著者は、実験を支援するエリンFennernに感謝したいと思います。この作品は、ノルウェーの研究評議会（180504、185306及び191699）、ピュー慈善トラスト、および国立老化研究所（NIA P01 AG22500）によって賄われていた。

パート1：RNAiによる二重の遺伝子ノックダウン 1。 dsRNAを合成<ol><li> VG、USPとミツバチのゲノムに含まれていない緑色蛍光タンパク質（GFP）をコードする制御遺伝子を標的dsRNAをの設計プライマー：プライマーはオンラインフリーソフトウェアPrimer3のを（使用して設計された<a href="http://frodo.wi.mit.edu/" target="_blank">http://frodo.wi.mit.edu/</a> ） 。 </li><li> VG、USPとGFPのためのdsRNA合成：in vitro転写用プロメガからの使用RiboMax T7 RNAの生産システム。 </li><li> dsRNAの精製： <ol><li>変性および再生：85への熱のdsRNAを℃で5分間、それを1時間室温で冷ましてみましょう。 </li><li>のDNase I処理：それぞれのdsRNA反応に1μlのDNアーゼIを追加し、チューブをフリックでそれらをミックス。 37℃で15分インキュベート</li><li>各反応にLS、そして優しくINVでそれらをミックス - 150μlのヌクレアーゼフリー水と750μlのトリゾールを追加チューブerting。 30℃で5分のためのサンプルをインキュベート</li><li>各サンプルに200μlのクロロホルムを追加します。 20秒間激しく混ぜる。 4℃12,000×gで15分間サンプルを遠心℃に</li><li>各新しいチューブに上清位相を移し、各チューブに500μlのイソプロピルアルコールを追加します。反転チューブでそれらをミックス。 -20℃で20分間混合物をインキュベート4℃で12,000×gで10分間、それを遠心</li><li>上清を除去し、ペレットを洗浄するために1,000μlの75％エタノールを使用しています。 C、空気乾燥したdsRNAはペレット4℃で7,500×gで5分間チューブを遠心分離した後、ペレットを溶解するヌクレアーゼフリーの水を使用しています。遺伝子の効能ダウンレギュレーションを確保するためには、dsRNA濃度は9 -10μgの/μlの周りでなければなりません。 </li></ol></li></ol> 2。 dsRNAは腹部注射 1）単回注射：ミックス二つの遺伝子のdsRNAと麟蹄二重遺伝子ノックダウンのために、2つの戦略がありますカラットは、混合物、2）2日間注射：初日に一つの遺伝子を標的とする最初のdsRNAを注入し、二日目の同じ蜂に第二のdsRNAを注入。 <ol><li>寒さは、新たに1〜2分のために4℃冷蔵庫にミツバチを浮上した。ミツバチが固定化されている場合、それらの腹部とthoraxの複数形の間で交差虫ピンで固形ワックスの完全ペトリ皿上で並列に3-4ミツバチをマウントします。 </li><li> 4℃の冷蔵庫に再びミツバチを冷やす。ミツバチは完全に不動であることを確認してください。それは、約1〜2分かかります。ミツバチが緩んでいるはずです。ミツバチが丸まったりねじ曲げなった場合、それは彼らがあまりにも長い間避けるべきであるどの冷やしていることを意味します。なごむはあまり高い死亡率の原因となるが、どんな動きが傷や死亡のサイズが大きくなりますように、完全な固定化は重要です。 </li><li>ハミルトンマイクロシリンジで使い捨て30 G針（BD）を入れます。 3μlのdsRNAのを取ると、シリンジ内に気泡がないことを確認してください。針は玉を避けるために、腹部の側面に挿入される内臓パトン。 dsRNAは、吸収されるように徐々にシリンジプランジャを押す。 dsRNAは、非常に粘性があるので、それは完全にシリンジから追放する2-3秒かかります。完全にプランジャーを押し下げた後、4,5秒のために傷口に針を残す。注射後3-5秒間ミツバチを観察します。傷口から血リンパ液滴が漏れた場合は、蜂を捨てる。 </li><li>さまざまな治療法に対応した彼らのthoraxの複数形をマークするために異なる色を使用してください。シングル注入戦略が使用された場合、この時点で、蜂、1時間の観察の後にコロニーに戻って配置することができる。しかし、2日間の注入戦略は最初のdsRNAを注射された後、使用された場合、ミツバチは蜂蜜シリンダメッシュケージに置かれるべき側に供給され、34のインキュベーターで保たれる℃、湿度80％ 。 </li><li>二日目は、メッシュケージにミツバチを冷やし、ワックスプレートにマウントするためのステップ2.2および2.3に従ってください。それはdescriあるように、第2のdsRNAで注射を行うステップ2.4​​のベッド。 </li><li>ミツバチが1時間室温で回復し、植民地にそれらを紹介しましょう​​。 </li></ol>注：ノックダウン効果を定量的RT-PCR（定量RT-PCR）およびウェスタンブロットによって検出することができる。一般的には、ノックダウン効果が変化し、多くの要因に依存する。我々の研究では、二重の遺伝子ノックダウンは7日、シングル注入と二日間の注射後に検出することができます。 パート2：テングザル拡張応答（PER）アッセイ 1。ハイブから集めるミツバチ注射後6日目に、喫煙者を使用せずに、コロニーからの処理ミツバチを収集します。金属メッシュシリンダーケージでミツバチを収集します。各ケージに3ミツバチを保つ。 2。 PER用取付ミツバチ彼らが完全に固定化されている°Cまで4でミツバチを冷やす。特別にPERのために設計プラスチックチューブに垂直各蜂をマウントします。ボイジャーによって管内腹部を保つgの二つの小さなテープ片、チューブから突き出頭を維持。小さなプラスチック製のカラムの2行を持つラックにチューブを入れて、ID番号で各チューブ（蜂）を割り当てます。その後、インキュベーター（34℃、湿度80％）にラックで治療ミツバチを入れて、2時間インキュベーターに保管してください。 3。ショ糖水溶液のシリーズの準備 0％、0.1％、0.3％、1％、3％、10％及び30％スクロース水溶液を調製し、15〜20 G針とシリンジにソリューションを読み込む。 4。アッセイPER 0.1％、0.3％、1％、3％、10％および30％スクロース水溶液に続いて0％溶液（水）の液滴と蜂のアンテナをタッチします。最初に水ですべてのミツバチをテストし、各ソリューション間の間隔として、少なくとも2分が使用されていることができます。したがって、我々は、通常、各試験のために少なくとも20蜂を持っている。ハチが完全に彼女の口吻を拡張する場合、肯定応答がカウントされます。 5。データ管理味覚応答スコア（GRS）のために、個々の総陽性反応をまとめる。データに適切な統計分析を実行します。 パート3。遺伝子ノックダウンの検証脂肪体は7日齢扱わミツバチの別のセットから解剖されています。標準トリゾール手順は、DNアーゼI処理24が行われるRNA抽出のために使用される。Vgとし、USPの遺伝子発現は二段階定量RT-PCRによって分析される。 アクチンは、それが、異なる組織で安定した式を有するので、参照遺伝子として用いられるミツバチの。 VG及びUSP遺伝子の公表リアルタイムPCRプライマーは、24本実験で使用されている。データは、デルタデルタCT法25を用いて分析される。

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+genomics+of+honeybee+foraging+and+nest+defense.">Behavioral genomics of honeybee foraging and nest defense.</a> Naturwissenschaften. 94, 247-267 (2007).</li><li>Pankiw, T., Waddington, K. D., 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=Modulation+of+sucrose+response+thresholds+in+honey+bees+(Apis+mellifera+L.):+influence+of+genotype,+feeding,+and+foraging+experience.">Modulation of sucrose response thresholds in honey bees (Apis mellifera L.): influence of genotype, feeding, and foraging experience.</a> J. Comp. Physiol. A. 187, 293-301 (2001).</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. Neuroethol. Sens. Neural. Behav. Physiol. 189, 675-684 (2003).</li></ol>

We have nothing to disclose.

我々の研究は、同時に成虫に二つの遺伝子をノックダウンするための最初の取り組みです。我々の結果は、dsRNAを注射（単回注射と二日間の注射）の2つの戦略は、二重遺伝子抑制に有効であることを示している、とVGとUSPの同時遺伝子サイレン蜂8,9に6日間dsRNAを注射した後にテストすることができます。しかし、遺伝子ノックダウンの効果は、細胞や臓器によって標的遺...

RNA干渉（RNAi）は、真核生物の多種多様に発生するRNAベースの転写後遺伝子サイレンシングである。 RNAiのプロセスは、内因性又は外因性の二本鎖RNA（dsRNA）前駆物質によって引き起こされる。 dsRNAは、結合して小さな断片（20〜25塩基対）へのdsRNAを切断するリボヌクレアーゼタンパク質ダイサーを活性化する。 dsRNAのガイドの、小さな断片アルゴノートタンパク質による補完的なmRNAの認識と切断、RNA誘導サイレンシング複合体（RISC）1の触媒成分。哺乳類では、30ヌクレオチドより長いdsRNAはは、RNA転写産物2の非特異的な劣化につながる抗ウイルス応答（インターフェロン応答、IFN）を活性化する。しかし、長いdsRNAは、このIFN 3の不足しているため、昆虫に有効かつ特異的であることが証明されている。 長いdsRNAは、異なる昆虫種の標的遺伝子の下方制御に使用されてきた。ミツバチは、パイオンの一つです発達や行動の多くの重要な遺伝子の機能がdsRNAは4,5を使用することによって明らかにされているEER昆虫生物。いくつかのdsRNAの発送方法はミツバチで行われています：dsRNAをインジェクションはミツバチの胚4と成体ミツバチ7,8の遺伝子ノックダウンのための効果的なアプローチであるのに対し、dsRNAを送り、効率的に、ミツバチの幼虫6に標的遺伝子の発現を下方制御する。 昆虫へのdsRNAを適用することにより、展示遺伝子ノックダウン効果は一過性とローカライズされています。研究では、腹部のdsRNA注射胸部のdsRNA注射の両方を効果的に昆虫9,10の腹部脂肪体細胞において標的遺伝子の発現を抑制することが示されている。 dsRNAは、細胞が10を浴びている血リンパからのdsRNAを取ることができている腹部や胸部の空洞や脂肪体細胞内に注入される。しかし、そのような卵巣や脳などの他の臓器、の遺伝子のいずれかが対象とすることはできません腹部や胸部注射。ミツバチの脳内の遺伝子を標的とするためには、dsRNAの脳の注入は効果的局所脳領域11内の標的遺伝子の発現に影響を及ぼす、行われている。ここで、我々は唯一の、より一般的に成人のミツバチで使用されている腹部のdsRNA注入を文書化します。 RNAiは、主に単一遺伝子を標的とするために使用されており、遺伝子の機能を明らかにするための強力なツールとなっている。しかし、任意の遺伝子は他から絶縁されていません、それは複雑な制御ネットワークである。生物学的プロセスを理解するための鍵は、遺伝子が複数の遺伝子の同時操作ではなく、単一の遺伝子ノックダウンを必要とする、どのように相互作用するかを詳細に分析することである。哺乳動物細胞株において、科学者は、送達システム12またはマルチマイクロRNA（miRNAの）ヘアピンのデザイン13を用いて同時に阻害する二、三の遺伝子に成功した。しかし昆虫で、複数の遺伝子ノックダウンはまだ未検証です。ここでは、プレゼンテーション二重の遺伝子ノックダウンを達成することができますトンの異なる注入戦略。卵黄タンパク質前駆体をコードしてビテロゲニン（VG）、及び幼若ホルモン（JH）のため推定受容体をコードし、ミツバチにJH 14への応答を仲介する転写因子として働くことができるピラクル（USP）：我々は2つの遺伝子をターゲットにしています。 VGとJHは、フィードバックループ15でお互いを調節し、ミツバチの行動規制9に関与している。二重の遺伝子ノックダウンを使用して、我乱すVgを両方とJH経路、そして、彼らが共同でミツバチの行動と生理機能とどのようにVG、USPとJHは9対話にどのように影響するかを発見する。 味覚認識はミツバチ社会的行動のための16の行動予測因子である。高い味覚認知と行動の発達、巣のミツバチ行動成熟速く、通常人生の早い段階で飼料の観点と花粉16,17を収集することを好む。ものの規制M味覚認識を根底echanismsはまだ不明で、研究は味覚知覚は内部エネルギー代謝9、ホルモン分泌18,19及び生物遺伝アミンにリンクされていることが示されている20の経路。 VgはとJHの両方が変調味覚認知7,21重要なホルモンのレギュレータです。研究室では、ミツバチで味覚知覚の変化が異なるショ糖溶液に口先拡張応答（PER）をテストすることによって評価することができます。各蜂、0.1、0.3、1、3、10、30％スクロースの上昇濃度系列に続いて、水の液滴と彼女のアンテナの両方に触れることによってテストされる。水またはスクロースの液滴は、各アンテナに触れたときのハチが完全に彼女の口吻を拡張する場合は正の応答が注目される。ソリューションに対する肯定応答の数に基づいて、各個体の味覚知覚レベルが16を決定することができる。しかし、PERの適用はグラム測定に限定されるものではないustatory認識。 PERはまた、このような飽食対飢餓としてミツバチの代謝状態をテストするための有効な方法である。ショ糖へのより大きな応答とミツバチは、一般的にハングリー（王とAmdam、未発表データ）である。また、PERパラダイムはミツバチに連想学習と記憶にも使用することができます。このケースではミツバチは臭気とショ糖水の存在を関連付けるために訓練されます。ミツバチが関連を学ぶとき、臭気の唯一の存在は、スクロース22,23とそれらを報いることなく、正口先応答を呼び起こすことができます。このビデオでは、我々は以前の研究9でVGとUSPダブルノックダウンで接続された味覚の知覚を評価するために、PERを実行する方法を示しています。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>GE Healthcare PCR beads in 96 well plate</td>
			<td>GE Healthcare</td>
			<td>27-9557-01</td>
			<td></td>
		 </tr>
		 <tr>
			<td>QIAquick PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td></td>
		 </tr>
		 <tr>
			<td>RiboMax T7 RNA production system</td>
			<td>Promega</td>
			<td>P1300</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trizol LS</td>
			<td>Invitrogen</td>
			<td>10296028</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Chloroform:Isoamyl alcohol 24:1</td>
			<td>Sigma-Aldrich</td>
			<td>C0549</td>
			<td></td>
		 </tr>
		 <tr>
			<td>isopropyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>I9516</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Hamilton micro syringe</td>
			<td>Hamilton</td>
			<td>80301</td>
			<td></td>
		 </tr>
		 <tr>
			<td>30G BD disposable needles</td>
			<td>BD Biosciences</td>
			<td>305106</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>84097</td>
			<td></td>
		 </tr>
		 <tr>
			<td>1 ml Syringe</td>
			<td>BD Biosciences</td>
			<td>30960</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Stereo dissection microscope</td>
			<td>Leica Microsystems</td>
			<td>S6D</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Insect pins</td>
			<td>Fine Science Tools</td>
			<td>26000-25</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Wax plate</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

rnai-mediated double gene knockdown and gustatory perception measurement in honey bees (apis mellifera)

このビデオでは、二本鎖RNA（dsRNA）注射を用いたミツバチで同時に二つの遺伝子をダウンレギュレートするRNA干渉（RNAi）の新規技術を示しています。また、味覚の知覚を測定するための口先拡張応答（PER）アッセイのプロトコルを提示します。 RNAiによる遺伝子ノックダウンは、標的遺伝子の発現をダウンレギュレーション有効な技術である。この技術は、通常、単一の遺伝子操作に用いられるが、それは相互作用と遺伝子との接合効果を検出するための限界​​を有している。このビデオの最初の部分で、我々は、同時に2つの遺伝子を（二重遺伝子ノックダウンと呼ばれる）をノックダウンするための2つの戦略を提示する。我々は両方の戦略が効果的に規制のフィードバックループにある2つの遺伝子、ビテロゲニン（VG）とピラクル（USP）を抑制することができますを示しています。この二重の遺伝子ノックダウンのアプローチは、遺伝子間の相互関係を分析するために使用することができ、容易に適用することができ異なる昆虫種。 このビデオの第二部は、二重遺伝子ノックダウンの治療後のミツバチで口先拡張応答（PER）アッセイのデモンストレーションです。 PERアッセイは、ミツバチ、ミツバチの行動の成熟がどれほど速いの鍵な予測因子であるの味覚の知覚を測定するための標準的なテストです。巣ミツバチの大味覚認識は、多くの場合、花粉の特化を餌と餌の発症に早い年齢に関連付けられて増加した行動の発展を示している。また、PERアッセイはミツバチに飽食か飢餓の代謝状態を識別するために適用することができます。最後に、1アッセイあたりミツバチも広くミツバチの学習と記憶研究のために使用されているエアコンに異なる匂い刺激をペアリングと組み合わせる。

シングル注入と2日間注入戦略大幅VG（一方向ANOVA、P &lt;0.001）（ 図2A）とUSP（一方向ANOVA、P &lt;0.001）（ 図2B）ミツバチにおける転写レベルを減少させ、両方のdsRNAを注入した6日後。 dsRNAの混合物およびVG第一および第二のUSP（VG / USP）と2日間の注射で単回注射を使用してUSP転写の抑制がUSP第一および第二のVG（USP / ...

Watch this Scientific Journal Video about RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera at JoVE.com

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

このプロトコルでは、我々は同時に、ミツバチの二つの遺伝子（二重遺伝子ノックダウン）を抑制の2つの戦略について説明します。その後私たちはミツバチ味覚知覚に二重遺伝子ノックダウンの効果を研究するために口先拡張応答（PER）アッセイを使用する方法を提示します。

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 ...

rnai-mediated-double-gene-knockdown-gustatory-perception-measurement

Research

JoVE Journal

Neuroscience

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

14.8K ビュー.  Arizona State University. このプロトコルでは、我々は同時に、ミツバチの二つの遺伝子（二重遺伝子ノックダウン）を抑制の2つの戦略について説明します。その後私たちはミツバチ味覚知覚に二重遺伝子ノックダウンの効果を研究するために口先拡張応答（PER）アッセイを使用する方法を提示します。

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

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

二重蛍光その場で</em新鮮な脳切片における>ハイブリダイゼーション

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

神経科学

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 ...

Creating Objects and Object Categories for Studying Perception and Perceptual Learning

In order to quantitatively study object perception, be it perception by biological systems or by machines, one needs to create objects and object categories with precisely definable, preferably naturalistic, properties1. Furthermore, for studies on perceptual learning, it is useful to create novel objects and object categories (or object classes) with such properties2.
Many innovative and useful methods currently exist for creating novel objects and object categories3-6 (also see refs. 7,8). However, generally speaking, the existing methods have three broad types of shortcomings.
First, shape variations are generally imposed by the experimenter5,9,10, and may therefore be different from the variability in natural categories, and optimized for a particular recognition algorithm. It would be desirable to have the variations arise independently of the externally imposed constraints.
Second, the existing methods have difficulty capturing the shape complexity of natural objects11-13. If the goal is to study natural object perception, it is desirable for objects and object categories to be naturalistic, so as to avoid possible confounds and special cases.
Third, it is generally hard to quantitatively measure the available information in the stimuli created by conventional methods. It would be desirable to create objects and object categories where the available information can be precisely measured and, where necessary, systematically manipulated (or 'tuned'). This allows one to formulate the underlying object recognition tasks in quantitative terms.
Here we describe a set of algorithms, or methods, that meet all three of the above criteria. Virtual morphogenesis (VM) creates novel, naturalistic virtual 3-D objects called 'digital embryos' by simulating the biological process of embryogenesis14. Virtual phylogenesis (VP) creates novel, naturalistic object categories by simulating the evolutionary process of natural selection9,12,13. Objects and object categories created by these simulations can be further manipulated by various morphing methods to generate systematic variations of shape characteristics15,16. The VP and morphing methods can also be applied, in principle, to novel virtual objects other than digital embryos, or to virtual versions of real-world objects9,13. Virtual objects created in this fashion can be rendered as visual images using a conventional graphical toolkit, with desired manipulations of surface texture, illumination, size, viewpoint and background. The virtual objects can also be 'printed' as haptic objects using a conventional 3-D prototyper.
We also describe some implementations of these computational algorithms to help illustrate the potential utility of the algorithms. It is important to distinguish the algorithms from their implementations. The implementations are demonstrations offered solely as a 'proof of principle' of the underlying algorithms. It is important to note that, in general, an implementation of a computational algorithm often has limitations that the algorithm itself does not have.
Together, these methods represent a set of powerful and flexible tools for studying object recognition and perceptual learning by biological and computational systems alike. With appropriate extensions, these methods may also prove useful in the study of morphogenesis and phylogenesis.

We describe a novel methodology for creating naturalistic 3-D objects and object categories with precisely defined feature variations. We use simulations of the biological processes of morphogenesis and phylogenesis to create novel, naturalistic virtual 3-D objects and object categories that can then be rendered as visual images or haptic objects.

知覚と知覚学習を研究するためのオブジェクトとオブジェクトのカテゴリを作成する

In order to quantitatively study object perception, be it perception by biological systems or by machines, one needs to create objects and object ...

Using Affordable LED Arrays for Photo-Stimulation of Neurons

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single cells in response to electrical or pharmacological manipulations1,2. With the invention of methods to optically control genetically targeted neurons (optogenetics), researchers now have an unprecedented level of control over specific groups of neurons in the standard slice preparation. In particular, photosensitive channelrhodopsin-2 (ChR2) allows researchers to activate neurons with light3,4. By combining careful calibration of LED-based photostimulation of ChR2 with standard slice electrophysiology, we are able to probe with greater detail the role of adult-born interneurons in the olfactory bulb, the first central relay of the olfactory system. Using viral expression of ChR2-YFP specifically in adult-born neurons, we can selectively control young adult-born neurons in a milieu of older and mature neurons. Our optical control uses a simple and inexpensive LED system, and we show how this system can be calibrated to understand how much light is needed to evoke spiking activity in single neurons. Hence, brief flashes of blue light can remotely control the firing pattern of ChR2-transduced newborn cells.

Adult-born neurons expressing ChR2 can be manipulated in slice electrophysiological preparations in order to examine their contribution towards the function of olfactory neural circuits.

ニューロンの光刺激のために手ごろな価格のLEDアレイを使用する

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single ...

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

慢性のためのアダルト生まれの嗅覚ニューロンの選択的なウイルス形質導入 in vivoで Optogenetic刺激

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

MPI CyberMotion Simulator: Implementation of a Novel Motion Simulator to Investigate Multisensory Path Integration in Three Dimensions

Path integration is a process in which self-motion is integrated over time to obtain an estimate of one's current position relative to a starting point 1. Humans can do path integration based exclusively on visual 2-3, auditory 4, or inertial cues 5. However, with multiple cues present, inertial cues - particularly kinaesthetic - seem to dominate 6-7. In the absence of vision, humans tend to overestimate short distances (&lt;5 m) and turning angles (&lt;30&deg;), but underestimate longer ones 5. Movement through physical space therefore does not seem to be accurately represented by the brain.
Extensive work has been done on evaluating path integration in the horizontal plane, but little is known about vertical movement (see 3 for virtual movement from vision alone). One reason for this is that traditional motion simulators have a small range of motion restricted mainly to the horizontal plane. Here we take advantage of a motion simulator 8-9 with a large range of motion to assess whether path integration is similar between horizontal and vertical planes. The relative contributions of inertial and visual cues for path navigation were also assessed.
16 observers sat upright in a seat mounted to the flange of a modified KUKA anthropomorphic robot arm. Sensory information was manipulated by providing visual (optic flow, limited lifetime star field), vestibular-kinaesthetic (passive self motion with eyes closed), or visual and vestibular-kinaesthetic motion cues. Movement trajectories in the horizontal, sagittal and frontal planes consisted of two segment lengths (1st: 0.4 m, 2nd: 1 m; &plusmn;0.24 m/s2 peak acceleration). The angle of the two segments was either 45&deg; or 90&deg;. Observers pointed back to their origin by moving an arrow that was superimposed on an avatar presented on the screen.
Observers were more likely to underestimate angle size for movement in the horizontal plane compared to the vertical planes. In the frontal plane observers were more likely to overestimate angle size while there was no such bias in the sagittal plane. Finally, observers responded slower when answering based on vestibular-kinaesthetic information alone. Human path integration based on vestibular-kinaesthetic information alone thus takes longer than when visual information is present. That pointing is consistent with underestimating and overestimating the angle one has moved through in the horizontal and vertical planes respectively, suggests that the neural representation of self-motion through space is non-symmetrical which may relate to the fact that humans experience movement mostly within the horizontal plane.

An efficient way to gain insight into how humans navigate themselves in three dimensions is described. The method takes advantage of a motion simulator capable of moving observers in ways unattainable by traditional simulators. Results confirm that movement in the horizontal plane is underestimated, while vertical movement is overestimated.

MPI Cyber​​Motionシミュレータ：三次元的に多感覚パスの統合を調査するための新しいモーションシミュレータの実装

Path integration is a process in which self-motion is integrated over time to obtain an estimate of one's current position relative to a starting point 1. ...

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 ...

Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular concentration of all major ions. As these ion shifts bear significant functional consequences, their quantitative determination is often required to understand the function and dysfunction of neural networks under physiological and pathophysiological conditions. In the present study, we demonstrate the fabrication and calibration of double-barreled ion-selective microelectrodes, which have proven to be excellent tools for such measurements in brain tissue. Moreover, so-called &#8220;concentric&#8221; ion-selective microelectrodes are also described, which, based on their different design, offer a far better temporal resolution of fast ion changes. We then show how these electrodes can be employed in acute brain slice preparations of the mouse hippocampus. Using double-barreled, potassium-selective microelectrodes, changes in the extracellular potassium concentration ([K+]o) in response to exogenous application of glutamate receptor agonists or during epileptiform activity are demonstrated. Furthermore, we illustrate the response characteristics of sodium-sensitive, double-barreled and concentric electrodes and compare their detection of changes in the extracellular sodium concentration ([Na+]o) evoked by bath or pressure application of drugs. These measurements show that while response amplitudes are similar, the concentric sodium microelectrodes display a superior signal-to-noise ratio and response time as compared to the double-barreled design. Generally, the demonstrated procedures will be easily transferable to measurement of other ions species, including pH or calcium, and will also be applicable to other preparations.

We demonstrate the fabrication, calibration and properties of two types of ion-selective microelectrodes (double-barreled and concentric) for measurement of ion concentrations in brain tissue. These are then used in the mouse hippocampal slice preparation to show that excitatory activity changes both extracellular potassium and sodium concentrations.

脳組織における測定細胞外イオンの信号のための、二連と同心微小電極

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular ...

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees (Bombus terrestris)

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from flowering plants. Only a few studies have described the gustatory responses of bees toward toxins in food, and these experiments have mainly used the proboscis extension response on restrained honey bees. Here, a new behavioral assay is presented for measuring the feeding responses of freely-moving, individual worker bumble bees to nutrients and toxins. This assay measures the amount of solution ingested by each bumble bee and identifies how tastants in food influence the microstructure of the feeding behavior.
The solutions are presented in a microcapillary tube to individual bumble bees that have been previously starved for 2-4 hr. The behavior is captured on digital video. The fine structure of the feeding behavior is analyzed by continuously scoring the position of the proboscis (mouthparts) from video recordings using event logging software. The position of the proboscis is defined by three different behavioral categories: (1) proboscis is extended and in contact with the solution, (2) proboscis is extended but not in contact with the solution and (3) proboscis is stowed under the head. Furthermore the speed of the proboscis retracting away from the solution is also estimated.
In the present assay the volume of solution consumed, the number of feeding bouts, the duration of the feeding bouts and the speed of the proboscis retraction after the first contact is used to evaluate the phagostimulatory or the deterrent activity of the compounds tested.
This new taste assay will allow researchers to measure how compounds found in nectar influence the feeding behavior of bees and will also be useful to pollination biologists, toxicologists and neuroethologists studying the bumble bee&#39;s taste system.

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees Bombus terrestris

A novel behavioral assay is described for investigating the short term gustatory responses of the mouthparts of freely-moving bumble bees (Bombus terrestris) toward nutrients and toxins in solution.

小説行動アッセイ個人の味覚応答を調査するために、自由に移動バンブル蜂（<em&gt;セイヨウオオマルハナバチ</em&gt;）

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from ...

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 ...