している管理するための比較的簡単な試験である。アダルトゼブラフィッシュは、ロバスト制御条件の下で6,9,13アリーナのダークサイドを好む。不安の増大は、運用上、暗いゾーンで過ごしたかなり多くの時間として定義され、魚が光ゾーンで過ごした、比較的多くの時間を費やしている時に不安を想定することができます減少する。モーショントラッキングソフトウェアを使用すると、他の有益な変数も平均速度、不動、蛇行を含め、定量することができる、そしてゾーンは14に移行します。 我々の研究室で開発された投与方法は、水溶性化合物は、1つ以上のzebrafiに投与される任意の研究に適用することができるSH。この方法論から利益を得ることができる他の多くの薬理学的薬剤は、現在、ゼブラフィッシュで検証されています。一般的に試験された化合物は、エタノールと同様の方法で溶解させ、ニコチン、クロルジアゼポキシド、ブスピロン、およびスコポラミンが挙げられる。水に化学物質の適切な量を混合することによって。したがって、この手順の一般的な範囲ははるかに広いとエタノールに限定されるものではない。さらに、複数の日のための薬物を投与した後、明/暗タスクを使用することができる多くの行動試験の1つにすぎません。薬物投与後、または引き抜きの間、利用できる他の一般的なアッセイは新しいタンクダイビングテスト15など16の浅水として社会的行動のテストが含まれています。次の手順を繰り返し、目的の薬理学的な化合物を含む溶液に魚や個々の魚のグループを転送する効率的な方法を概説します。さらに、明/暗テストとテスト不安のプロセスアルコール投与の長いスケジュールにさらさ​​れた後に撤退している魚のグループに説明する。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Three Shelf benchtop housing system</td>
			<td>Aquatic Habitats</td>
			<td>N/A</td>
			<td><a href="http://aquatichabitats.com/research-systems/z-hab-system/">http://aquatichabitats.com/research-systems/z-hab-system/</a></td>
		</tr>
		<tr>
			<td>1.5L Spawning tank w/400 micron baffle</td>
			<td>Aquatic Habitats</td>
			<td>N/A</td>
			<td><a href="http://aquatichabitats.com/consumables/tanks-lids-and-baffles/info/nursery-tank-and-baffles/">http://aquatichabitats.com/consumables/tanks-lids-and-baffles/info/nursery-tank-and-baffles/</a></td>
		</tr>
		<tr>
			<td>Pure Grain Ethanol</td>
			<td>Luxco, INC</td>
			<td>N/A</td>
			<td><a href="http://www.fishersci.com/ecomm/servlet/fsproductdetail?storeId=10652&amp;aid=3759149&amp;store=Scientific">http://www.luxco.com/public/brands/brands.asp?brandid=21</a></td>
		</tr>
		<tr>
			<td>Ethovision XT Motion tracking software</td>
			<td>Noldus Information Technology</td>
			<td></td>
			<td><a href="http://www.noldus.com/animal-behavior-research/products/ethovision-xt">http://www.noldus.com/animal-behavior-research/products/ethovision-xt</a></td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Eppendorf Canada</td>
			<td></td>
			<td><a href="http://www.eppendorf.ca/int/index.php?l=211&amp;pb=e396f9e705e754a3&amp;action=products&amp;contentid=1&amp;catalognode=9477.">http://www.eppendorf.ca/int/index.php?l=211&#38;pb=e396f9e705e754a3&#38;action=products&#38;contentid=1&#38;catalognode=9477.&#160;</a></td>
		</tr>
		<tr>
			<td>Light/Dark Arena</td>
			<td>Custom</td>
			<td></td>
			<td>Construct as per procedure description. 9.5cm wide, 9.5cm deep, 55cm long</td>
		</tr>
	</tbody>
</table>

a novel method of drug administration to multiple zebrafish (danio rerio) and the quantification of withdrawal

Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely netted and transported between home aquaria and dosing tanks. In order to enhance the ease of compound administration, a novel method for transferring fish between tanks for drug administration was developed. Inserts that are designed for spawning were used to transfer groups of fish into the drug solution, allowing accurate dosing of all fish in the group. This increases the precision and efficiency of dosing, which becomes very important in long schedules of repeated drug administration. We implemented this procedure for use in a study examining the behavior of zebrafish in the light/dark test after administering ethanol with differing 21 day schedules. In fish exposed to daily-moderate amounts of alcohol there was a significant difference in location preference after 2 days of withdrawal when compared to the control group. However, a significant difference in location preference in a group exposed to weekly-binge administration was not observed. 
This protocol can be generalized for use with all types of compounds that are water-soluble and may be used in any situation when the behavior of fish during or after long schedules of drug administration is being examined. The light/dark test is also a valuable method of assessing withdrawal-induced changes in anxiety.

上記のようにゼブラフィッシュと薬理学的研究の精度および制御を維持するためには、一貫して正確にエタノール投与の持続時間が重要である。我々の手順では、投与手順の容易性とスループットを向上させることができる。毎週-過食や毎日の中等度のスケジュールのいずれかに対するエタノールの投与は、対照と比較して、明/暗テストで測定し、変化した不安レベルをもたらした。二日?...

Watch this Scientific Journal Video about A Novel Method of Drug Administration to Multiple Zebrafish Danio rerio and the Quantification of Withdrawal at JoVE.com

A Novel Method of Drug Administration to Multiple Zebrafish Danio rerio and the Quantification of Withdrawal

A novel method for reducing variability when exposing fish to drugs is explained. Fish exposed to various patterns of ethanol exposure were found to have altered anxiety levels during withdrawal in a light/dark scototaxic assay.

複数のゼブラフィッシュへの医薬品の新規方法（<em&gt;ゼブラフィッシュ</em&gt;）と撤退の定量

Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely ...

a-novel-method-drug-administration-to-multiple-zebrafish-danio-rerio

Research

JoVE Journal

Neuroscience

A Novel Method of Drug Administration to Multiple Zebrafish (Danio rerio) and the Quantification of Withdrawal

12.0K ビュー.  MacEwan University. A novel method for reducing variability when exposing fish to drugs is explained. Fish exposed to various patterns of ethanol exposure were found to have altered anxiety levels during withdrawal in a light/dark scototaxic assay.

ビデオ: A Novel Method of Drug Administration to Multiple Zebrafish Danio rerio and the Quantification of Withdrawal

Novel Apparatus and Method for Drug Reinforcement

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction. Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in animal research to model various components of drug reinforcement, consumption, and addiction in humans. For this study, we used a novel approach to studying drug reinforcement in rats by combining traditional CPP and self-administration methodologies. We assembled an apparatus using two Med Associate operant chambers, sensory stimuli, and a Plexiglas-constructed neutral zone. These modifications allowed our experiments to encompass motivational aspects of drug intake through self-administration and drug-free assessment of drug/cue conditioning strength with the CPP test. In our experiments, rats self-administered cocaine (0.75 mg/kg/inj, i.v.) during either four (e.g., the "short-term") or eight (e.g., the "long-term") alternating-day sessions in an operant environment containing distinctive sensory cues (e.g., olfactory and visual). On the alternate days, in the other (differently-cued) operant environment, saline was available for self-infusion (0.1 ml, i.v.). Twenty-four hours after the last self-administration/cue-pairing session, a CPP test was conducted. Consistent with typical CPP findings, there was a significant preference for the chamber associated with cocaine self-administration. In addition, in animals undergoing the long-term experiment, a significant positive correlation between CPP magnitude and the number of cocaine-reinforced lever responses. In conclusion, this apparatus and approach is time and cost effective, can be used to examine a wide array of topics pertaining to drug abuse, and provides more flexibility in experimental design than CPP or self-administration methods alone.

Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in research to model various components of drug reinforcement, consumption, and addiction in humans. In this report, we combined traditional CPP and self-administration methods as a novel approach to studying drug reinforcement and addiction in rats.

薬物の強化のための新規装置および方法

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction.  Operant drug ...

神経科学

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

ラットの薬物自己投与時の超音波発声の評価

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

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

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

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

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

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

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

ゼブラフィッシュ成体終脳の刺し傷傷害：脊椎動物の脳神経発生·再生を調査する方法

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.

This protocol describes how to accurately measure neuronal viability using Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research.

小脳顆粒ニューロン培養におけるFluorescein Diacetate-Propidium Iodide二重染色を用いたニューロン生存率の評価

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic Brain Injury (mTBI). The mechanisms responsible for its complex pathological changes remain largely elusive, despite a recent consensus to define the neuropathological criteria. Here, we describe a novel method to develop a model of CTE in Drosophila melanogaster&#160;(Drosophila&#160;)&#160;in an attempt to identify the key genes and pathways that lead to the characteristic hyperphosphorylated tau accumulation and neuronal death in the brain. Adjustable-strength impacts to inflict mild closed injury are delivered directly to the fly head, subjecting the head to rapid acceleration and deceleration. Our method eliminates the potential problems inherent with other Drosophila mTBI models (e.g.,animal death might be induced by damage to other parts of the body or to internal organs). The less labor- and cost-intensive animal care, short life span, and extensive genetic tools make the fruit fly ideal to study CTE pathogenesis and make it possible to perform large-scale, genome-wide forward genetic and pharmacological screens. We anticipate that the ongoing characterization of the model will generate important mechanistic insights on disease prevention and therapeutic approaches.

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Here, we describe a new approach to inflict closed-head traumatic brain injury in Drosophila melanogaster. Our method has the advantage of directly delivering repetitive impacts with adjustable strength to the head alone. Further exploration of the invertebrate system will help to illuminate the pathogenesis of chronic traumatic encephalopathy.

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 ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Sod1 突然変異体蛋白質集計における細胞の高いコンテンツ定量化のための分析法の開発

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

A Rat Model of Central Fatigue Using a Modified Multiple Platform Method

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed as a water tank with narrow platforms on the bottom. The model rats were put into the tank and stood on the platforms for 14 h (18:00 - 8:00) per day for a consecutive 21 days, with a blank control group set for contrast. At the end of modeling, rats in the model group showed an obvious fatigued appearance. To assess the model, we performed several behavioral tests, including the open field test (OFT), the elevated plus maze (EPM) test, and the exhaustive swimming (ES) test. The results showed that anxiety, spatial cognition impairment, poor muscle performance, and declined voluntary activity presented in model rats confirm the diagnosis of&#160;central fatigue. Changes of the central neurotransmitters also verified the result. In conclusion, the model successfully simulated central fatigue, and future study with the model may help reveal the pathological mechanism of the disease.

Here, we present a protocol to introduce a rat model of central fatigue using the modified multiple platform method (MMPM).

ラット モデル改良を使用して中枢性疲労の複数プラットフォーム メソッド

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed ...

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor determining patient outcome and is therefore frequently taken as a study endpoint. However, in small animal studies on SAH, quantification of cerebral vasospasm is a major challenge. Here, an ex vivo method is presented that allows quantification of volumes of entire vessel segments, which can be used as an objective measure to quantify cerebral vasospasm. In a first step, endovascular casting of the cerebral vasculature is performed using a radiopaque casting agent. Then, cross-sectional imaging data are acquired by micro computed tomography. The final step involves 3-dimensional reconstruction of the virtual vascular tree, followed by an algorithm to calculate center lines and volumes of the selected vessel segments. The method resulted in a highly accurate virtual reconstruction of the cerebrovascular tree shown by a diameter-based comparison of anatomical samples with their virtual reconstructions. Compared with vessel diameters alone, the vessel volumes highlight the differences between vasospastic and non-vasospastic vessels shown in a series of SAH and sham-operated mice.

The goal of this article is to present a method that allows a 3-dimensional reconstruction of the cerebrovascular tree in mice after micro computed tomography and determination of volumes of entire vessel segments that can be used to quantify cerebral vasospasm in murine models of subarachnoid hemorrhage.

くも膜下出血のマウスモデルにおける脳血管攣縮の定量化法の体積

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor ...

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity &#947;-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

シンプルかつ強力な可視化と薬理学的ターゲットの評価法として放射線写真法

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...