These studies were supported by grants from the National Institutes of Health, National Institute for Alcoholism and Alcohol Abuse to M.G. (P20AA017828, R01AA020634, P50 AA022537). The authors thank Jill Bettinger for helpful discussions and Jacqueline DeLoyht for technical assistance.

アッセイの前に1日<ol><li> 11（シングル性別）短い下（1-5分）CO 2のグループにおける生鮮食品バイアルにハエを収集します。 </li><li>ハエは、環境的に制御された空間（典型的には25°C、相対湿度60％、12時間の明/暗サイクル）において食物バイアル中にO / Nを回復することを可能にする。 </li><li>計画された実験のための適切な最終濃度（複数可）に精製された（≥18MΩ）水中の純粋な（100％）エタノールを希釈することによりエタノール溶液（単数または複数）を準備する。溶液（S）は、RT O / Nに戻ることができるようにします。 注：エタノールの希釈は、発熱性である。 </li></ol>アッセイの2日目<ol><li>ハエの各バイアルを試験するために、きれいな、空の食品バイアル（i）を調製する。すなわち、（ 表3参照）（ii）の新規セルロースアセテートプラグ、（iii）のシリコーンストッパー及び（iv）のエタノール溶液1mlを、バイアルを試験する。 </li><li>百分の55から65に20〜25℃の温度および相対湿度を調整することにより、試験室を準備します。 </li><li>別の労働者を持っているバイアルの各グループに一意のコードを割り当て、後でコードを記録する。少数分間順応さ試験室にハエでコード化されたバイアルを置きます。 </li><li> 2.3からフライバイアル上のコードと一致するように空のテストバイアルにラベルを付けます。 </li><li> 表1に似てスプレッドシート内の列（1列/バイアル）にコードを入力することで、ハードコピーのテストログを構築します。 </li><li>ガイドとして、テストログを使用して、試験室で配列をマッチングにハエや空のテスト用バイアルにコード化された食品のバイアルを手配。 注：テストするバイアルの合理的な最大数は24である（ すなわち、4バイアルそれぞれの6セット）。 </li><li>転送がマッチした/ラベル空のテストバイアルに食品バイアルから飛ぶと、すぐにセルロースアセテートプラグが2センチメートルバイアルのトップスを下回るまで、テストのバイアルにセルロースアセテートプラグを挿入します。 </li><li>以下、千鳥1分間隔でセットとして4バイアルの各行を処理します。 </li><li>時間0評価のために、wは個別に各バイアルを把握i番目の親指と人差し指、バイアルの底にハエをノック30秒待ってから、死んだ/不動であるハエの数をカウントするテーブルの上に静かに3回タップします。ハードコピーテスト·ログに一度に0分を各バイアルのための不動/死んだハエの数を記録します。 </li><li>タイマーが時間0で連続的にカウントアップを起動し、すぐに4バイアルの最初の行/セット用バイアル中にセルロースアセテートプラグにエタノール1mlを加え始める。それらがテストされるために、5秒間隔で、バイアル中のセルロースアセテートプラグにエタノール1mlを加える。エタノールは、セルロースアセテートプラグ全体に均一に吸収されるように円を描くようにセルロースアセテートプラグにエタノールを加える。エタノールは、セット内のすべての4試験バイアルに追加されたとき、それを密封するために、各バイアルシリコーンプラグを差し込む。 </li><li>時間1、2、3、4、5分で、それぞれ、4つのバイアルの第二、第三、第四、第五及び第六のセットにエタノール1mlを加える。後にシリコーン栓を挿入し続けます4バイアルの各セットにエタノールを添加すること。 </li><li>時間6分で、親指と人差し指で各バイアルを把持し、バイアルの底にハエをノックテーブルに静かに三回タップ、30秒待ち、その後の合計数をカウントし、記録することによって、4つのバイアルの一組をテストする鎮静されているハエ。スコアは、彼ら（i）は、バイアルの床の上に立ったが、あるいは彼らの羽ばたくことなく、彼らの背中の上を歩く又は（ii）嘘をつかない場合は、鎮静さとして飛ぶ。 </li><li> 表2のスケジュールを使用して5秒間隔でセット内の各バイアルを処理します。 </li><li>最初のセットのために行われたように7倍、8、9、10および11分の時点で、それぞれのバイアルの第2、第3、第4、第5および第6のセットをテスト。 </li><li>時間12分で2.12に記載のように、再び4つのバイアルの最初のセットをテストし、それぞれ、13、14、15、16及び17分でのバイアルの第二、第三、第四、第五及び第六のセットをテストし続ける。 </li><li> 2.12で説明したように、すべてのハエがSになるまで、テストのハエを続行edated。 </li><li>ハードコピー·テスト·ログの各バイアルにハエの合計数を入力します。ハエの総数から時間0で不動/死んだハエを検閲。 </li><li>時間（x軸）対％活性ハエ（y軸）として、各評価時点でのパーセントの活性ハエプロットデータを算出する。三次多項式またはシグモイド曲線から50値（ST50、50％の鎮静までの時間）が収まるまたは曲線下面積を計算鎮静時間を補間することにより、エタノールの鎮静を定量する。 </li><li>デコードされたバイアルからデータをコンパイルし、統計分析を行う（ 例えば、一方向ANOVAボンフェローニの多重比較検定付き）実験計画のために、必要に応じて。 </li></ol>

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The authors have nothing to disclose.

再現可能に意味のある表現型を定量簡単なアッセイは、行動の分析のために大きな価値がある。ここで説明する作業は、エタノールの鎮静感度とショウジョウバエの急速な許容範囲を測定するためのアッセイのいくつかの実用的な側面に対応しています。この作品の焦点では​​ないが、行動の分析は研究の中の被験者のための環境と遺伝的背景を一定に維持することによって促進さ?...

アルコール使用障害（AUD）（1に概説される）世界的な巨大な健康問題である。 AUDの開発を駆動するメカニズムは複雑ですが、これらの疾患は、主要な遺伝的要素を持っている（ 例えば、2）。 AUDと保存された行動反応の大遺伝はより良いの分子基盤を理解するためのエタノール関連行動における特定の遺伝子の関与を調査するために、遺伝的モデル生物を使用することに強い関心を生成している（3,4で検討）は、多くの種にわたってエタノールへAUD。ミバエ、キイロショウジョウバエは、（3,4で見直 ​​し）エタノール関連行動の分子遺伝学的メカニズムを探索するための主要なモデル生物として浮上している。ハエの研究は（5で検討）のエタノールへの行動反応のいくつかのシグナル伝達経路のための役割を強調している。興味深いことに、遺伝子および経路のいくつかの行動RESPONSに影響を与えることハエでエタノールのESはまたげっ歯類エタノール関連行動および/ ​​またはヒトAUD（ 例えば、6-14）に関与している。 ショウジョウバエモデル系で利用可能な遺伝学的ツールのスイートと相まって種にわたってエタノール関連行動を駆動するメカニズムの保全は、エタノールへの行動反応の遺伝学を調査するためのショウジョウバエモデルの有用性を強調する。 感度15,16及びヒトでエタノールに（17に概説）耐性はAUDの開発にリンクされている。エタノールへのこれらの行動反応の両方が（3,4で検討）実験室の種々のアッセイを経由してハエにモデル化することができます。著者らに知られているフライアッセイのすべては、時間依存エタノール誘発鎮静/協調不能またはエタノールの鎮静からの時間依存性の回復のいずれかに基づいている。 エタノール感度とrの遺伝学上の私たちのグループから前回の記事でショウジョウバエ APID耐性が、ハエのエタノール蒸気誘導性の鎮静に基づいて行動アッセイは、18を使用した。このアッセイにおける試験は、ライブ大人を転送することによって開始されたセルロースアセテートプラグの上部（ すなわち、非フライ側）にエタノールを加え、酢酸セルロースプラグ付きバイアル中のハエトラップ、餌バイアルを空に麻酔なしハエ、およびシリコーンストッパー（18を参照すると、 図S3に概略参照）ハエ、セルロースアセテートプラグおよびエタノールを含有するバイアルを密封する。ハエの異なるグループを表す複数のバイアルは、このアッセイのスループットを向上させる、並行して評価した。バイアルを匿名コードを与えられたと実験者は鎮静の評価において意図しない偏りを防ぐために、処置群を知らされた。標準実験では、バイアルにハエは6分間隔で穏やかにタッピングし、30秒の回復後、各バイアル中の鎮静さハエの数を数え、converteパーセントの活性ハエにD。ハエは、内部エタノール18および鎮静（本報告書でのCF参照18および図1Aおよび1B）の漸進的増加を引き起こし、時間依存的に酢酸セルロースプラグからエタノール蒸気を吸収した。このアッセイにおける鎮静は操作上ハエ（I）歩行の不在に立っまたは（ii）で、またはそれらの羽ばたくことなく、彼らの背中に横たわっていると定義した。ここで、このエタノールの鎮静作用アッセイを詳細に記載され、それを使用に関連するさらなる操作上の最適化が提供され、そしてアッセイは、フライ鎮静感度に対する食物補充オプションの寄与をアドレス指定するために使用される。

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			<td>111000200</td>
			<td>200 proof</td>
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</table>

an inexpensive, scalable behavioral assay for measuring ethanol sedation sensitivity and rapid tolerance in drosophila

アルコール使用障害（AUD）深刻な健康上の課題です。 AUDへの大規模な遺伝性の構成要素にもかかわらず、いくつかの遺伝子が明確に彼らの病因に関与している。ミバエ、 キイロショウジョウバエは 、アルコール関連行動の根底にある分子遺伝学的メカニズムを探索するための強力なモデルであるため、識別し、AUDに影響を与える遺伝子の機能を理解するための非常に有望である。研究は、これらのタイプのためのショウジョウバエモデルの使用を確実にエタノールへの行動応答を測定するアッセイの利用可能性に依存する。このレポートでは、ハエでエタノールの感度と迅速な寛容性を評価するための適切なアッセイを説明しています。このアッセイで測定されたエタノール感受性ハエ、試験の直前に食物なしに収容されているボリュームと使用のエタノール濃度、以前に報告された遺伝子操作で、さまざまな時間の長さも影響される。対照的に、エタノールのこのアッセイで測定されたensitivityフライ取り扱い、ハエの性別、および抗生物質または生酵母と成長培地の補充の活力に影響されない。エタノール感受性を定量するための3つの異なる方法は、すべて本質的に区別できないエタノール感度の結果につながる、記載されている。比較的低経費アップを設定するには、その全体的なシンプルさと組み合わせたこのアッセイのスケーラブルな性質は、エタノール感度やショウジョウバエの急速な寛容の小規模および大規模な遺伝子解析に適したもの。

このエタノールの鎮静作用アッセイからの生データは、エタノール蒸気暴露時間の関数として鎮静化されたハエの数である。生データは、時間の関数としてパーセント活性ハエに変換される（一次データ1Aの図、B、D - F）。曲線がフィットするから、一次データからのエタノール鎮静に対する感度が補間を経由して、鎮静時間50（ST50）として曲線（AUC）下の鎮静さま?...

Watch this Scientific Journal Video about An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila at JoVE.com

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-inexpensive-scalable-behavioral-assay-for-measuring-ethanol

Research

JoVE Journal

Neuroscience

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

10.2K ビュー.  Virginia Commonwealth University. 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.

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

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

行動内分泌学と実験経済学を組み合わせること：テストステロンと社会的意思決定

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

神経科学

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 ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

ラットのショ糖の渇望のインキュベーションを評価するための一般的な方法

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain perception in rodents. Withdrawal thresholds can be measured first at baseline and then following drug, venom, injury, allergen,&#160;or otherwise evoked inflammation by applying an accurate force on very specific areas of the skin. An electronic von Frey apparatus allows precise assessment of mouse hind paw withdrawal thresholds that are not limited by the available filament sizes in contrast to classical von Frey measurements. The ease and rapidity of measurements allow for incorporation of assessment of tactile sensitivity outcomes in diverse models of rapid-onset inflammatory and neuropathic pain as multiple measurements can be taken within a short time period. Experimental measurements for individual rodent subjects can be internally controlled against individual baseline responses and exclusion criteria easily established to standardize baseline responses within and across experimental groups. Thus, measurements using an electronic von Frey apparatus represent a useful modification of the well-established classical von Frey filament-based assays for rodent mechanical allodynia that may also be applied to other nonhuman mammalian models.

Electronic von Frey measurements are an effective and improved alternative to classical von Frey filaments, as they allow rapid and precise measurement of changes in rodent mechanical withdrawal thresholds to continuously applied pressure stimuli before and after an inflammatory event.

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain ...

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 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 ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

でMARCMベースのクローンのMechanosensationための行動アッセイ<em&gt;キイロショウジョウバエ</em

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

の記録細胞内の電圧応答のための電気生理学的手法<em&gt;ショウジョウバエ</em&gt;光刺激に感光体と介在ニューロン<em&gt;インビボ</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Measuring and Altering Mating Drive in Male Drosophila melanogaster

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, reductionist, and scalable system for in-depth investigation of motivation using the mating drive of male Drosophila&#160;melanogaster (Drosophila), the methods for which we detail here. The behavioral paradigm centers on the finding that male mating drive decreases alongside fertility over the course of repeated copulations and recovers over ~3 d. In this system, the powerful neurogenetic tools available in the fly converge with the genetic accessibility and putative wiring diagram available for sexual behavior. This convergence allows rapid isolation and interrogation of small neuronal populations with specific motivational functions. Here we detail the design and execution of the satiety assay that is used to measure and alter courtship motivation in the male fly. Using this assay, we also demonstrate that low male mating drive can be overcome by stimulating dopaminergic neurons. The satiety assay is simple, affordable, and robust to influences of genetic background. We expect the satiety assay to generate many new insights into the neurobiology of motivational states.

Measuring and Altering Mating Drive in Male Drosophila melanogaster

This article describes a behavioral assay that uses male mating drive in Drosophila melanogaster to study motivation. Using this method, researchers can utilize advanced fly neurogenetic techniques to uncover the genetic, molecular, and cellular mechanisms that underlie this motivation.

測定とマレの変更交尾ドライブ<em&gt;キイロショウジョウバエ</em

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.