この作品は、NSFの助成IOSの1121692で、ワシントンの研究財団の大学によってサポートされていました。

1。揮発性Follection <ol><li>この例では、Mから揮発性のサンプルを使用するlewisii花-カリフォルニア州に高山野草ネイティブ。揮発性物質をRiffell らに従って動的吸着法を用いて収集されます14。簡単に説明すると、このメソッドは花がテフロン袋に囲まれている閉ループ捕捉システムを採用しています。不活性の真空ポンプを使用して、花の周りの空気がPorapak Q行列でいっぱいパスツールピペットからなる &quot;トラップ&quot;を介して吸引される。ポンプからの戻り空気は活性炭によってフィルタリングされます。所定時間後、私たちの場合24時間で、Porapak Q行列は、濃縮された抽出物を収集するために、非極性溶媒、典型的にはヘキサンで溶出させる。抽出液を-80℃·分析まで保存されます。必要であれば、サンプルが窒素ガス気流下で、分析の前に濃縮することができる。サンプルは、既に十分に特徴付けられていない限り、それを介してのアリコートを実行サンプルを使用する前に、揮発性成分を識別するために、ガスクロマトグラフ質量分析計（GC-MS）。 </li></ol> 2。電気生理学的準備<ol><li>千μlのピペットチップの先端から約1cmを切った。ピペットチップのベースにマルハナバチ（ マルハナバチインパチェンス ） を配置し、頭だけが露出するまでそっと先端の反対側の端に向かって押します。 </li><li>歯科用ワックスを溶かすとセキュリティのための複眼の上にワックスが付着していることを確認して、露出した頭部のまわりでそれを成形し、完全に不動ハチの頭を作るために。ハチのアンテナの上に任意のワックスを取得しないようにしてください。 </li><li>ヘッドが固定されたら、カミソリの刃ブレーカまたはキューティクルをカットするメス適切なサイズを使用し、頭部カプセルに角、窓状、切り込みを入れる。ブレードブレーカを使用して、直ちにアンテナと複眼の1つに隣接後ろ、頭部カプセルの背側から開始します。カット1複眼から反対側の複眼に直線。反対複眼に直線をカットした後、その胸部に近い頭部カプセルカーブと終了するまで背側切開を作り始める。この時点で、頭部カプセルの反対側の端に向かってカットし始める。最後に、一度ラインが反対側の端にカットされており、初期の切開の開始位置に戻ってラインをカット。これは電極の挿入が妨げになるとして、それは、アンテナに隣接しているキューティクルを除去することが重要である。 </li><li>キューティクルがカットされたら、その後蜂の脳と、もっと重要なのは、触角葉を公開する必要があります蜂のキューティクルを除去するためにピンセットを使用しています。直ちに脳がdessicatedにならないように、昆虫生理食塩水で脳をsuperfusing始める。脳が露出した後、慎重に直ちに触角ローブの上に神経周囲シースを除去するために、非常に微細なピンセットを使用しています。非常に慎重にされないように鉗子で穿刺蜂の脳を。 </li></ol> 3。マルチチャンネル録音付きガスクロマトグラフィ<ol><li>蜂 &quot;準備&quot; - その脳が露出してチューブ内に固定 - 今電気生理学的記録の準備ができています。エアテーブル上に配置された磁気ベースに固定し、クランプで蜂を置きます。 </li><li>生理食塩水が継続的に脳をsuperfusesように、IVバッグ、フローコントローラ、チューブ（昆虫生理食塩水で満たされた）に配置します。 </li><li>蜂の目に、タングステン線で作られたマイクロマニピュレータ、insertareference電極を使用。 </li><li>独立したマイクロマニピュレータを使用して、このようなコイル状のワイヤーテトロード、またはシリコンマルチチャネル電極（Neuronexusテクノロジーズ）として、多チャンネル電極を挿入 ハチの触角葉に。この電極は、TDT系のZバスbioampプロセッサへのTDTのシステムのS-3のZシリーズのように、前置増幅器に接続されています。ガスクロマトからの出力シールドBNCケーブルを経由してクロマトグラムの検出器は、神経とGC信号の両方が同期されているアンプやデータ·アクイジション·システムと接続することができます。 </li><li>安定させるための神経録音の約30〜60分待ちます。自発的な活動と記録チャネルの単位の波形の形状が一貫になったら、蜂を刺激し、臭気に録音チャンネルの応答を観察するために臭気のシリンジを使用しています。 </li><li>個々の記録チャンネル上の信号の3.5から5シグマによる自動しきい値記録チャネルによってニューロンから細胞外スパイクを記録します。マニュアルしきい値処理は、電気的ノイズからの汚染を避けるために、いくつかのチャネルが必要な場合があります。ニューロンからの活動電位は記録チャネルの電圧スパイクとして表示されます。チャネルの電圧がしきい値を超えると、システムはそれによってターキン、しきい値への到達前と後数ミリ秒をバッファし、保存しますgaは波形のスナップショット、またはスパイク。 </li><li>直ちに空気テーブルの隣には、GCです。 GCに花のエキスを注入する前に、GC分析の温度上昇のための方法が正しいことを確認します。私たちの例では、10℃/分の速度で温度上昇が続く4分間50から始まる温度法°Cを使用します。 220℃我々は追加の6分のGCを保持し、その時点で、C。我々は、キャリアガスとしてヘリウムを、DB-5のGCカラム（J＆Wサイエンティフィック、フォルサム、カリフォルニア州、米国）を使用します。入口は、200℃に設定した温度と、スプリットレスです水素炎イオン化検出器の温度を230℃に設定されている</li><li> GCカラムに吸着した揮発性物質を解放するために、GCの加熱された注入口に花のヘッドスペースの抽出サンプルを注入します。カラムからの流出液は水素炎イオン化検出器（FID）とガラスの &quot;Y&quot;コネクター（J＆W Scientific）を用いたミツバチアンテナとの間に1:1に分割されます。 RECを開始は、GCにサンプルを注入するように電極からディング。 </li><li> GCの実行が終了した後、5〜15分間準備休止してください。次に、いずれかGCに別のサンプルを注入したり、単一の揮発性化合物又は化合物の混合物を使用して準備を刺激する。準備を刺激するこの後者の方法では、一定の空気の流れから空気のパルスは、化合物が堆積されている上に濾紙を入れたガラスシリンジを介して転送されます。匂い刺激は、ソフトウェアによって制御ソレノイド活性化バルブによってパルスした。 </li><li>ユニット活動が突然停止し、または変更された場合は、生理的食塩水の点滴をチェックして、15分間の準備休止してください。自発的な活動はその前のレベルを回復しない場合は可能であればその後の準備は破棄され、別の蜂が使用されるべきである。 </li><li>実験後、まだ組織内プローブを用いた20分間5％ホルマリンで脳を固定します。次に、消費税の脳そして4時間2％グルタルアルデヒドに置き、その後、段階的なエタノール脱水系列を行うとサリチル酸メチルと脳をクリアします。固定に基づいており、組織のクリア、電極が組織を穿刺アラバマ州の場所は、共焦点顕微鏡によって明確に識別する必要があります。 </li></ol> 4。データ解析<ol><li>記録された神経単位を分離し、同定するための実験の後に収集されたデータを分析します。このようなピークまたは谷振幅、ピーク半値幅などのスパイク形状、または減圧措置に基づいて別々の波形、または&quot;スパイク&quot;（主成分）17に代表的なソフトウェアプログラムを（オフラインソーター、MClust、そしてSClust）を使用し、18（ 図2）。三次元空間で分離された（PC1〜PC3）と、互いに統計的に異なっているスパイクのだけそれらのクラスタを使用して（多変量ANOVA、P &lt;0.05）（ 図2）、さらなる分析のため。を参照してくださいテトロード記録とスパイクソーティング方法論の完全な説明については、引用＃S 17から19。 </li><li>各クラスタ内のタイム·スタンプスパイク、ラスタープロットと発火率応答（ 図2、図3（a））を作成するためのMATLABまたはNeuroexplorer（NEXテクノロジーズウィンストンセーラム、ノースカロライナ州）を用いて、分析のためにこれらのデータをエクスポートします。 </li><li>同時に記録GCデータを用いて揮発性成分の保持時間を特定します。それらの時点でユニットの応答を調べるために、クロマトグラムからピークの頂点によって決定揮発性物質の保持時間を使用します。 </li><li> GC分析を介して個々のユニットの応答を調べるために、100ミリ秒間隔でスパイクの数をbinおよび揮発性物質を溶出する保持時間を参照して、速度応答を焼成の時間経過を調べる。 100ミリ秒間隔でスパイクのビニングは、GCから溶出​​する匂い物質への神経応答の時間経過について、十分な詳細、または信号を提供します。 </li><li> EXAに異なる溶出揮発性物質への人口応答を採掘、前、3秒のサンプリング·ウィンドウの上に1.5秒を個々のユニットの発火率応答を統合し、1.5秒後の保持時間揮発性（ 図3）。この期間は、GCから揮発性溶出の期間の典型です。我々は、色分けして（赤が高発射レート応答であり、青は低い応答です）によってユニットの速度応答を発射示し、（列）のGC排水にアンサンブル応答を表す各行が活性マトリックスとして、それらを配置する（ 図3）。 </li></ol>

<ol>
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昆虫嗅覚媒介行動は、再生、ホストサイトの選択、適切な食糧資源の識別を含む多くの異なるプロセスを駆動する。これらのプロセスの研究では、光源から出射された揮発性物質と同様に、行動を媒介されるそれらの化合物を同定するための能力を識別する能力を必要とします。ややこしいことは臭いが一緒に個々の成分6,7,13,19,20とは異なる知覚される独特の香りを作成し、個々の?...

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identification of olfactory volatiles using gas chromatography-multi-unit recordings (gcmr) in the insect antennal lobe

すべての生物は、その環境への彼らの行動や生理的な応答を決定する感覚刺激に満ちた世界に生息しています。嗅覚はに反応し、間で、複雑な香りの刺激を区別するために彼らの嗅覚システムを使用して昆虫、特に重要である。これらの臭気はこのような再生や生息地の選択1月3日のようなプロセスを仲介する行動を誘発する。食用作物の受粉4-6、草食7、病気8,9の伝送を含む、農業と人間の健康のために非常に重要である昆虫を媒介行動によってさらに、化学センシング。嗅覚信号と昆虫行動における役割の同定は、生態学的なプロセスおよびヒューマン·食糧資源と幸福の両方を理解するためにこのように重要です。 現在までに、昆虫行動を駆動揮発性物質の同定は困難で、しばしば退屈であった。現在の技術には、ガスクロマトグラフィー結合electroantennogram記録（GC-EAG）、およびガスクロマトグラフィー-結合されたシングル感覚子の録音（GC-SSR）10-12。これらの技術は、生物活性化合物の同定に不可欠であることが判明した。 13,14、我々は、触角葉のニューロン（昆虫の主嗅覚中枢AL）からマルチチャンネルの電気生理学的記録（ &#39;GCMR &quot;と呼ばれる）に結合し、ガスクロマトグラフィーを用いた方法を開発しました。この最先端の技術は、私たちは匂いの情報は昆虫脳の中でどのように表現されるかを調べることが可能になります。嗅覚処理のこのレベルの匂いに対する神経反応がALニューロンへのアンテナの受容ニューロンの収束の度合いに非常に敏感なせいであるので、さらに、ALの録音は自然な臭気の活性成分の検出を効率的かつ高感度にできるようになります。ここでは、GCMRを説明し、その使用例を与える。 いくつかの一般的な手順はinvolアール生理揮発性物質と昆虫応答の検出にVED。揮発性物質は、最初に興味のある情報源から収集します（この例では、我々は属Mimulus（Phyrmaceae）から花を使用）および標準のGC-MS技術を14-16を使用して、必要に応じて特徴づけする必要があります。昆虫は記録電極が神経録音が始まる触角葉およびマルチチャネルに挿入された後、最小限の切開を用いた研究のために準備されます。神経データの後処理はその後、特定の匂い物質は、昆虫の神経系で重要な神経反応を起こしているか明らかにする。 ここでお見せする例では、受粉の研究に固有のものですが、GCMRは試験生物と揮発性ソースの広い範囲に拡大することができます。たとえば、このメソッドは媒介昆虫と作物の害虫を集めたり反発臭気物質の識別に使用することができます。また、GCMRはまた、POと益虫に対して誘引を識別するために使用することができますllinators。技術は同様に非昆虫被験者に拡張される可能性があります。

Mを使用GCMRアッセイでlewisiiフローラルの香りは、GCに抽出物の3μlを注入します。 GCを介して溶出する揮発性物質の総数は、一般的に60から70揮発性物質である。 Mの香りlewisiiは、主に 6 -炭素例えば、2 -ヘキサノールなどの揮発性物質、およびヘッドスペースの&lt;1％を含むセスキテルから成る香りの残りの部分とは、β-ミルセン（非環式）およびα-<...

Watch this Scientific Journal Video about Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe at JoVE.com

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe

嗅覚は、昆虫の多くの異なった行動を媒介し、よく揮発性化合物の数十〜数百から構成される複雑な混合物である。昆虫の触角葉でマルチチャンネル録音でガスクロマトグラフィーを用いて、我々は、生物活性化合物の同定のための方法を説明します。

昆虫の触角葉でガスクロマトグラフィー - マルチユニットレコーディング（GCMR）を使用して、嗅覚揮発性物質の同定

All  organisms inhabit a world full of sensory stimuli that determine their  behavioral and physiological response to their environment. Olfaction is ...

identification-olfactory-volatiles-using-gas-chromatography-multi

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JoVE Journal

Neuroscience

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

14.2K ビュー.  University of Washington. 嗅覚は、昆虫の多くの異なった行動を媒介し、よく揮発性化合物の数十〜数百から構成される複雑な混合物である。昆虫の触角葉でマルチチャンネル録音でガスクロマトグラフィーを用いて、我々は、生物活性化合物の同定のための方法を説明します。

ビデオ: Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe

Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and other social interactions. The electroolfactogram (EOG) recording is an informative, easy to conduct, and reliable method to assay olfactory function at the level of the olfactory epithelium. Since the 1956 description of the EOG by Ottoson in frogs1, the EOG recording has been applied in many vertebrates including salamanders, rabbits, rats, mice, and humans (reviewed by Scott and Scott-Johnson, 2002, ref. 2). The recent advances in genetic modification in mice have rekindled interest in recording the EOG for physiological characterization of olfactory function in knock-out and knock-in mice. EOG recordings have been successfully applied to demonstrate the central role of olfactory signal transduction components3-8, and more recently to characterize the contribution of certain regulatory mechanisms to OSN responses9-12.
Odorant detection occurs at the surface of the olfactory epithelium on the cilia of OSNs, where a signal transduction cascade leads to opening of ion channels, generating a current that flows into the cilia and depolarizes the membrane13. The EOG is the negative potential recorded extracellularly at the surface of the olfactory epithelium upon odorant stimulation, resulting from a summation of the potential changes caused by individual responsive OSNs in the recording field2. Comparison of the amplitude and kinetics of the EOG thus provide valuable information about how genetic modification and other experimental manipulations influence the molecular signaling underlying the OSN response to odor. 
Here we describe an air-phase EOG recording on a preparation of mouse olfactory turbinates. Briefly, after sacrificing the mouse, the olfactory turbinates are exposed by bisecting the head along the midline and removing the septum. The turbinate preparation is then placed in the recording setup, and a recording electrode is placed at the surface of the olfactory epithelium on one of the medial turbinates. A reference electrode is electrically connected to the tissue through a buffer solution. A continuous stream of humidified air is blown over the surface of the epithelium to keep it moist. The vapor of odorant solutions is puffed into the stream of humidified air to stimulate the epithelium. Responses are recorded and digitized for further analysis.

The electroolfactogram (EOG) recording is an informative, easy-to-conduct, and reliable way of assessing olfactory function at the level of the olfactory epithelium. This protocol describes a recording setup, mouse tissue preparation, data collection, and basic data analysis.

空気相Electroolfactogram記録を用いたマウス嗅覚神経細胞の分析応答

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and ...

神経科学

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 ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

吸引ピペット技術を用いた嗅覚受容ニューロンから記録匂い物質誘発性応答

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

急性海馬スライスにおけるシナプス誘発アストログリアとニューロン活動の二重の電気生理学的記録

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

ローカスの神経活動を特徴づけるためにマルチユニット記録方法（<em&gt; Schistocercaアメリカーナ</em&gt;）嗅覚回路

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Using Insect Electroantennogram Sensors on Autonomous Robots for Olfactory Searches

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects foraging for food or searching for mates3: the olfactory search is constrained by the physics of turbulent transport4. The concentration landscape of wind borne odors is discontinuous and consists of sporadically located patches. A pre-requisite to olfactory search is that intermittent odor patches are detected. Because of its high speed and sensitivity5-6, the olfactory organ of insects provides a unique opportunity for detection. Insect antennae have been used in the past to detect not only sex pheromones7 but also chemicals that are relevant to humans, e.g., volatile compounds emanating from cancer cells8 or toxic and illicit substances9-11. We describe here a protocol for using insect antennae on autonomous robots and present a proof of concept for tracking odor plumes to their source. The global response of olfactory neurons is recorded in situ in the form of electroantennograms (EAGs). Our experimental design, based on a whole insect preparation, allows stable recordings within a working day. In comparison, EAGs on excised antennae have a lifetime of 2 hr. A custom hardware/software interface was developed between the EAG electrodes and a robot. The measurement system resolves individual odor patches up to 10 Hz, which exceeds the time scale of artificial chemical sensors12. The efficiency of EAG sensors for olfactory searches is further demonstrated in driving the robot toward a source of pheromone. By using identical olfactory stimuli and sensors as in real animals, our robotic platform provides a direct means for testing biological hypotheses about olfactory coding and search strategies13. It may also prove beneficial for detecting other odorants of interests by combining EAGs from different insect species in a bioelectronic nose configuration14 or using nanostructured gas sensors that mimic insect antennae15.

We describe a protocol for using insect antennae in the form of electroantennograms (EAGs) on autonomous robots. Our experimental design allows stable recordings within a day and resolves individual odor patches up to 10 Hz. The efficiency of EAG sensors for olfactory searches is demonstrated in driving a robot toward an odor source.

嗅覚検索用自律型ロボットに昆虫Electroantennogramセンサを用いた

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

無傷の鋤鼻器官と副嗅球のex vivoでの準備

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular recording has historically been a common technique used to study sensitivities of individual photoreceptor cells to different light stimuli that is still being used today. However, there remains a dearth of detailed methodology in the literature for researchers wishing to replicate intracellular recording experiments in the eye. Here we present the insect as a model for examining eye physiology more generally. Insect photoreceptor cells are located near the surface of the eye and are therefore easy to reach, and many of the mechanisms involved in vision are conserved across animal phyla. We describe the basic procedure for in vivo intracellular recording of photoreceptor cells in the eye of a butterfly, with the goal of making this technique more accessible to researchers with little prior experience in electrophysiology. We introduce the basic equipment needed, how to prepare a live butterfly for recording, how to insert a glass microelectrode into a single cell, and finally the recording procedure itself. We also explain the basic analysis of raw response data for determining spectral sensitivity of individual cell types. Although our protocol focuses on determining spectral sensitivity, other stimuli (e.g., polarized light) and variations of the method are applicable to this setup.

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

The electrophysiological technique of intracellular recording is demonstrated and used to determine spectral sensitivities of single photoreceptor cells in the compound eye of a butterfly.

昆虫モデルにおける視細胞分光感度の決意から<em&gt;インビボ</em&gt;細胞内記録

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular ...

Insect-controlled Robot: A Mobile Robot Platform to Evaluate the Odor-tracking Capability of an Insect

Robotic odor source localization has been a challenging area and one to which biological knowledge has been expected to contribute, as finding odor sources is an essential task for organism survival. Insects are well-studied organisms with regard to odor tracking, and their behavioral strategies have been applied to mobile robots for evaluation. This "bottom-up" approach is a fundamental way to develop biomimetic robots; however, the biological analyses and the modeling of behavioral mechanisms are still ongoing. Therefore, it is still unknown how such a biological system actually works as the controller of a robotic platform. To answer this question, we have developed an insect-controlled robot in which a male adult silkmoth (Bombyx mori) drives a robot car in response to odor stimuli; this can be regarded as a prototype of a future insect-mimetic robot. In the cockpit of the robot, a tethered silkmoth walked on an air-supported ball and an optical sensor measured the ball rotations. These rotations were translated into the movement of the two-wheeled robot. The advantage of this "hybrid" approach is that experimenters can manipulate any parameter of the robot, which enables the evaluation of the odor-tracking capability of insects and provides useful suggestions for robotic odor-tracking. Furthermore, these manipulations are non-invasive ways to alter the sensory-motor relationship of a pilot insect and will be a useful technique for understanding adaptive behaviors.

The capability to localize an odor source is necessary for insect survival and is expected to be applicable to artificial odor-tracking. The insect-controlled robot is driven by an actual silkmoth and enables us to evaluate the odor-tracking capability of insects through a robotic platform.

昆虫制御のロボット：移動ロボットプラットフォーム昆虫の匂い追跡能力を評価します

Robotic odor source localization has been a challenging area and one to which biological knowledge has been expected to contribute, as finding odor ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

嗅覚神経軸索分子識別コードの視覚化のための嗅球の四重免疫染色

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...