私たちは、原稿の以前のバージョン上で貴重なコメントをランドルフメンツェルに感謝学習の神経基盤（B​​FNL）DEへ：この作品は、バーンスタインフォーカス内教育研究省（BMBF）のドイツ連邦環境省からの助成金によって支えられて。

1。ハイブからキャッチ蜂<ol><li>実験は、午後2〜4を開始する前に、ある日、ハイブを残してミツバチがキャッチされています。そのためには、頂点および基部で開閉可能なUV光透過性のプレキシガラスのピラミッドは、（高さ= 30センチ、頂点3,5 × 3、5 cmのベース18 × 18センチ）、、20で開催される - ベースのオープンと頂点を持つハイブの入り口の前に30cmの距離は、ハイブを残してミツバチがピラミッドの底辺を入力するように閉鎖。ベースはその後閉鎖され、キャプチャされたミツバチは、さらなる処理のための研究室に持ち込まれている。 </li></ol> 2。ガラスバイアルにピラミッドから蜂を転送<ol><li>ラボでは、ピラミッドはそのベースに配置されます。ピラミッドの壁面は暗くなります（タオルなど）はしかし、頂点が発見されたままになります。開いたときのため、その正の走光性を、ミツバチが頂点を通じてピラミッドを残す。一つ一つは、ミツバチがオープン頂点上のバイアルを保持することによって、ガラスバイアルにピラミッドから転送されます。 1バイアルは、蜂ごとに使用されます。したがって、頂点は1つのミツバチは、バイアルに入ったときに閉じられます。 </li></ol> 3。チューブの活用の蜂<ol><li>ミツバチは、2.5から3.5分間氷上でガラスバイアルに、それらを冷却することによって固定化される。蜂を見るとすぐそれが変化しないように氷からそれを削除することをお勧めします。 </li><li>単一の固定されたミツバチは、それが自由ではなく、その頭部、胸部や脚の長い鼻を移動することになるように粘着テープ付きの小さなプラスチック製のチューブ、で活かされている。首が圧縮されていないことが重要です。 </li><li>プラスチックチューブで修正すべての蜂がより良いハンドリングと識別するためのラックの番号のボーリング孔に配置されます。それはコンディショニングまたはメモリ取得のために削除された後にチューブを常に正確に同じ孔に返されます。 </li></ol> 4。餌蜂<ol><li>最初の晩（4-6時）に、キャッチされた後、蜂はショ糖溶液（0,88 M、水道水に溶解した白洗練された家庭用砂糖）で満腹に供給されています。蜂を養うために、そのPERは、ショ糖溶液と動物とのアンテナがショ糖溶液の一滴（4μL）を消費するために許可されて触れることで誘発される。自分のアンテナがショ糖溶液で触れているとき、彼らはもはやPER高速かつ信頼性を示さないまで、ミツバチは、繰り返し次々に供給されています。 </li><li>実験の蜂の後続の各​​夜（4-6時）にショ糖溶液の1滴（4μL、0,88 M、水道水で解決白い精製された砂糖）を使用して別の4倍々に供給されています。それは、ショ糖溶液を摂食中にミツバチのアンテナやテングザルに塗抹されていないことと、チューブがショ糖溶液で汚染されていないことが重要です。ミツバチは、ショ糖刺激によるトレーニングのコンテキストを関連付けるの可能性を排除するコンディショニングの部位またはその近くに供給されるべきではない。 </li></ol> 5。活かした蜂が一晩に保つ<ol><li>ミツバチは、室温でプラスチック製のボウルで一晩保持されます。水道水は、ボウル（約0,5-1センチの高さ）に充填される。活かしたミツバチが付いているラックは、プラットフォームとしてのELISAプレートを使用して水線の上のボウルに配置されます。最後に、ボールは段ボールで覆われている。 </li></ol> 6。嗅覚コンディショニング実験の最初の朝（午前10時）に嗅覚調整が行われる。条件刺激（CS）が臭気である、無条件刺激（米国）は、ショ糖溶液です。 <ol><li>臭気（例えばクローブオイル）の4μLを20 mLシリンジに挿入される円形の濾紙（1,3 cmの直径）にピペットで。臭気は、ボンネットの下ピペットされ、フィルタのヒントは、ピペットの汚染を防止するために使用されています。 </li><li>活かしたミツバチとラックは、コンディショニングの手順を開始する前にコンディショニングのサイトで30分の近くに配置し、しかし、単一のミツバチが調整されている排気管の前にある場所までの距離にあります。 </li><li>エアコンは10分の期日間間隔（ITI）を持つ3つの香りの組み合わせ（条件刺激、CS）とショ糖溶液（無条件刺激、米国、1,25 M）で構成されています。オーディオプレーヤーを介して実験者に配信される音響信号は、刺激開始、刺激オフセット、刺激の持続時間と配置の正確なタイミングが保証されます。買収の裁判は、排気の前で動物の10秒の位置から始まります。間もなく10秒の終わりの前に臭いを含む注射器は、ミツバチのアンテナを対象としたハチの目の前に3センチに配置されます。続いて、匂いは注射器を介して20mLの空気を押すことで、5秒間表示されます。最初の3秒後に両方のアンテナの遠位鞭毛は、つまようじと動物が4秒moistend爪楊枝をなめるために許可されているソリューションは、湿らせたショ糖で触れています。 13秒は、CSの刺激の終了後に、ミツバチは、トレーニングの文脈から取り出し、ラックに戻って配置されます。完全にグループレッスンの試験では、28秒間継続。 ハチの触角も長い鼻のどちらも調整後のショ糖で覆われていることが重要です。したがって、つまようじが唯一のスクロース溶液と爪楊枝でのショ糖溶液の形のない滴その湿らせていることを確保する必要があります。 </li><li>コンディショニング後にラックをボウルに戻って配置されます。 </li><li>動物実験時の動作、配置とCSの間にPERの発生と米国のプレゼンテーションをすなわち、実験者によって監視され、下に記載されています。 </li></ol> 7。記憶維持<ol><li>保持テストは、任意の間隔（日数の分）で行うことができます。メモリテストが開始される前にミツバチを持つラックは、コンディショニングのサイトで30分の近くに配置されます。 </li><li>メモリテストは、米国のプレゼンテーションすることなく5秒CSのプレゼンテーションで構成されています。テストは、排気の前で動物の10秒の位置から始まります。前述のように続いて、臭いを5秒間表示されます。動物の行動は、配置時にPERの発生、すなわちとCSのプレゼンテーションは、実験中にダウンして注目される。 </li><li>実験の終わりに口先延長の応答は、再び動物はまだその口吻を拡張できることを保証するためにショ糖溶液でアンテナに触れることで誘発される。 </li></ol> 8。全身注射全身注射の時点では、実験計画に依存します。 <ol><li>小さ ​​な穴は、使い捨て注射針（21 G）を使用して、飛翔筋17は、上記のscutumの割れ目の横のたて座の後部のクチクラで作られています。 </li><li>ガラス毛細管を使用して、1μLのソリューションは、飛翔筋へのクチクラの穴から噴射される。訓練を受けた実験者は、注射やコンディショニングの正確なタイミングを可能にする、30秒ごとone蜂を注入することができます。 </li></ol> 9。実験中に餌蜂<ol><li>コンディショニング後の夜（4-6時）に、ミツバチは、ショ糖溶液を1滴（4μL、0,88 M、水道水に溶解して白い精製された砂糖）を使用して別の4倍後のものを供給している。それは、ショ糖溶液を摂食中にミツバチのアンテナやテングザルに塗抹されていないことと、チューブがショ糖溶液で汚染されていないことが重要です。ミツバチは、ショ糖刺激によるトレーニングのコンテキストを関連付けるの可能性を排除するコンディショニングの部位またはその近くに供給されるべきではない。 </li></ol> 10。データ収集とデータ解析<ol><li>実験時の口先拡張応答の発生が監視されます。それは、CSの発症と米国のプレゼンテーション（研修中）の間またはCSのプレゼンテーション中に、その長い鼻を拡張する場合ハチが開いて顎の間に仮想回線を経由する、（テストフェーズで）正の得点が記録されます。 </li><li>分析の動物に含まれるようにするには、2つの条件を満たす必要があります：彼らは、実験全体を生き残る必要で、それらは実験終了時にショ糖溶液に無条件口先拡張応答を（PER）を示す必要があります。獲得と保持テストの間にPERを示す蜂の割合は、各CSのプレゼンテーションのためにプロットされます。 </li></ol> 11。代表的な結果： 二つの実験はここに表示されます。 最初の実験では、リン酸緩衝生理食塩水（PBSの全身注射の影響を見て、内のMM：137のNaCl、2,7のKCl、10,1のNa 2 HPO 4、1,8 KH 2 PO 4、pHが7、 2）学習と長期記憶の形成に。無処理群、トレーニング前に1μLPBSで30分間で注入した群、および治療を偽注射群：ミツバチの3つのグループは10分の期日間間隔で3 CS - USペアで訓練されたPBS注入群としてではなく、PBSを注入することなく、同じ方法で。トレーニングのメモリの24時間後は3つのグループすべてが1つのCSのプレゼンテーションでテストされています。コンディショニング時にエアコンの応答を示す動物の割合は、3つのトレーニングの試験にわたって大幅に増加する（図1、係数の時間を繰り返し測定するための分散分析F 2378 = 340456、P &lt;0.05）。 PERの差は調整中に、三つのグループ（因子グループの繰り返し測定のための分散分析F2、189 = 1299、P&gt; 0,05）との間で認められなかった。 PBS注入し、偽注射動物と非投与動物を比較するときに24時間の保持テスト（F2、187 = 0752、P&gt; 0,05）の場合にも当てはまります。 タンパク質合成阻害剤のアニソマイシン（10 mm）を全身3つのC - USペアリング後に30分を注入したときに2番目の実験では、3、4日間のトレーニング後のメモリ保持の阻害を示す。 PBS注入群とアニソマイシン注入群の比較では、保持さtの間に群間で有意差を明らかにEST（図2、ファクターグループF1、94の繰り返し測定のANOVA = 8,86、P &lt;0.05）。主な違いは、3日と4日目のメモリテストで観察することができます。これはWüstenberg らの結果に似ている。18また、別のプロトコルではあるが、三CS - US組み合わせで蜂を条件付け人。 <img src="/files/ftp_upload/2282/2282fig1.jpg" alt="図1" /> 図1。 PBSのクチクラの病変や注射は、買収と記憶の保持が変更されることはありません。動物は、3つのCS - USの組み合わせ（買収）で訓練された。 24時間後、メモリは（メモリ保持）試験された。トレーニング前の30分、穴が二つのグループのクチクラ（病変、PBS）で行われ、これらのグループの一つであった1μLPBS（PBS）を注入した。第三のグループは、（無処理）そのまま残されました。発表は各グループごとに臭気のプレゼンテーションに対応するための動物のパーセンテージです。 ANOVAは、アクイジションフェーズ（F2、189 = 1.299、P&gt; 0.05）またはメモリ保持のグループ（F2、187 = 0.752、P&gt; 0.05）の間に有意な違いは示されません。 <img src="/files/ftp_upload/2282/2282fig2.jpg" alt="図2" /> 図2。最後のCS - USのペアリング後にアニソマイシン40分の注入は、メモリの保持三日と四日間の訓練の後を削減。動物は、3つのCS - USの組み合わせ（買収）で訓練を受け、1μLの10mMアニソマイシン（異方性）または1μLのPBSを注入した最初のトレーニング試験1時間後。メモリは後72時間および96時間、（記憶保持）24時間、48時間をテストした。 PERと臭気のプレゼンテーションへの応答をグループごとに動物の割合は、され提示。アニソマイシンの注射は、ANOVAによって明らかにトレーニング（F1、94 = 8.86、p &lt;0.05）が後の記憶保持72時間と96時間の大幅な削減につながります。

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anatomy of the honeybee. , (1956).</li><li>Wustenberg, D., Gerber, B., Menzel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+communication:+long-+but+not+medium-term+retention+of+olfactory+memories+in+honeybees+is+impaired+by+actinomycin+D+and+anisomycin.">Short communication: long- but not medium-term retention of olfactory memories in honeybees is impaired by actinomycin D and anisomycin.</a> Eur. J. Neurosci. 10, 2742-2745 (1998).</li><li>Vollbehr, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zur+Orientierung+junger+Honigbienen+bei+ihrem+1.">Zur Orientierung junger Honigbienen bei ihrem 1.</a> Orientierungsflug. Zool. Jb. 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Physiol. 194, 861-869 (2008).</li><li>Menzel, R., Manz, G., Menzel, R., Greggers, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massed+and+spaced+learning+in+honeybees:+the+role+of+CS,+US,+the+intertrial+interval,+and+the+test+interval.">Massed and spaced learning in honeybees: the role of CS, US, the intertrial interval, and the test interval.</a> Learn. Mem. , 198-208 (2001).</li><li>Friedrich, A., Thomas, U., Muller, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+at+different+satiation+levels+reveals+parallel+functions+for+the+cAMP-protein+kinase+A+cascade+in+formation+of+long-term+memory.">Learning at different satiation levels reveals parallel functions for the cAMP-protein kinase A cascade in formation of long-term memory.</a> J. Neurosci. 24, 4460-4468 (2004).</li></ol>

利害の衝突は宣言されません。

<ol><li>蜂キャッチ：経験豊富な採餌蜂を収集するには、ミツバチは、ハチの子は向きのフライト19日にある時間を避けて、午前中か午後のいずれかで捕捉されます。飼料収穫機は花粉や蜜のいずれかを収集する。花粉と蜜の飼料収穫機の違いは、それらの糖応答性とその学習能力20によれば観察されている。唯一の蜜の飼料収穫機をキャッチするために、ショ糖溶液で満たされた人工的な食料源は、ハイブと動物の近くに設置することができますそこに収集することができる。ハイブに戻っ花粉飼料収穫機は、その後ろ足20日に花粉籠によって識別することができます。 </li><li>修正と処理：それは実験の最後まで学習すると生存能力に影響を与える可能性があるため、氷上で蜂の拡張冷却は避けてください。足根は、ショ糖溶液による偶発的な刺激を避けるために、チューブ内に慎重に固定する必要があります。ミツバチがsuccroseソリューション3,21と足根刺激によって習得することが示されているので、これは、重要です。 </li><li>嗅覚コンディショニング：コンディショニングの試行回数、期日間の間隔、およびテスト時間のポイントは、研究3,22の焦点に応じて変化させることができる。 </li><li>餌：ミツバチの飽食のレベルは、その学習性能とメモリの保持3,23に影響を与えることが知られている。これは、実験を計画する際に考慮する必要があります。 </li><li>注入：空気嚢は、ミツバチの胸部17に配置されています。呼吸器系のこれらの重要な部分は、生存のために必要です。したがって、注意を注入中に空気嚢を傷つけないように注意してください。気泡の放出は、空気嚢への損傷を示している。 </li></ol>

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<td>Syringe Omnifix 20 ml</td>
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<td>White refined household sugar</td>
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<td>Kaisers Supermarket, Berlin, Germany</td>
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<td>Clove oil</td>
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<td>Bombastus, Freital, Germany </td>
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<td>Rack for 30 bee tubes</td>
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<td>UV light-permeable plexiglass pyramid</td>
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<td>height = 30cm, 
apex 3.5 x 3.5cm, 
base 18x18cm</td>
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<td>Sticky tape</td>
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<td>Filter paper 0.16-0.2 mm thick</td>
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<td>MN 616 or MN615</td>
<td>Adjusted to circular pieces with a 1.3 cm diameter</td>
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<td>Disposable hypodermic needle</td>
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<td>B. Braun, Melsungen, Germany</td>
<td>4657683</td>
<td>0.45 mm diameter</td>
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behavioural pharmacology in classical conditioning of the proboscis extension response in honeybees (apis mellifera)

ミツバチ（ セイヨウミツバチ ） がよく彼らのコミュニケーションと向きのスキルのためにと彼らの印象的な学習能力の1,2のための知られている。ミツバチのコロニーの生存は、食料源の利用に依存するため、飼料収穫機ミツバチは、変数の花のサイトだけでなく、その収益性を学び、記憶する。飼料収穫機ミツバチは簡単に自然な設定で訓練することができますどこに彼らが餌場での飼料​​とそのような臭いや色など、関連する信号を学ぶ。食欲の連合学習は、エアコン、個別に活かさミツバチ3,4のテングザルの拡張子の応答（PER）によって実験室で制御された条件下で研究することができる。この学習のパラダイムでは、シンプルかつ信頼性の高い方法5-12に学習や記憶の形成の根底にある神経細胞および分子メカニズムの研究が可能になります。行動薬理学的なアプローチは、分子メカニズムを研究するために使用されます。薬物は、学習と記憶形成の13から16までの間または後に特定の分子の機能を妨害するために全身的に注入されています。 ここでは、PERコンディショニングに活かさミツバチを訓練する方法と、ミツバチの飛翔筋に全身に注入することにより薬物を適用する方法を示します。

Watch this Scientific Journal Video about Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees Apis mellifera at JoVE.com

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees Apis mellifera

我々は、薬物の全身アプリケーションによってミツバチの食欲の嗅覚コンディショニングのパラダイム（セイヨウミツバチ）の行動薬理学のメソッドを実装する方法を示します。 、このメソッドでは、メカニズムの基礎となる学習や、シンプルで信頼性の高い方法で、記憶形成の調査を可能にします。

ミツバチのテングザルの拡張レスポンスの古典的条件における行動薬理学（セイヨウミツバチ）

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the ...

behavioural-pharmacology-classical-conditioning-proboscis-extension

Research

JoVE Journal

Neuroscience

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees (Apis mellifera)

19.2K ビュー.  Freie Universität Berlin. 我々は、薬物の全身アプリケーションによってミツバチの食欲の嗅覚コンディショニングのパラダイム（セイヨウミツバチ）の行動薬理学のメソッドを実装する方法を示します。 、このメソッドでは、メカニズムの基礎となる学習や、シンプルで信頼性の高い方法で、記憶形成の調査を可能にします。

ビデオ: Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees Apis mellifera

Recording Large-scale Neuronal Ensembles with Silicon Probes in the Anesthetized Rat

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of behaviors from the spiking activity of its neurons. One of the most effective methods to monitor spiking activity from a large number of neurons in multiple local neuronal circuits simultaneously is by using silicon electrode arrays1-3.
Action potentials produce large transmembrane voltage changes in the vicinity of cell somata. These output signals can be measured by placing a conductor in close proximity of a neuron. If there are many active (spiking) neurons in the vicinity of the tip, the electrode records combined signal from all of them, where contribution of a single neuron is weighted by its 'electrical distance'. Silicon probes are ideal recording electrodes to monitor multiple neurons because of a large number of recording sites (+64) and a small volume. Furthermore, multiple sites can be arranged over a distance of millimeters, thus allowing for the simultaneous recordings of neuronal activity in the various cortical layers or in multiple cortical columns (Fig. 1). Importantly, the geometrically precise distribution of the recording sites also allows for the determination of the spatial relationship of the isolated single neurons4. Here, we describe an acute, large-scale neuronal recording from the left and right forelimb somatosensory cortex simultaneously in an anesthetized rat with silicon probes (Fig. 2).

Extracellular recordings of neuronal activity using silicon probes in the anesthetized rat will be described. This technique allows information to be obtained across multiple brain areas from more than 100 neurons simultaneously. It provides information with single cell resolution about neuronal ensembles dynamics in multiple local circuits.

麻酔ラットのシリコンプローブで大規模なニューロンアンサンブルの録音

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of ...

神経科学

Behavioral Determination of Stimulus Pair Discrimination of Auditory Acoustic and Electrical Stimuli Using a Classical Conditioning and Heart-rate Approach

Acute animal preparations have been used in research prospectively investigating electrode designs and stimulation techniques for integration into neural auditory prostheses, such as auditory brainstem implants1-3 and auditory midbrain implants4,5. While acute experiments can give initial insight to the effectiveness of the implant, testing the chronically implanted and awake animals provides the advantage of examining the psychophysical properties of the sensations induced using implanted devices6,7.
Several techniques such as reward-based operant conditioning6-8, conditioned avoidance9-11, or classical fear conditioning12 have been used to provide behavioral confirmation of detection of a relevant stimulus attribute. Selection of a technique involves balancing aspects including time efficiency (often poor in reward-based approaches), the ability to test a plurality of stimulus attributes simultaneously (limited in conditioned avoidance), and measure reliability of repeated stimuli (a potential constraint when physiological measures are employed).
Here, a classical fear conditioning behavioral method is presented which may be used to simultaneously test both detection of a stimulus, and discrimination between two stimuli. Heart-rate is used as a measure of fear response, which reduces or eliminates the requirement for time-consuming video coding for freeze behaviour or other such measures (although such measures could be included to provide convergent evidence). Animals were conditioned using these techniques in three 2-hour conditioning sessions, each providing 48 stimulus trials. Subsequent 48-trial testing sessions were then used to test for detection of each stimulus in presented pairs, and test discrimination between the member stimuli of each pair. 
This behavioral method is presented in the context of its utilisation in auditory prosthetic research. The implantation of electrocardiogram telemetry devices is shown. Subsequent implantation of brain electrodes into the Cochlear Nucleus, guided by the monitoring of neural responses to acoustic stimuli, and the fixation of the electrode into place for chronic use is likewise shown.

The application of a classical fear conditioning behavioral paradigm for auditory prosthetic research in rats is described. This paradigm provides a mechanism for identifying both detection of, and discrimination between, distinct acoustic and electrical stimuli using heart-rate as an outcome measure.

古典的条件とハートレート·アプローチを用いた聴覚の音響および電気刺激の刺激ペアの差別の行動決定

Acute animal preparations have been used in research prospectively investigating electrode designs and stimulation techniques for integration into neural ...

The Mouse Forced Swim Test

The forced swim test is a rodent behavioral test used for evaluation of antidepressant drugs, antidepressant efficacy of new compounds, and experimental manipulations that are aimed at rendering or preventing depressive-like states. Mice are placed in an inescapable transparent tank that is filled with water and their escape related mobility behavior is measured. The forced swim test is straightforward to conduct reliably and it requires minimal specialized equipment. Successful implementation of the forced swim test requires adherence to certain procedural details and minimization of unwarranted stress to the mice. In the protocol description and the accompanying video, we explain how to conduct the mouse version of this test with emphasis on potential pitfalls that may be detrimental to interpretation of results and how to avoid them. Additionally, we explain how the behaviors manifested in the test are assessed.

The forced swim test is validated as an experimental approach to assess potential antidepressant efficacy in rodents. Experimental animals are placed in a tank of water and escape-related mobility behavior is quantified. The common procedures for the mouse version of this test are described.

マウス強制水泳試験

The forced swim test is a rodent behavioral test used for evaluation of antidepressant drugs, antidepressant efficacy of new compounds, and experimental ...

The Tail Suspension Test

The tail-suspension test is a mouse behavioral test useful in the screening of potential antidepressant drugs, and assessing of other manipulations that are expected to affect depression related behaviors. Mice are suspended by their tails with tape, in such a position that it cannot escape or hold on to nearby surfaces. During this test, typically six minutes in duration, the resulting escape oriented behaviors are quantified. The tail-suspension test is a valuable tool in drug discovery for high-throughput screening of prospective antidepressant compounds. Here, we describe the details required for implementation of this test with additional emphasis on potential problems that may occur and how to avoid them. We also offer a solution to the tail climbing behavior, a common problem that renders this test useless in some mouse strains, such as the widely used C57BL/6. Specifically, we prevent tail climbing behaviors by passing mouse tails through a small plastic cylinder prior to suspension. Finally, we detail how to manually score the behaviors that are manifested in this test.

The tail-suspension test is validated as an experimental procedure to assess antidepressant efficacy of drug treatments in mice. Mice are suspended by their tails for six minutes and escape-related behaviors are assessed. We describe procedures used in conducting the tail suspension test.

尾懸垂テスト

The tail-suspension test is a mouse behavioral test useful in the screening of potential antidepressant drugs, and assessing of other manipulations that ...

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees (Apis mellifera L.)

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker bees spend the first half of their life inside the dark hive, where they are nursing the larvae or building the regular hexagonal combs for food (e.g. pollen or nectar) and brood3. The antennae are extraordinary multisensory feelers and play a pivotal role in various tactile mediated tasks4, including hive building5 and pattern recognition6. Later in life, each single bee leaves the hive to forage for food. Then a bee has to learn to discriminate profitable food sources, memorize their location, and communicate it to its nest mates7. Bees use different floral signals like colors or odors7, 8, but also tactile cues from the petal surface9 to form multisensory memories of the food source. Under laboratory conditions, bees can be trained in an appetitive learning paradigm to discriminate tactile object features, such as edges or grooves with their antennae10, 11, 12, 13. This learning paradigm is closely related to the classical olfactory conditioning of the proboscis extension response (PER) in harnessed bees14. The advantage of the tactile learning paradigm in the laboratory is the possibility of combining behavioral experiments on learning with various physiological measurements, including the analysis of the antennal movement pattern.

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees Apis mellifera L.

In this protocol we show how to condition harnessed honey bees to tactile stimuli and introduce a 2D motion capture technique for analyzing the kinematics of fine-scale antennal sampling pattern.

蜂蜜の蜂で触角サンプリング戦略の触覚エアコンと運動解析（<em&gt;ミツバチ</em&gt; L.）

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker ...

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.	
	RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.	
	The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception.

ミツバチにおけるRNAiによる二重遺伝子ノックダウンと味覚知覚測定（<em&gt;ミツバチ</em&gt;）

This video demonstrates novel techniques of RNA  interference (RNAi) which downregulate two genes simultaneously in honey bees  using double-stranded RNA ...

A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides.
We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.

The Proboscis Extension Response (PER) conditioning protocol, developed for the honey bee (Apis mellifera), provides an ecologically-relevant and easily quantifiable means for studying several different mechanisms of learning in many insect species.

昆虫に行動可塑性を調査するためのテングザルの拡張レスポンスプロトコル：基本、生物医学および農業研究への応用

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). ...

Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent and divergent anatomical connections including feed forward and feedback wiring. Furthermore, information of the same origin is partially sent via parallel pathways to different and sometimes into the same brain areas. To understand the evolutionary benefits as well as the computational advantages of these wiring strategies and especially their temporal dependencies on each other, it is necessary to have simultaneous access to single neurons of different tracts or neuropiles in the same preparation at high temporal resolution. Here we concentrate on honeybees by demonstrating a unique extracellular long term access to record multi unit activity at two subsequent neuropiles1, the antennal lobe (AL), the first olfactory processing stage and the mushroom body (MB), a higher order integration center involved in learning and memory formation, or two parallel neuronal tracts2 connecting the AL with the MB.&#160;The latter was chosen as an example and will be described in full. In the supporting video the construction and permanent insertion of flexible multi&#160;channel wire electrodes is demonstrated. Pairwise differential amplification of the micro&#160;wire electrode channels drastically reduces the noise and verifies that the source of the signal is closely related to the position of the electrode tip. The mechanical flexibility of the used wire electrodes allows stable invasive long&#160;term recordings over many hours up to days, which is a clear advantage compared to conventional extra&#160;and intracellular in vivo recording techniques.

Simultaneous extracellular long term recordings from two different brain neuropiles or two different anatomical tracts were established in honeybees. These recordings allow the investigation of temporal aspects of neuronal processing across different brain areas at the single neuron as well as at the ensemble level in a behaving animal.

行動するミツバチの二つの神経の処理段階での同時長期レコーディング

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent ...

Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the surface of the cornea, electrical activity generated in response to light can be measured and used to assess the activity of retinal cells in vivo. This manuscript describes the use of the ERG to measure visual function in zebrafish. Zebrafish have long been utilized as a model for vertebrate development due to the ease of gene suppression by morpholino oligonucleotides and pharmacological manipulation. At 5-10 dpf, only cones are functional in the larval retina. Therefore, the zebrafish, unlike other animals, is a powerful model system for the study of cone visual function in vivo. This protocol uses standard anesthesia, micromanipulation and stereomicroscopy protocols that are common in laboratories that perform zebrafish research. The outlined methods make use of standard electrophysiology equipment and a low light camera to guide the placement of the recording microelectrode onto the larval cornea. Finally, we demonstrate how a commercially available ERG stimulator/recorder originally designed for use with mice can easily be adapted for use with zebrafish. ERG of larval zebrafish provides an excellent method of assaying cone visual function in animals that have been modified by morpholino oligonucleotide injection as well as newer genome engineering techniques such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, all of which have greatly increased the efficiency and efficacy of gene targeting in zebrafish. In addition, we take advantage of the ability of pharmacological agents to penetrate zebrafish larvae to evaluate the molecular components that contribute to the photoresponse. This protocol outlines a setup that can be modified and used by researchers with various experimental goals.

We present a method for the electroretinographic (ERG) analysis of zebrafish larvae utilizing micromanipulation and electroretinography techniques. This is a simple and straightforward method for assaying visual function of zebrafish larvae in vivo.

ゼブラフィッシュ幼虫における視覚反応の電位図解析

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the ...

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize and to study. Therefore, bees have long been a favored model amongst neurobiologists and neuroethologists for studying the neural basis of social and natural behavior. It is important, however, that the experimental techniques used to study bees do not interfere with the behaviors being studied. Because of this, it has been necessary to develop a range of techniques for pharmacological manipulation of honey bees. In this paper we demonstrate methods for treating restrained or free-flying honey bees with a wide range of pharmacological agents. These include both noninvasive methods such as oral and topical treatments, as well as more invasive methods that allow for precise drug delivery in either systemic or localized fashion. Finally, we discuss the advantages and disadvantages of each method and&#160;describe common hurdles and how to best overcome them. We conclude with a discussion on the importance of adapting the experimental method to the biological questions rather than the other way around.

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

This manuscript describes several protocols for administering pharmacological agents to honey bees, including simple noninvasive methods for free-flying bees, as well as more invasive variants that allow precise localized treatment of restrained bees.

拘束と自由飛行ミツバチの神経薬理学的操作、<em&gt;アピスミツバチ</em

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize ...