この研究は、NIH / NIAAA（AA11852およびAA007471）からの補助金によって支えられている。

<ol>
	<li>Geiger, B. M., Frank, L. E., Caldera-Siu, A. D., Pothos, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survivable+stereotaxic+surgery+in+rodents.">Survivable stereotaxic surgery in rodents.</a> J. Vis. Exp. (20), e880 (2008).</li><li>Howard, E. C., Schier, C. J., Wetzel, J. S., Gonzales, 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=The+dopamine+response+in+the+nucleus+accumbens+core-shell+border+differs+from+that+in+the+core+and+shell+during+operant+ethanol+self-administration.">The dopamine response in the nucleus accumbens core-shell border differs from that in the core and shell during operant ethanol self-administration.</a> Alcohol Clin. Exp. Res. 33, 1355-1365 (2009).</li><li>Doyon, W. M., York, J. L., Diaz, L. M., Samson, H. H., Czachowski, C. L., Gonzales, 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=Dopamine+activity+in+the+nucleus+accumbens+during+consummatory+phases+of+oral+ethanol+self-administration.">Dopamine activity in the nucleus accumbens during consummatory phases of oral ethanol self-administration.</a> Alcohol Clin. Exp. Res. 27, 1573-1582 (2003).</li><li>Pettit, H. O., Justice, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+dose+on+cocaine+self-administration+behavior+and+dopamine+levels+in+the+nucleus+accumbens.">Effect of dose on cocaine self-administration behavior and dopamine levels in the nucleus accumbens.</a> Brain Res. 539, 94-102 (1991).</li><li>Paxinos, G., Kus, L., Ashwell, K. W. S., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chemoarchitectonic Atlas of The Rat Forebrain. , (1998).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Rat Brain in Stereotaxic Coordinates. , (1986).</li><li>Skoog, D. A., Holler, F. J., Nieman, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of Instrumental Analysis. , (1998).</li><li>Doyon, W. M., Anders, S. K., Ramachandra, V. S., Czachowski, C. L., Gonzales, 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=Effect+of+operant+self-administration+of+10%+ethanol+plus+10%+sucrose+on+dopamine+and+ethanol+concentrations+in+the+nucleus+accumbens.">Effect of operant self-administration of 10% ethanol plus 10% sucrose on dopamine and ethanol concentrations in the nucleus accumbens.</a> J Neurochem. 93, 1469-1481 (2005).</li><li>Carrillo, J., Gonzales, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+exposure+to+voluntary+ethanol+self-administration+produces+adaptations+in+ethanol+consumption+and+accumbal+dopamine+signaling.">A single exposure to voluntary ethanol self-administration produces adaptations in ethanol consumption and accumbal dopamine signaling.</a> Alcohol. 45, 559-5566 (2011).</li><li>Howard, E. C., Schier, C. J., Wetzel, J. S., Duvauchelle, C. L., Gonzales, 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=The+shell+of+the+nucleus+accumbens+has+a+higher+dopamine+response+compared+with+the+core+after+non-contingent+intravenous+ethanol+administration.">The shell of the nucleus accumbens has a higher dopamine response compared with the core after non-contingent intravenous ethanol administration.</a> Neuroscience. 154, 1042-1053 (2008).</li><li>Robinson, D. L., Lara, J. A., Brunner, L. J., Gonzales, 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=Quantification+of+Ethanol+Concentrations+in+the+Extracellular+Fluid+of+the+Rat+Brain.">Quantification of Ethanol Concentrations in the Extracellular Fluid of the Rat Brain.</a> J. Neurochem. 75, 1685-1693 (2000).</li><li>Gonzales, R. A., McNabb, J., Yim, H. J., Ripley, T., Bungay, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Microdialysis+of+Ethanol+in+Rat+Striatum.">Quantitative Microdialysis of Ethanol in Rat Striatum.</a> Alcohol Clin. Exp. Res. 22, 858-867 (1998).</li></ol>

この記事は、メッドアソシエイツ株式会社によって資金を供給された

アプリケーションと制限薬物自己投与は、モデル薬物中毒にげっ歯類で使用されています。この方法でモデル化され、虐待の多くの薬物は、薬物が中心コンパートメントに直接配信されて、静脈内に投与することができる。これは、自己管理セッションを介して投与量の綿密なモニタリングを可能にします。エタノールは経口通常自記式であるので、それは吸収と代謝の個体差に起因する薬物濃度を監視することは非常に困難です。興味のある脳領域からサンプルにマイクロダイアリシスを用いて、我々は唯一の領域に到達するエタノールのパターンをモニタすることはできませんが、我々はまた、同時に各々自己管理フェーズで時間をかけて同じ領域内の神経伝達物質の変化を監視することができます。 脳内の神経化学的な変化と薬物誘発性応答は薬物乱用と依存性に関連付けられている、したがって、同時に測定する能力を具体的な自己管理のフェーズで確認神経化学と薬物濃度は非常に強力でユニークなツールを提供しています。行動と検体の透析液の濃度を相関させるために心に留めておくべき一つの問題は、マイクロダイアリシス配管の物理的な特性である。具体的には、マイクロダイアリシスプローブの内腔からのコレクションチューブに転送する流体にかかる時間は重要であり、短い時間では優れています。私たちの研究室では、時間は約90秒になるようにプローブを構築。これらの条件を用いて、我々は、透析液のエタノール濃度の増加に伴って、ドリンクやポストドリンク期間の経過とともに減少し、エタノールの消費、開始時のaccumbalドーパミンの増加があることを発見した。2,3,8,9これらの実験は、 、薬理学的研究からのデータと組み合わせたとき、私たちはCHにエタノールと自己管理に伴う環境手がかりの厳密薬理作用を解析することができました神経伝達物質濃度のアンジェス。 これは、結合された行動 - マイクロダイアリシス技術のこの特定のアプリケーションは、我々の研究室の現在の研究対象に適していることに留意すべきである。それは、我々は同期自己管理行動にこれらの措置を関連付けることができるように、同じ領域内の神経伝達物質の変化のパターンと比較して脳に到達するエタノールの時系列パターンを評価するために設計されています。派生した透析液のエタノール濃度は、in vivoでのプローブ回復のために修正され、脳のエタノール組織中濃度のほんの一部ではありませんされています。エタノールの定量的な微小透析が必要な場合は、プローブへの細胞外空間から拡散することをエタノール抽出画分は、実験的に決定されるべきである。メソッドとさらなる議論のために我々の研究室から以前の資料を参照してください。10,11,12 このプロトコルは、ガスchの使い方を示していますが微小透析試料のヘッドスペースからのエタノールの固相マイクロ抽出と共にromatographyは、マイクロダイアリシスサンプルのエタノール含有量の分析のための他の方法を用いることができる。しかし、代替の方法は、いくつかの欠点に苦しむことがあります。例えば、機密性の低い分析方法は、ここに示されて5月7日分未満のサンプリング倍必要とサンプル量を増やす必要がある場合があります。ここで説明するシステムのタイプは、バイアルのヘッドスペースに配置ファイバへの吸収を可能にすることにより、密封されたサンプル瓶内の気相中のエタノールを濃縮固相マイクロ抽出を使用しています。これは、通常の蒸気の50から100マイクロリットルを注入することができます直接ヘッドスペースサンプリングと比べて検出限界を向上させます。ヘッドスペース法のもう一つの大きな利点は、分析のために注入されたサンプルは非常にきれいで、塩の自由であるということです。液体微小透析試料の直接注入も高いsensitと共に使用されるかもしれませんivityが、これは注入された塩をきれいにするために必要な定期的なメンテナンスのためのダウンタイムより楽器が必要になります。 トラブルシューティングやその他の注意事項<ol><li>あなたの実験を開始する前に、あなたのマイクロダイアリシスのセットアップが正常に機能していることを確認するために、すべての問題をトラブルシュートするために自分の時間の多くを与える。私たちはあなたと一緒に切り替える準備、ACSFで灌流し、セットアップ余分を持っていることを示唆しているほとんどのオペラントセッションが毎日特定の時間に発生する傾向があるので、自分の時間を節約するためにセットアップ誤動作。 </li><li>マイクロダイアリシスプローブはプローブが脳を貫通などの組織の剪断によって生成される組織の損傷を最小限にするために、約5分間かけて挿入されていることを確認してください。 </li><li>オペラントチャンバーにラットを転送する場合は、2番目の人は、搬送ライン（マイクロダイアリシスのセットアップの一部）がもつれになっていないこと、およびオペラントプログラムがinitをすることができるように支援しておくと便利です時間にiated。 </li><li>あなたが適切な温度に達するまでの時間のオートサンプラの水槽をたくさん与えることを確認してください。我々のシステムは、通常は2時間、50℃に到達するために必要また、あなたのサンプルが完全にサンプルを分析する前に気化されることを保証する。我々は一般的に視覚的に、これはサンプル分析を開始する前に発生したことを確認します。 </li><li>標準の良い分析化学プラクティスに従うべきである。これらには、個人、機器、および手順の検証が、これらに限定されない。要するに、我々は、次のガイドラインを採用しています個々のユーザは、複数日にわたって標準濃度と線形標準曲線（R2≥0.99）の再現性、ピーク高さの値を生成する能力を実証する必要があります。 GC-FIDシステムが正常に動作していることを確認するには、標準的には、すべての透析液サンプルが注入される前に解析することが常にあるべきである。生成された標準曲線はR2≥0.99を持っている必要があります。 </li><li>我々が使用する機器のほとんど2010年にアジレント·テクノロジーズに買収されたバリアン社を通じて購入した。この時点では、ブルカー·サイエンティフィックインスツルメンツはVarianの実験室ガスクロマトグラフィー機器事業を取得しました。今後の購入については、どちらかの会社に相談してください。 </li><li>本プロトコルでは、ラットを用いたオペラント行動を示していますが、ビューアは動作をより混乱が小さいマウスモデルで発生する可能性があることに注意する必要があります。注意すべきもう一つの問題は、その内側前頭前野や側坐核以外の脳領域に微小透析プローブの配置がここに示されているよりも大きくオペラント行動に支障をきたす場合がありです。密接にプローブ配置による被害が行動に深刻な変化をもたらすかどうかを判断するために、多数の行動のパラメータを調べることが重要です。 </li></ol>

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td> 95％エタノール</td><td> AAPERアルコールと化学株式会社シェルビーヴィル、ケンタッキー州</td><td> E190、111000190 </td></tr><tr><td>超純ショ</td><td> MPバイオメディカル、LLCは、ソロン、オハイオ州</td><td> 821721 </td></tr><tr><td>超高純度ヘリウム</td><td>空気ガス</td><td> HE UHP300 </td></tr><tr><td>空気</td><td>空気ガス</td><td> AIZ300 </td></tr><tr><td>水素</td><td>空気ガス</td><td> AIZ300 </td></tr><tr><td> GCの2 mLバイアル</td><td>アジレント</td><td> ＃8010-0015 </td></tr><tr><td> PTFE /シリコンセプタム付きのGCバイアルキャップ</td><td>アジレント</td><td> ＃8010-0084 </td></tr></tbody></table> 表1に特異的な試薬

microdialysis of ethanol during operant ethanol self-administration and ethanol determination by gas chromatography

オペラント自己管理の方法は、一般的にエタノールを含む虐待の多くの薬剤の行動や薬理作用を研究するために使用されています。しかし、エタノールは、一般的に経口投与した自己ではなく、虐待の多くは、他の薬のように静脈内にされています。経口投与された薬物の薬物動態は、静脈内投与される薬よりも複雑です。エタノールの薬理学的および行動への影響の関係を理解することは、エタノールの経時的変化の知識が飲酒中や後に脳に到達する必要があるので、我々は時間をかけて脳透析液のエタノール濃度を監視するために水素炎イオン化検出器のin vivoマイクロダイアリシスとガスクロマトグラフィーに使用します。 組み合わせたマイクロダイアリシス - 行動実験は、いくつかの技術の使用を含む。自己投与と神経の多くをテストするために適合させることができ、この記事では、定位手術、行動訓練とマイクロダイアリシス、rochemical中心仮説は、彼らだけがサンプル採取および透析液エタノール分析の後続フェーズとどのように関係するかを説明するために含まれています。エタノールの研究に固有の炎イオン化検出付きガスクロマトグラフィーによるエタノール濃度分析を透析液、詳細に記載されている。これらのメソッドによって生成されたデータは、自己管理手順の間に脳に到達するエタノールのパターンを明らかにし、同じ透析液サンプルの神経化学的解析と組み合わせた場合、結論はエタノールの薬理と行動への影響についての定めをすることができます。

Watch this Scientific Journal Video about Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography at JoVE.com

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

オペラントエタノール自己投与時のラットの脳におけるエタノール濃度の経時変化を決定するための方法が記載されている。水素炎イオン化検出器付きガスクロマトグラフィーは、それが生成される小さなボリュームに必要な感度を持っているので、透析液サンプル中のエタノールを定量化するために使用されます。

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.  ...

microdialysis-ethanol-during-operant-ethanol-self-administration

Research

JoVE Journal

Neuroscience

17.4K ビュー.  The University of Texas at Austin. オペラントエタノール自己投与時のラットの脳におけるエタノール濃度の経時変化を決定するための方法が記載されている。水素炎イオン化検出器付きガスクロマトグラフィーは、それが生成される小さなボリュームに必要な感度を持っているので、透析液サンプル中のエタノールを定量化するために使用されます。

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

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 ...

神経科学

Operant Sensation Seeking in the Mouse

Operant methods are powerful behavioral tools for the study of motivated behavior. These 'self-administration' methods have been used extensively in drug addiction research due to their high construct validity. Operant studies provide researchers a tool for preclinical investigation of several aspects of the addiction process. For example, mechanisms of acute reinforcement (both drug and non-drug) can be tested using pharmacological or genetic tools to determine the ability of a molecular target to influence self-administration behavior1-6. Additionally, drug or food seeking behaviors can be studied in the absence of the primary reinforcer, and the ability of pharmacological compounds to disrupt this process is a preclinical model for discovery of molecular targets and compounds that may be useful for the treatment of addiction3,7-9. One problem with performing intravenous drug self-administration studies in the mouse is the technical difficulty of maintaining catheter patency. Attrition rates in these experiments are high and can reach 40% or higher10-15. Another general problem with drug self-administration is discerning which pharmacologically-induced effects of the reinforcer produce specific behaviors. For example, measurement of the reinforcing and neurological effects of psychostimulants can be confounded by their psychomotor effects. Operant methods using food reinforcement can avoid these pitfalls, although their utility in studying drug addiction is limited by the fact that some manipulations that alter drug self-administration have a minimal impact on food self-administration. For example, mesolimbic dopamine lesion or knockout of the D1 dopamine receptor reduce cocaine self-administration without having a significant impact on food self-administration 12,16. 
Sensory stimuli have been described for their ability to support operant responding as primary reinforcers (i.e. not conditioned reinforcers)17-22. Auditory and visual stimuli are self-administered by several species18,21,23, although surprisingly little is known about the neural mechanisms underlying this reinforcement. The operant sensation seeking (OSS) model is a robust model for obtaining sensory self-administration in the mouse, allowing the study of neural mechanisms important in sensory reinforcement24. An additional advantage of OSS is the ability to screen mutant mice for differences in operant behavior that may be relevant to addiction. We have reported that dopamine D1 receptor knockout mice, previously shown to be deficient in psychostimulant self-administration, also fail to acquire OSS24. This is a unique finding in that these mice are capable of learning an operant task when food is used as a reinforcer. While operant studies using food reinforcement can be useful in the study of general motivated behavior and the mechanisms underlying food reinforcement, as mentioned above, these studies are limited in their application to studying molecular mechanisms of drug addiction. Thus, there may be similar neural substrates mediating sensory and psychostimulant reinforcement that are distinct from food reinforcement, which would make OSS a particularly attractive model for the study of drug addiction processes. The degree of overlap between other molecular targets of OSS and drug reinforcers is unclear, but is a topic that we are currently pursuing. While some aspects of addiction such as resistance to extinction may be observed with OSS, we have found that escalation 25 is not observed in this model24. Interestingly, escalation of intake and some other aspects of addiction are observed with self-administration of sucrose26. Thus, when non-drug operant procedures are desired to study addiction-related processes, food or sensory reinforcers can be chosen to best fit the particular question being asked. 
In conclusion, both food self-administration and OSS in the mouse have the advantage of not requiring an intravenous catheter, which allows a higher throughput means to study the effects of pharmacological or genetic manipulation of neural targets involved in motivation. While operant testing using food as a reinforcer is particularly useful in the study of the regulation of food intake, OSS is particularly apt for studying reinforcement mechanisms of sensory stimuli and may have broad applicability to novelty seeking and addiction.

In this protocol we describe a method of operant learning using sensory stimuli as a reinforcer in the mouse. It requires no prior training or food restriction, and it allows the study of motivated behavior without the use of a pharmacological or natural reinforcer such as food.

マウスに求めてオペラントセンセーション

Operant methods are powerful behavioral tools for the study of motivated behavior.  These 'self-administration' methods have been used extensively in drug ...

A Simple Way to Measure Ethanol Sensitivity in Flies

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain is correlated with that strain s ethanol preference, and therefore sedation is a highly relevant measure to study the genetics of alcohol responses and drinking. We demonstrate a simple way to expose flies to ethanol and measure its intoxicating effects. The assay we describe can determine acute sensitivity, as well as ethanol tolerance induced by repeat exposure. It does not require a technically involved setup, and can therefore be applied in any laboratory with basic fly culture tools.

A simple assay to measure the sedating effects of ethanol on Drosophila flies, based on the loss of righting reflex, is described.

ハエでエタノールの感度を測定するために簡単

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain ...

Methods for Intravenous Self Administration in a Mouse Model

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages of using an IVSA paradigm to study the reinforcing properties of drugs of abuse such as cocaine include the fact that the drug is self-administered instead of experimenter-administered, the schedule of reinforcement can be altered, and accurate measurement of the quantities of drug consumed as well as the timing and pattern of IV injections can be obtained. Furthermore, the intravenous route of administration avoids potential confounds related to first pass metabolism or taste, and produces rapid increases in blood and brain drug levels. As outlined in this video, intravenous self-administration can be obtained without prior food restriction or prior drug training following careful catheter placement during surgery and meticulous daily catheter flushing and maintenance. Experimental procedures outlined in this paper include a description of animal housing and acclimation methods, operant training using sweetened milk solutions, and catheter implantation surgery.

The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.

マウスモデルにおける静脈内自己投与方法

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages ...

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

All organisms inhabit a world full of sensory stimuli that determine their behavioral and physiological response to their environment. Olfaction is especially important in insects, which use their olfactory systems to respond to, and discriminate amongst, complex odor stimuli. These odors elicit behaviors that mediate processes such as reproduction and habitat selection1-3. Additionally, chemical sensing by insects mediates behaviors that are highly significant for agriculture and human health, including pollination4-6, herbivory of food crops7, and transmission of disease8,9. Identification of olfactory signals and their role in insect behavior is thus important for understanding both ecological processes and human food resources and well-being.
	To date, the identification of volatiles that drive insect behavior has been difficult and often tedious. Current techniques include gas chromatography-coupled electroantennogram recording (GC-EAG), and gas chromatography-coupled single sensillum recordings (GC-SSR)10-12. These techniques proved to be vital in the identification of bioactive compounds. We have developed a method that uses gas chromatography coupled to multi-channel electrophysiological recordings (termed 'GCMR') from neurons in the antennal lobe (AL; the insect's primary olfactory center)13,14. This state-of-the-art technique allows us to probe how odor information is represented in the insect brain. Moreover, because neural responses to odors at this level of olfactory processing are highly sensitive owing to the degree of convergence of the antenna's receptor neurons into AL neurons, AL recordings will allow the detection of active constituents of natural odors efficiently and with high sensitivity. Here we describe GCMR and give an example of its use.
	Several general steps are involved in the detection of bioactive volatiles and insect response. Volatiles first need to be collected from sources of interest (in this example we use flowers from the genus Mimulus (Phyrmaceae)) and characterized as needed using standard GC-MS techniques14-16. Insects are prepared for study using minimal dissection, after which a recording electrode is inserted into the antennal lobe and multi-channel neural recording begins. Post-processing of the neural data then reveals which particular odorants cause significant neural responses by the insect nervous system.
	Although the example we present here is specific to pollination studies, GCMR can be expanded to a wide range of study organisms and volatile sources. For instance, this method can be used in the identification of odorants attracting or repelling vector insects and crop pests. Moreover, GCMR can also be used to identify attractants for beneficial insects, such as pollinators. The technique may be expanded to non-insect subjects as well.

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe

Olfactory cues mediate many different behaviors in insects, and are often complex mixtures comprised of tens to hundreds of volatile compounds. Using gas chromatography with multi-channel recording in the insect antennal lobe, we describe a method for the identification of bioactive compounds.

昆虫の触角葉でガスクロマトグラフィー - マルチユニットレコーディング（GCMR）を使用して、嗅覚揮発性物質の同定

All  organisms inhabit a world full of sensory stimuli that determine their  behavioral and physiological response to their environment. Olfaction is ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

海馬のインスリンおよびマイクロインジェクション<emインビボ&gt;</em空間記憶テスト中&gt;マイクロダイアリシス

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in their etiology. The fruit fly, Drosophila melanogaster, is a powerful model for exploring molecular-genetic mechanisms underlying alcohol-related behaviors and therefore holds great promise for identifying and understanding the function of genes that influence AUD. The use of the Drosophila model for these types of studies depends on the availability of assays that reliably measure behavioral responses to ethanol. This report describes an assay suitable for assessing ethanol sensitivity and rapid tolerance in flies. Ethanol sensitivity measured in this assay is influenced by the volume and concentration of ethanol used, a variety of previously reported genetic manipulations, and also the length of time the flies are housed without food immediately prior to testing. In contrast, ethanol sensitivity measured in this assay is not affected by the vigor of fly handling, sex of the flies, and supplementation of growth medium with antibiotics or live yeast. Three different methods for quantitating ethanol sensitivity are described, all leading to essentially indistinguishable ethanol sensitivity results. The scalable nature of this assay, combined with its overall simplicity to set-up and relatively low expense, make it suitable for small and large scale genetic analysis of ethanol sensitivity and rapid tolerance in Drosophila.

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Straightforward assays for measuring ethanol sensitivity and rapid tolerance in Drosophila facilitate the use of this model organism for investigating these important ethanol-related behaviors. Here, a relatively simple, scalable assay for measuring ethanol sensitivity and rapid tolerance in flies is described.

エタノール鎮静感度との急速な寛容を測定するための安価な、スケーラブルな行動アッセイ<em&gt;ショウジョウバエ</em

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in ...

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

の歩行速度に対するエタノールの効果を測定するためのアッセイ<em&gt;線虫（Caenorhabditis elegans）</em

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

Custom-made Microdialysis Probe Design

Microdialysis is a commonly used technique in neuroscience research. Therefore commercial probes are in great demand to monitor physiological, pharmacological and pathological changes in cerebrospinal fluid. Unfortunately, commercial probes are expensive for research groups in public institutions. In this work, a probe assembly is explained in detail to build a reliable, concentric, custom-made microdialysis probe for less than $10. The microdialysis probe consists of a polysulfone membrane with a molecular cut-off of 30 kDa. Probe in vitro recoveries of substances with different molecular weight (in the range of 100-1,600 Da) and different physicochemical properties are compared. The probe yields an in vitro recovery of approximately 20% for the small compounds glucose, lactate, acetylcholine and ATP. In vitro recoveries for neuropeptides with a molecular weight between 1,000-1,600 Da amount to 2-6%. Thus, while the higher molecular weight of the neuropeptides lowered in vitro recovery values, dialysis of compounds in the lower range (up to 500 Da) of molecular weights has no great impact on the in vitro recovery rate. The present method allows utilization of a dialysis membrane with other cut-off value and membrane material. Therefore, this custom-made probe assembly has the advantage of sufficient flexibility to dialyze substances in a broad molecular weight range. Here, we introduce a microdialysis probe with an exchange length of 2 mm, which is applicable for microdialysis in mouse and rat brain regions. However, dimensions of the probe can easily be adapted for larger exchange lengths to be used in larger animals.

Microdialysis is a commonly used technique in neuroscience research. Therefore commercial probes are in great demand.In this work a probe assembly is explained in detail to build a reliable, concentric, custom-made microdialysis probe for less than $10.

カスタムメイドマイクロダイアリシスプローブの設計

Microdialysis is a commonly used technique in neuroscience research. Therefore commercial probes are in great demand to monitor physiological, ...

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson&#39;s disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases.
Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas.
DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay.
Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

高速液体クロマトグラフィー分析とシナプトソーム ドーパミンによるマウスにおけるドーパミン作動性恒常性の評価

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...