我々は、重要な原稿の読み取り、および有用な議論のための研究室のメンバーのためにAylinロダンに感謝。この研究は、NIH、R01AA019526によってサポートされています。

1。暴露室を作成する<ol><li>カミソリの刃を使用して半分にバイアルのプラグをカット。また、綿球を使用してください。 </li><li>ハエがログイン立ち往生しないように滑らかな表面を作るために食品のバイアルの底に、バイアルのプラグの半分、または綿のボールをプッシュ</li><li>複数の曝露チャンバーを作る場合、バイアルのプラグと食品が各チャンバー内の同じ容積を取ることを確認してください。バイアルの側面のマークはこれを容易にすることができます。 </li></ol> 2。ハエを公開<ol><li>酵母のない通常の餌をバイアルに露出する前に1つ性別、年齢1-5日、一日の8ハエを集める。 </li><li>実験者が実際に使用される遺伝子型/株に盲検化されるように、コード内でラベルを付けます。 </li><li>タップして、曝露チャンバー8つのハエを移す。麻酔しないでください。 </li><li>それは簡単に見えるように200プルーフのエタノールに食品染料、例えば、アシッドブルー9、少量のを追加。 </li><li> 0.5mlのエタノールとコートはバイアルのプラグの下部、または綿ボール、。 </li><li>露出のバイアル瓶にエタノールのプラグを差し込みます。アルコールは、バイアルに、ではなくバイアルの壁の方を向いていることを確認してください。 </li><li>カウントアップタイマーを開始します。 </li></ol> 3。記録エタノール誘発性の中毒<ol><li>タイマーが開始された後、各分には、ハエを驚愕し、10秒のためにそれらを観察するためにベンチに暴露室をタップします。 </li><li>毎分固定ハエの数を記録。ハエは、彼らが自分の背中から降りることができない場合は、1つの場所に残っている、または急速に全体の10秒の観測期間を通じて、その翼を振動さ固定です。これらのハエの大多数は急速な翼の運動の期間の間静止しているので、後者のハエは、静止とみなされます。ハエは、彼らが自分の背中から降りるとの距離および/またはバイアルの壁まで歩くことができれば静止していないです。 </li><li> 4つ以上のハエが固定として記録されるときの露光試験が終了しています。 </li><li>ハエを破棄し、後で再利用するために暴露室の空気を外に出して。 </li></ol> 4。 50％の鎮静に時間を決定する 50％の鎮静（ST50）までの期間は、8ハエの4つは静止したままエタノールの暴露の開始後の時間です。これは、次のように決定されます。 次のいずれかの操作を正確に4ハエが静止している最初の測定分。 または：以下つ以上のハエ（Y1）は1時点（分X）、そして次の測定時点においてつ以上（Y2）（X +1分）、式を使用して線形補間ST50で静止している場合： ST50 =（4 - Y1）/（Y2 - Y1）+ X 5。許容範囲を決定するために露出を繰り返してハエを公開<ol><li>あなたの実験で使用されている野生型の対照株のST50（重量）を決定します。 </li><li>前述のようにエタノールにハエの実験群を公開。 </li><li>彼らのST50（1）を測定しますが、時間2倍ST50（重量）に達するまで公開し続ける。 </li><li>バックの空の食品用バイアルにハエを移し、4時間保持する。 </li><li>ハエを再公開し、露出2のST50（2）を決定する。 </li></ol>許容範囲を決定する X100 - （1 ST50（2）/ ST50（1））のような％の許容誤差を計算する。 6。代表的な結果： ここに示されている、我々は遺伝子型と投与量あたりのハエの6-8バイアルで得られたいくつかの代表的な結果は次のとおりです。あなたの標準誤差が大幅に大きい場合、体系的な問題が可能性が発生しました。エタノールは、曝露チャンバーに直面する、とチャンバーが同じボリュームであることを確認してください。 <img src="/files/ftp_upload/2541/2541fig1.jpg" alt="図1" /> 野生型の図1。用量反応曲線は、エタノールの増加割合にさらさハエ。ハエの八バイアルは、投与量及び性別ごとに暴露された。用量間ST50が増加し、エタノールの投与量（P &lt;0.001）で有意に減少する男性と女性のハエは、（P&gt; 0.22、2ウェイANOVA）各用量で同じST50を示す。過密ボトルの便を運行する航空会社はノートでは、健康なボトルのものより大幅に小さくすることができ、このサイズの違いは、ST50に影響を与える可能性があります。 15から20人の女性と種子各ボトルは、ボトルごとに最適な幼虫の密度を得るために1-2日間敷設。 <img src="/files/ftp_upload/2541/2541fig2.jpg" alt="図2" /> 図2 90％、または100％エタノールのいずれかにハエを公開するにはmutation2白ウサギ（P &lt;0.001、2ウェイANOVA）によって引き起こされる前述のエタノール耐性を再現。 <img src="/files/ftp_upload/2541/2541fig3.jpg" alt="図3" /> 図3：4時間回復の間隔を隔て、100％エタノールに露出を繰り返し、秒露光（P &lt;0.001、対応のあるtの後にST50の大幅な増加を引き起こす- テスト）。ハエが最初に暴露（そのNAのVE ST50倍に相当）中に24分間暴露されたことに注意してください。短い時間（12分）のための暴露は、再暴露（データは示さず）によりST50の著しい増加を引き起こさなかった。

<ol>
	<li>Bainton, R. J., Tsai, L. T. -. Y., Singh, C. S., Moore, 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=.">.</a> Curr. Biol. 10, 187-194 (2000).</li><li>Rothenfluh, A., Threlkeld, R. J., Bainton, R. J., Tsai, L. T. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell. 127, 199-211 (2006).</li><li>Devineni, A. V., Heberlein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Curr. Biol. 19, 2126-2132 (2009).</li><li>Scholz, H., Ramond, J., Singh, C. S., Heberlein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuron. 28, 261-271 (2000).</li><li>Allen, M. J., Godenschwege, T. A., Tanouye, M. A., Phelan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Semin. Cell Dev. Biol. 17, 31-41 (2006).</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> Nature. 404, 394-398 (2000).</li></ol>

利害の衝突は宣言されません。

本発表では、我々はバイントンらによって記述された以前のアッセイに基づいて、エタノール誘発性中毒を判断する簡単な方法の概要を示している。 2000 1、およびRothenfluhら。 2006 2。アルコール誘発性の鎮静が飲酒の嗜好3と相関しているので、このアッセイは、アルコール使用障害の研究に関連しています。記載のアッセイは、驚愕誘発性負の走地性に基づいており、どのようにアルコール暴露は、この生得的反応に干渉されています。エクスポージャーを開始する前に、すべてあなたのハエが食品のバイアルでハエをタップした後、彼らはすべて10秒の観測時間内に下から上に移動する必要があるすなわち、そのような驚愕に正常な応答を示すことを確認してください。減少負の走地性を持つハエの例としては、ビッグバンに敏感な発作の変異体5と、ドーパミンニューロン6の変性による運動欠損ハエが含まれています。 記載のアッセイは比較的単純であるにもかかわらず、二つの変数は、再現性のある結果を確保するために最も注目に値する。最初に、すべてのエクスポージャーのバイアルは、同じボリュームにする必要があります。これは、バイアルの底部は食品+バイアルプラグ/コットンボールだけでなく、エタノールを含有するプラグインごとにバイアルに同じ距離を挿入するのと同じ量が含まれているかどうかの確認が行われます。完全に空のバイアルは、このアッセイに使用しているが、アッセイのスループットを減少させる、より長いST50の値を導くことができる。 このアッセイはまた、繰り返しのエタノール曝露によって開発公差のハエの量を決定するために適しています。繰り返しの曝露を行うときに変数の数を考慮する必要があります。最初に、間露光間隔は、ここで4時間として記述されています。この変数は変更可能ですが、我々はそれが最初の曝露からエタノールのクリアランスを確保するために、2時間よりも低くしないことをお勧めすることができます。ショルツらを参照してください。エタノール耐性の発達と減衰の速度論のための2000 4。考慮する2番目の変数は、最初の曝露の期間です。これは明らかに制御し、実験的なハエで同じでなければなりません。しかし、これは両方のこれらのハエの系統に供給されるエタノールの同量になる場合があります。鎮静ハエは、以前の後、鎮静株よりも高い線量を受けることがsedatesひずみしたがって、アクティブなハエ2より多くのエタノールを吸収するため、より高い初期投与に起因する多くの耐性を開発する。最初のST50（1）の違いを示す菌株の耐性の開発を比較するとき私達は強くリーダーを注意してください。 我々はここで説明するアッセイは、簡単にかつ再現性アルコールにハエ行動反応を決定するのに便利なツールです。

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a simple way to measure ethanol sensitivity in flies

エタノールの原因の低用量は高用量で鎮静している間、非常に活発になるために飛ぶ。与えられたハエ株のエタノール誘発性鎮静に対する感度は、その株のエタノールの嗜好と相関し、したがって、鎮静は、アルコールの反応と飲酒の遺伝学を研究するために、関連性の高い指標です。我々はその酔わせる効果を、エタノール及び測定するためにハエを公開する簡単な方法を示しています。我々が記述するアッセイは、繰り返しの曝露により誘発される急性感度だけでなく、エタノール耐性を決定することができます。それは技術的に関与してセットアップを必要としないため、基本的なフライの文化のツールを使っての検査に適用することができます。

Watch this Scientific Journal Video about A Simple Way to Measure Ethanol Sensitivity in Flies at JoVE.com

A Simple Way to Measure Ethanol Sensitivity in Flies

のエタノールの鎮静作用を測定する簡単なアッセイ<em&gt;ショウジョウバエ</em正向反射の消失に基づいて、&gt;ハエは、、説明されています。

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 ...

a-simple-way-to-measure-ethanol-sensitivity-in-flies

Research

JoVE Journal

Neuroscience

23.1K ビュー.  University of Texas Southwestern Medical Center. のエタノールの鎮静作用を測定する簡単なアッセイショウジョウバエハエは、、説明されています。

ビデオ: A Simple Way to Measure Ethanol Sensitivity in Flies

Hyponeophagia: A Measure of Anxiety in the Mouse

Before the present day, when fast-acting and potent rodenticides such as alpha-chloralose were not yet in use, the work of pest controllers was often hampered by a phenomenon known as "bait shyness". Mice and rats cannot vomit, due to the tightness of the cardiac sphincter of the stomach, so to overcome the problem of potential food toxicity they have evolved a strategy of first ingesting only very small amounts of novel substances. The amounts ingested then gradually increase until the animal has determined whether the substance is safe and nutritious. So the old rat-catchers would first put a palatable substance such as oatmeal, which was to be the vehicle for the toxin, in the infested area. Only when large amounts were being readily consumed would they then add the poison, in amounts calculated not to affect the taste of the vehicle. The poisoned bait, which the animals were now readily eating in large amounts, would then swiftly perform its function.
Bait shyness is now used in the behavioural laboratory as a way of measuring anxiety. A highly palatable but novel substance, such as sweet corn, nuts or sweetened condensed milk, is offered to the mice (or rats) in a novel situation, such as a new cage. The latency to consume a defined amount of the new food is then measured.
Robert M.J. Deacon can be reach at <a href="mailto:robert.deacon@psy.ox.ac.uk">robert.deacon@psy.ox.ac.uk</a>

Mice and rats, due to their innate cautiousness, are initially slow in consuming a novel food, particularly in a novel place. This hyponeophagia can readily be measured in the laboratory, even though laboratory animals are much less anxious than their wild counterparts

Hyponeophagia：マウス​​の​​不安の測定

Before the present day, when fast-acting and potent rodenticides such as alpha-chloralose were not yet in use, the work of pest controllers was often ...

神経科学

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.  ...

The FlyBar: Administering Alcohol to Flies

Fruit flies (Drosophila melanogaster) are an established model for both alcohol research and circadian biology. Recently, we showed that the circadian clock modulates alcohol sensitivity, but not the formation of tolerance. Here, we describe our protocol in detail. Alcohol is administered to the flies using the FlyBar. In this setup, saturated alcohol vapor is mixed with humidified air in set proportions, and administered to the flies in four tubes simultaneously. Flies are reared under standardized conditions in order to minimize variation between the replicates. Three-day old flies of different genotypes or treatments are used for the experiments, preferably by matching flies of two different time points (e.g., CT 5 and CT 17) making direct comparisons possible. During the experiment, flies are exposed for 1 hr&#160;to the pre-determined percentage of alcohol vapor and the number of flies that exhibit the Loss of Righting reflex (LoRR) or sedation are counted every 5 min. The data can be analyzed using three different statistical approaches. The first is to determine the time at which 50% of the flies have lost their righting reflex and use an Analysis of the Variance (ANOVA) to determine whether significant differences exist between time points. The second is to determine the percentage flies that show LoRR after a specified number of minutes, followed by an ANOVA analysis. The last method is to analyze the whole times series using multivariate statistics. The protocol can also be used for non-circadian experiments or comparisons between genotypes.

Drosophila has emerged as a significant model system for dissecting the cellular and molecular underpinnings of behavioral responses to alcohol. Here we present a protocol for the collection of alcohol sensitivity data in a circadian context that can be easily applied to other experiments and is well-suited for undergraduate research.

フライ：ハエにアルコールを管理

Fruit flies (Drosophila melanogaster) are an established model for both alcohol research and circadian biology. Recently, we showed that the circadian ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

脳スライスビオチン：<em&gt;生体外</emアダルトニューロンにおいて地域固有の細胞膜のタンパク質輸送を測定するために&gt;の取り組み

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

マウスにおける低温感受性と適応を決定するためのシンプルで安価な方法

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

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 ...

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.

MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

miRNAの脳特異的ノックダウンのための定位注射への単純な代替

MicroRNAs (miRNAs) are key regulators of gene expression.  In the brain, vital processes like neurodevelopment and neuronal functions depend on the ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

使用して報酬を求める行動の変化を測定するための簡単な方法<em&gt;キイロショウジョウバエ</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

大人のホールマウントの準備のためのシンプルなワンステップ解剖プロトコル<em&gt;ショウジョウバエ</em&gt;ブレインズ

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer&#039;s Mouse Models

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).