私たちはミツバチに触覚学習パラダイムをご紹介してヨアヒムErberに感謝します。この作品はドイツのエクセレンス·イニシアチブの枠組みの中で賄わエクセレンス277 CITECのクラスター、サポートされていました。

1。蜂の準備<ol><li>採餌旅行から帰国しながら花の蜜や花粉飼料収穫機は、スクロースフィーダから、または直接ハイブの入り口から、どちらかのフィールドでキャッチされます。各シングル蜂は泡栓で閉じ、さらなる処理のための実験室にすぐに取られるガラスバイアル中に取り込まれます。 </li><li>彼らは不動の最初の兆候を示し°Cまでの研究室では、キャプチャされたミツバチは簡潔に4℃冷蔵庫で冷却される。 </li><li>各シングル固定化されたハチは、頭部と胸部の間に粘着テープで、腹部の上に小さな金属管に装着されています。ケアは、口吻とアンテナは自由に移動可能であるように注意してください。 </li><li>ビジョンを閉塞する白色塗料（ 例えば無溶媒ティペックス）の固定ミツバチの複眼と単眼をペイント。 </li><li>録音中にヘッドの動きを防ぐために、頭部と胸部の間にテープにそれを修正するために蜂の頭の後ろに溶けたワックスの小滴を追加。 </li><li>識別しやすくするために、テープ上の番号で各単蜂をマークし、脱水を防ぐために、湿気のある大気中に固定されたハチとチューブを配置します。 </li><li>シリンジを用いて提示され、30％ショ糖溶液の液滴と5秒間、各単一蜂を養うために、触覚コンディショニングプロトコルで始まる前に、すべてのハチが30分間回復させる。 </li></ol> 2。触覚コンディショニング<ol><li>エアコンの前に、各単蜂がアンテナに適用し、30％スクロース刺激に対する口吻拡張応答（PER）のためにテストする必要があります。それによって、口吻の先端が開いた下顎骨の間に仮想回線を横断する必要があります。ショ糖刺激にPERで応答しないすべてのハチを捨ててください。 </li><li>触覚のコンディショニングのために条件刺激（CS）のように、150μmの波長と回折格子を形成し、 例えば 、平滑または刻まれたパターンが水平方向または垂直方向の溝と真鍮のキューブ（ 例えば 3×5 mm）を使用しています。 Fまたは無条件刺激（US）は、30％ショ糖溶液（家庭用の砂糖、水で希釈した）を使用します。 </li><li>真鍮キューブ（CS）は、コンディショニング手順の間に正確な位置決めを確実にするために（ 例えば 、MärzhäuserMM33）マイクロマニピュレータにホルダーに置かれます。米国は、30％ショ糖溶液が充填されたシリンジを持つ蜂に提示されます。 </li><li>条件付けの手続きは5分の間トライアル間隔（ITI）と触覚刺激（CS）とショ糖溶液（米国）の5ペアで構成されています。マウントされた触覚刺激（CS）を持つマイクロマニピュレータの前に単一蜂を置きます。触覚刺激の表面がハチの頭（ 図1およびB）に平行になるようにゆっくりと、CSを配置します。動物と触覚物体の間の距離は、試験ハチの触角の作業半径の範囲内である必要があり、ミツバチが快適な位置wに触覚刺激をスキャンすることができる必要があり、すなわちi番目のアンテナの両方。 図1に示す例では、距離は3mmであった。蜂が5秒間触覚刺激（CS）をスキャンしてみましょう。最初の3秒後に、30％ショ糖（米国）長い鼻の下にシリンジで溶液の液滴を提示します。優しく口吻を上げるために注射器の先端を使用しています。長い鼻の下スクロース刺激が15％無条件を引き出すだろう。蜂はスクロース報酬をなめるすることができます。空調時の正確な時間間隔を維持するためにアラーム信号とストップウォッチを使用しています。 </li><li>エアコンPERはミツバチの学習成功のための尺度として用いられる。第一報わペアリングした後に、ミツバチは、彼らが今度の報酬を期待することを示し、それらの口吻の伸長によってCSのプレゼンテーションへの対応を開始します。完全に伸びた口吻は、触覚刺激提示の3秒の時間窓の間、ショ糖プレゼンテーションが肯定的な反応として採点される前に、いつでも観察した。応答が負の計測されません。 OCCPERのurrenceは、実験者が留意しなければならない。 CSのプレゼンテーション中にPERを示すミツバチの割合は、各試験ごとにプロットされています。 </li></ol> 3。キネマ録音<ol><li>シングル活かしハチの触角の動きは、適切なマクロレンズ（ 例えば TECHSPEC VZM 200装備バスラーA602f-2は、火災のワイヤ接続を経由して50 fpsで運営）を使用して、デジタルビデオカメラで記録されます。カメラはトップダウンビューでは、動物（ 図1）の上方に位置している。 図1では、録音の空間分解能は0.02mm /ピクセルです。 </li><li>カメラの目標の下、異なる向きから1mmの辺の長さと10×10mmの市松模様の画像をまとめて記録することにより、カメラのキャリブレーションを行います。キャリブレーションは、カメラキャリブレーションツールボックスMatlabの16で行うことができます。 </li><li>カメラレンズの下に単一の固定された蜂を置きます。マニピュレーターに固定され、触覚刺激を提示、動物に。触覚のコンディショニングのために説明したのと同じ方法で進行し、蜂がオブジェクトをスキャンしている間に触角の動きを記録。それは完全な触角作業範囲と触覚刺激が目に見えるものであることが重要です。刺激パターンの選択肢がテストされ、試験番号は、実験的なデザインによって異なります。 </li></ol> 4。データ解析<ol><li>画像背景の計算は、Matlabで行われます。まず、時間をかけて中央値グレースケール値は、画素毎に算出する必要があります。静的オブジェクトは、ハチと触覚刺激の固定ヘッドのように、画像の背景を構成することになる。移動するオブジェクトは、二つのアンテナのように、背景の一部にはなりません。 </li><li>記録されたビデオの各フレームを順次ロードする必要があり、現在のフレームと画像の背景の違いは計算しなければならない。この減算の結果は、フレームからフレームへと移動している画像の部分を強調している。イドeally、ハチの触角は、ゼロ以外の値（ 図1C）の唯一の領域である。 </li><li>さらに処理するために、ゼロ以外のピクセル値を持つ2つの最大の領域はアンテナであると想定されます。両方のアンテナ用のバイナリマスクピクセルがアンテナと値以外の場合は0に属している場合、各画素ごとに値1を含む生成されるべきである。このマスクは後で触角ヒントをローカライズするための基礎としての役割を果たす。画像全体の予備のマスクを得るために、差分画像の各ピクセルのグレイ値は、事前に定義されたしきい値と比較されます。我々はノイズの世話をするので、後でこのしきい値は非常に低くなるように選択される。まず、画像ノイズ小領域に起因まだマスクの一部です：予備マスクがまだエラーの2種類を搭載しています。第二に、アンテナに属している領域は、必ずしも完全に接続されない場合があります。背景はほぼアンテナと同じ発光を持っている場合、後者は主に発生します。これらの影響を排除するために、標準画像処理モルフォロジー演算が適用され、標準のMATLAB関数はimerodeと（詳細な説明については、17頁158から205を参照）をそれぞれIMDILATE頼る、画像の侵食と拡張の組み合わせ、 すなわち 。ノイズ除去処理の後にアンテナの仮説は、まだ同じマスク内に含まれています。したがって、次のステップとしてバイナリーマスクは標準のMATLAB関数bwlabel（画像セグメンテーションアルゴリズムの詳細については17頁40から48を参照）を使用してばらばらの分野では、クラスタ化されています。 </li><li>クラスタあたりのピクセル数がカウントされ、2つの最大のクラスタが選択されています。重心は左右のアンテナ（ 図1D）を区別するために計算されます。 </li><li>各アンテナの触角の先端が近位から遠位方向（ 図1D参照 ）の中で最も大きい数値を使用してクラスタ内のピクセルとして定義することができます。 </li></ol>

<ol>
	<li>Page, R. E., Scheiner, R., Erber, J., Amdam, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+and+evolution+of+division+of+labor+and+foraging+specialization+in+a+social+insect+(Apis+mellifera++L.).">The development and evolution of division of labor and foraging specialization in a social insect (Apis mellifera L.).</a> Curr. Top. Dev. Biol. 74, 253-286 (2006).</li><li>Menzel, R., M&#252;ller, 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+and+memory+in+honeybees:+From+behavior+to+neural+substrates.">Learning and memory in honeybees: From behavior to neural substrates.</a> Annu. Rev. Neurosci. 19, 379-404 (1996).</li><li>Seeley, T. D. <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 wisdom of the hive. , (1995).</li><li>Staudacher, E., Gebhardt, M. J., D&#252;rr, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antennal+movements+and+mechanoreception:+neurobiology+of+active+tactile+sensors.">Antennal movements and mechanoreception: neurobiology of active tactile sensors.</a> Adv. Insect Physiol. 32, 49-205 (2005).</li><li>Martin, H., Lindauer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sinnesphysiologische+Leistungen+beim+Wabenbau+der+Honigbiene.">Sinnesphysiologische Leistungen beim Wabenbau der Honigbiene.</a> Z. Vergl. Physiol. 53, 372-404 (1966).</li><li>Kevan, P. G., Menzel, R., Mercer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Texture+sensitivity+in+the+life+of+honeybees.">Texture sensitivity in the life of honeybees.</a> Neurobiology and behavior of honeybees. , 96-101 (1987).</li><li>von Frisch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Tanzsprache und Orientierung der Bienen. , (1965).</li><li>Waser, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flower+constancy:+definition,+cause,+and+ceasurement.">Flower constancy: definition, cause, and ceasurement.</a> Am. Nat. 127, 593-603 (1986).</li><li>Kevan, P. G., Lane, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flower+petal+microtexture+is+a+tactile+cue+for+bees.">Flower petal microtexture is a tactile cue for bees.</a> Proc. Natl. Acad. Sci. U.S.A. 82, 4750-4752 (1985).</li><li>Erber, J., Kierzek, S., Sander, E., Grandy, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tactile+learning+in+the+honeybee.">Tactile learning in the honeybee.</a> J. Comp. Physiol. A. 183, 737-744 (1998).</li><li>Kisch, J., Erber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Operant+conditioning+of+antennal+movements+in+the+honey+bee.">Operant conditioning of antennal movements in the honey bee.</a> Behav. Brain. Res. 99, 93-102 (1999).</li><li>Scheiner, R., Erber, J., Page, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tactile+learning+and+the+individual+evaluation+of+the+reward+in+honey+bees+(Apis+mellifera+L.).">Tactile learning and the individual evaluation of the reward in honey bees (Apis mellifera L.).</a> J. Comp. Physiol. A. 185, 1-10 (1999).</li><li>Erber, J., Scheiner, R., Adelman, G., Smith, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Honeybee+learning.">Honeybee learning.</a> Encyclopedia of Neuroscience. , (2004).</li><li>Bitterman, M. E., Menzel, R., Fietz, A., Sch&#228;fer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classical+conditioning+of+proboscis+extension+in+honeybees+(Apis+mellifera).">Classical conditioning of proboscis extension in honeybees (Apis mellifera).</a> J. Comp. Psychol. 97, 107-119 (1983).</li><li>Dacher, M., Gauthier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+NO-synthase+and+nicotinic+receptors+in+learning+in+the+honey+bee.">Involvement of NO-synthase and nicotinic receptors in learning in the honey bee.</a> Physiol. Behav. 95, 200-207 (2008).</li><li>Haralick, R. M., Shapiro, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Computer and Robot Vision. I, (1992).</li><li>Erber, J., Galizia, C. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tactile+antennal+learning+in+the+honey+bee.">Tactile antennal learning in the honey bee.</a> Honeybee Neurobiology and Behavior. , 439-455 (2012).</li><li>Pankiw, T., Page, R. E. <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+pheromones,+hormones,+and+handling+on+sucrose+response+thresholds+of+honey+bees+(Apis+mellifera+L.).">Effect of pheromones, hormones, and handling on sucrose response thresholds of honey bees (Apis mellifera L.).</a> J. Comp. Physiol. A. 189, 675-684 (2003).</li><li>Harris, J. W., Woodring, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+stress,+age,+season+and+source+colony+on+levels+of+octopamine,+dopamine+and+serotonine+in+the+honey+bee+(Apis+mellifera+L.)+brain.">Effects of stress, age, season and source colony on levels of octopamine, dopamine and serotonine in the honey bee (Apis mellifera L.) brain.</a> J. Insect. Physiol. 38, 29-35 (1992).</li><li>Scheiner, R., Kuritz-Kaiser, A., Menzel, R., Erber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+responsiveness+and+the+effects+of+equal+subjective+rewards+on+tactile+learning+and+memory+of+honeybees.">Sensory responsiveness and the effects of equal subjective rewards on tactile learning and memory of honeybees.</a> Learn. Mem. 12, 626-635 (2005).</li><li>Mujagic, S., Sarkander, J., Erber, B., Erber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sucrose+acceptance+and+different+forms+of+associative+learning+of+the+honey+bee+(Apis+mellifera+L.)+in+the+field+and+laboratory.">Sucrose acceptance and different forms of associative learning of the honey bee (Apis mellifera L.) in the field and laboratory.</a> Front. Behav. Neurosci. 4, 46 (2010).</li><li>Scheiner, R., Barnert, M., Erber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+in+water+and+sucrose+responsiveness+during+the+foraging+season+affects+proboscis+extension+learning+in+honey+bees.">Variation in water and sucrose responsiveness during the foraging season affects proboscis extension learning in honey bees.</a> Apidologie. 34, 67-72 (2003).</li><li>Mujagic, S., Erber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sucrose+acceptance,+discrimination+and+proboscis+responses+of+honey+bees+(Apis+mellifera+L.)+in+the+field+and+the+laboratory.">Sucrose acceptance, discrimination and proboscis responses of honey bees (Apis mellifera L.) in the field and the laboratory.</a> J. Comp. Physiol. A. 195, 325-339 (2009).</li><li>Friedrich, A., Thomas, U., M&#252;ller, 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><li>Erber, J., Pribbenow, B., Grandy, K., Kierzek, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tactile+motor+learning+in+the+antennal+system+of+the+honeybee+(Apis+mellifera+L.).">Tactile motor learning in the antennal system of the honeybee (Apis mellifera L.).</a> J. Comp. Physiol. A. 181, 355-365 (1997).</li><li>Lambin, M., D&#233;glise, P., Gauthier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antennal+movements+as+indicators+of+odor+detection+by+worker+honeybees.">Antennal movements as indicators of odor detection by worker honeybees.</a> Apidologie. 36, 119-126 (2005).</li></ol>

特別な利害関係は宣言されません。

ミツバチの調製：ミツバチの収集と固定は、動物の低応力レベルを維持するために、迅速に行われるべきである。ストレスがPER-反応性に影響を与え、したがって、蜂19、20の学習パフォーマンスに間接的な影響を及ぼす可能性があります。ストレスレベルはすぐにそれらを固定するために収集した直後に直接氷の上でハチとガラスバイアルを配置することによって減少さ...

<table border="1"><tbody><tr><td> 試薬/材料の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td>花粉飼料収穫機、 ミツバチ L. </td><td></td><td></td><td>プライベートじんましん</td></tr><tr><td>ガラスバイアル（22×45ミリメートル） </td><td>カール·ロート社＆株式会社</td><td> X655.1 </td><td></td></tr><tr><td>黄銅管、10×30ミリメートル</td><td></td><td></td><td>自分自身で製作した</td></tr><tr><td>真鍮キューブ、3×5ミリメートル</td><td></td><td></td><td>自分自身で製作した</td></tr><tr><td>ティップ-EX（無溶剤型） </td><td>ティペックス社＆CO KG </td><td></td><td></td></tr><tr><td>ショ糖溶液（30％） </td><td></td><td></td><td>水で希釈した家庭用砂糖</td></tr><tr><td>エタノール（70％） </td><td>カール·ロート社＆株式会社</td><td> 9065.3 &lt;/ TD&gt; <td></td></tr><tr><td>シリンジ（1ml）と針（0.6×25） </td><td>カール·ロート社＆株式会社</td><td> 59.1 </td><td></td></tr><tr><td>ストップウォッチ</td><td>カール·ロート社＆株式会社</td><td> L423.1 </td><td></td></tr><tr><td>極微操作装置</td><td> MärzhäuserMM33 </td><td> 00-42-101-0000 </td><td></td></tr><tr><td>デジタルビデオカメラ</td><td>バスラーA602f-2 </td><td></td><td></td></tr><tr><td>カメラ用マクロレンズ</td><td> TECHSPEC VZM 200 </td><td></td><td></td></tr><tr><td> MATLAB R2009b </td><td> MathWorks社</td><td></td><td></td></tr></tbody></table>

tactile conditioning and movement analysis of antennal sampling strategies in honey bees (apis mellifera l.)

ミツバチ（ セイヨウミツバチ L.）は真社会性昆虫であるとよく労働と連想学習能力1、2のその複雑な除算で知られています。働き蜂は、彼らが幼虫の介護や食品（ 例えば花粉や花の蜜）とひな3の正六角形の櫛を構築している暗いハイブ、内側に自分の人生の最初の半分を費やしています。アンテナは臨時五感触手あり、ハイブビル5と6パターン認識を含む様々な触覚媒介タスク4、極めて重要な役割を果たす。その後の人生で、それぞれ単一の蜂が食べ物をあさるにハイブを残す。すると蜂は、収益性の高い食品のソースを区別その場所を記憶し、7その巣の仲間にそれを伝えるために学ばなければならない。蜂は食料源の多感覚記憶を形成するために、花弁表面9から色や臭い、7,8のような別の花の信号だけでなく、触覚的手がかりを使用しています。研究室の共同の下nditionsは、ミツバチは、それらのアンテナ10、11、12、13とエッジや溝などの触覚のオブジェクト機能を、区別するために食欲の学習パラダイムに訓練することができます。この学習パラダイムは密接に活かさ蜂14でテングザルの拡張応答（PER）の古典的な嗅覚のコンディショニングに関連しています。実験室における触覚学習パラダイムの利点は、触角運動パターンの分析を含む様々な生理学的測定と学習に行動実験を組み合わせることの可能性である。

以下の実験では、触覚学習がミツバチで触角サンプリング動作にどのように影響するかを検討した。このため、我々は触覚刺激のプレゼンテーションの実行前と実行中ナイーブおよびエアコン蜂で触角の先端の動きを監視した。 まず、花粉飼料収穫機のグループの自発的な触角の動き（N = 42）は、1分間記録した。ミツバチの半分（N = 21）は、その後、30％スクロース報?...

Watch this Scientific Journal Video about Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees Apis mellifera L. at JoVE.com

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

このプロトコルでは、条件に触覚刺激にミツバチを活かしたとファインスケール触角サンプリングパターンの運動を解析するための2Dモーションキャプチャ技術を導入する方法を示しています。

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 ...

tactile-conditioning-movement-analysis-antennal-sampling-strategies

Research

JoVE Journal

Neuroscience

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

10.5K ビュー.  Bielefeld University. このプロトコルでは、条件に触覚刺激にミツバチを活かしたとファインスケール触角サンプリングパターンの運動を解析するための2Dモーションキャプチャ技術を導入する方法を示しています。

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

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

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the survival of a honeybee colony depends on the exploitation of food sources, forager bees learn and memorize variable flower sites as well as their profitability. Forager bees can be easily trained in natural settings where they forage at a feeding site and learn the related signals such as odor or color. Appetitive associative learning can also be studied under controlled conditions in the laboratory by conditioning the proboscis extension response (PER) of individually harnessed honeybees3,4. This learning paradigm enables the study of the neuronal and molecular mechanisms that underlie learning and memory formation in a simple and highly reliable way5-12. A behavioral pharmacology approach is used to study molecular mechanisms. Drugs are injected systemically to interfere with the function of specific molecules during or after learning and memory formation13-16.
Here we demonstrate how to train harnessed honeybees in PER conditioning and how to apply drugs systemically by injection into the bee flight muscle.

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

We demonstrate how to implement a behavioral pharmacology method in an appetitive olfactory conditioning paradigm in honeybees (Apis mellifera) by systemic application of drugs. This method allows investigation of the mechanisms underlying learning and memory formation in a simple and reliable way.

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 ...

神経科学

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

複数のスケールでデジタル化された神経形態の自動化されたショールの分析

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned to completely random. In addition, fibers can be spun from a variety of materials, including biodegradable polymers such as poly-L-lactic acid (PLLA). These characteristics make electrospun fibers suitable for a variety of scaffolding applications in tissue engineering. Our focus is on the use of aligned electrospun fibers for nerve regeneration. We have previously shown that aligned electrospun PLLA fibers direct the outgrowth of both primary sensory and motor neurons in vitro. We maintain that the use of a primary cell culture system is essential when evaluating biomaterials to model real neurons found in vivo as closely as possible. Here, we describe techniques used in our laboratory to electrospin fibrous scaffolds and culture dorsal root ganglia explants, as well as dissociated sensory and motor neurons, on electrospun scaffolds. However, the electrospinning and/or culture techniques presented here are easily adapted for use in other applications.

Aligned electrospun fibers direct the growth of neurons in vitro and are a potential component of nerve regeneration scaffolds. We describe a procedure for preparing electrospun fiber substrates and the serum-free culture of primary rat E15 sensory (DRG) and motor neurons. Visualization of neurons by immunocytochemistry is also included.

一次運動とエレクトロポリ- L -乳酸ナノファイバー足場上で定義されているメディアの感覚ニューロンの文化

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned ...

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

の臭気誘発応答のカルシウムイメージングショウジョウバエ触角葉

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

Electrophysiological Measurements and Analysis of Nociception in Human Infants

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are based on behavioural and physiological body reactions, such as crying, body movements or changes in facial expression. While these measures demonstrate that infants mount a response following noxious stimulation, they are limited: they are based on activation of subcortical somatic and autonomic motor pathways that may not be reliably linked to central sensory processing in the brain. Knowledge of how the central nervous system responds to noxious events could provide an insight to how nociceptive information and pain is processed in newborns.
The heel lancing procedure used to extract blood from hospitalised infants offers a unique opportunity to study pain in infancy. In this video we describe how electroencephalography (EEG) and electromyography (EMG) time-locked to this procedure can be used to investigate nociceptive activity in the brain and spinal cord.
This integrative approach to the measurement of infant pain has the potential to pave the way for an effective and sensitive clinical measurement tool.

The assessment and treatment of pain in infants is difficult because infants cannot verbally report their experience. In this video we describe quantitative electrophysiological methods and analysis techniques that can be used to measure the response to noxious events from the infant nervous system.

電気生理学的測定とヒト幼児における痛覚の分析

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are ...

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by B&ouml;lcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 &deg;C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of B&ouml;lcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Alm&aacute;si et al 5 and B&ouml;lcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

糖尿病ラットの熱侵害受容閾値の迅速定量

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

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 ...

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 ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

胎児アルコール スペクトルの無秩序のモデルラットにおける海馬機能障害を評価するためにトレース瞬目反射条件づけの使用

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Drosophila Courtship Conditioning As a Measure of Learning and Memory

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model organisms such as the fruit fly, Drosophila melanogaster. Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male&#39;s copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to na&#239;ve flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized here. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms.

Drosophila Courtship Conditioning As a Measure of Learning and Memory

This protocol describes a Drosophila learning and memory assay called courtship conditioning. This classic assay is based on a reduction of male courtship behavior after sexual rejection by a non-receptive premated female. This natural form of behavioral plasticity can be used to test learning, short-term memory, and long-term memory.

ショウジョウバエの訓練と記憶の尺度としてのコンディショニング

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model ...