Name: whole mount labeling of cilia in the main olfactory system of mice
Uploaded: 2014-12-27T14:00:00.000+00:00
Duration: 8 min 42 s
Description: Watch this Scientific Journal Video about Whole Mount Labeling of Cilia in the Main Olfactory System of Mice at JoVE.com

This work was funded by the Deutsche Forschungsgemeinschaft DFG (Exc257, SFB958).

注：すべての動物の手順は、動物の不当な苦しみを避けドイツの動物管理の法律と一致してシャリテまたは大学クリニックイエナで取り扱った。 1.準備ソリューションおよび解剖職場<ol><li>ソリューション 注：上皮の解剖を開始する前に、以下のソリューションを準備します。 <ol><li>解剖手続きのためのソリューション： <ol><li> 140 NaCl、5mMのKClを、10mMのHEPES、2mMのCaCl 2を 、1mMのMgCl 2、および10mMのグルコース濃度でリンゲル液（pH7.4）を調製。 </li><li> 2.68のKCl、1.47 mMのKH 2 PO 4、136mMのNaCl、および8.1のNa 2 HPO 4の濃度の液（pH 7.4） - / - PBS準備。 </li><li> - / - 、0.2mMのCaCl 2を 4％スクロース、4％パラホルムアルデヒド固定液1×PBSの濃度の液（pH7.2）を調製。固定液中のスクロースは、クライオを改善切片と凍結切片のアップを拾う。 -20℃で保管してください。 </li></ol></li><li>染色手順のためのソリューション<ol><li> - / - 、0.48のMgCl 2、0.9のCaCl 2 1×PBSの濃度のPBS + / +溶液を調製する。 </li><li> 1X PBS + / +および0.1％トリトンX-100の濃度のPBST + / +溶液を調製する。 </li><li> 1X PBST + / +および1％ゼラチンの濃度のブロッキング溶液を準備します。 </li></ol></li></ol></li><li>職場<ol><li>解剖職場の場合は、明るい照明、並びに液体ブロッカーペンで解剖顕微鏡を使用しています。 </li><li>リンゲル液で満たされたペトリ皿、および接着剤のガラススライドを準備します。 </li><li>以下の手術器具を入手：シャープ1と1鈍い先端を有する外科ハサミ、ストレート先端形状と春のハサミ、細かい湾曲した先端形状とカミソリの刃を持つ2鉗子（ <strong&gt;図1A）。 </li></ol></li></ol>鼻中隔の調製<ol><li>食物および水を定期的にアクセスし、適切な昼/夜サイクルに承認されたケージ内ハウス動物。 </li><li>リア足の反射神経をテストすることによって、100％のイソフルラン及びモニタ鎮痛に浸したガーゼを含有する密閉容器に麻酔を行います。頸椎脱臼が鼻腔内の血液を引き起こす可能性がありますので、直接、麻酔したマウスを首を切る。 </li><li>全体の頭蓋骨と鼻の骨を露出する肌を外し、徹底的にペーパータオルを使用して残りの血液や組織を拭き取る。下顎と前歯を削除します。 </li><li>上顎（横方向）の隔壁（内側）の一方の側を分離するために、縫合線1〜2 mmの距離の平行な左右の鼻の背側骨を切開する。シングルカットで鼻を分割します。骨の残骸と甲介がまだ隔壁に接続されている場合、完全にセプタムを露出させるために慎重にこれらを除去するそれに触れることなく。 </li><li>中隔と鼻の骨の間、中隔の背側に沿って鉗子の罰金曲がった先端部をスライドさせて、背側鼻の骨を削除します。 、骨にわずかな圧力を適用し、それを押し上げて取り外します。 </li><li> 2中隔組織、各キャビティから1へのアクセスを提供するには、慎重に頭の反対側の上顎を削除します。高齢の動物（P&gt; 28）を調製する場合、嗅球をカバーする前頭骨の先端を取り除く。 </li><li>そのわずかに黄色（ 図1B）で嗅上皮を特定します。境界気道上皮は、白色であり、解剖顕微鏡下で見ることができ、運動性繊毛の動きを示している。細かい春のハサミで嗅覚と気道上皮の境界に沿って切断する。 </li><li>カットを拡張し、鋤骨の骨への腹側の接続からセプタムを分離する。その後、中隔とOSの篩骨の篩板との間の境界に沿って切断E。セプタムは、現在は完全頭部へのすべての接続から隔離されている。 図1Cに示す切断位置を使用してください。 </li></ol>免疫染色のための嗅上皮3.絶縁<ol><li>リンゲル液で満たされたペトリ皿に接着剤ガラススライドを置きます。ガラスの半分がリンゲル液（ 図1A）にあるように、ペトリ皿の端にそれを置きます。 </li><li>慎重にペトリ皿に嗅上皮で篩骨を転送するために鉗子を使用してください。鉗子のヒントでそれをつかんでなく、それを持ち上げます。 </li><li> 1鉗子で篩骨の垂直プレートをつかみ、慎重に中隔の一方の側から上皮を除去するための第二1を使用しています。上皮を剥離し垂直プレ​​ートと上皮の間で曲がった先端部をスライドさせます。 </li><li>上皮はリンゲルsolutiに反転させた場合に、常に毛様側を識別するようにしてください上。上皮が反転する場合は、その後、毛様側を識別することはほとんど不可能である。主に、上皮は内側毛様体表面にロールアップします。 </li><li> 図1Cに示す切断位置に在籍した組織形状を比較してください。国境での位置で上皮をつかみ、スライドガラス上に引き出します。組織の病変を避けるために、ピンセットで毛様側には手を触れないでください。 </li><li>セプタムを回し、反対側から上皮で手順を繰り返します。 </li><li>ペーパータオルで嗅上皮の両方の部分の周りにスライドガラスを乾燥させ、液体遮断ペンで組織を取り囲む。 </li><li> RTで10分間、150μlの固定剤溶液を用いて組織を固定してください。繊毛の安定性と完全性については、試験した抗体の様々な短い固定時間を使用しています。これらの10分の繊毛内の固定が、おそらく全体ではなく、上皮組織されている。このため、即時のは、Sの後に顕微鏡で可視化する手順をtaining。しかし、固定液の時間は個々の抗体のために異なる場合があります。 </li></ol> 4.染色プロトコル注：嗅細胞の繊毛の構造を維持するために非常に慎重に組織を処理します。ピペッティングによりソリューションを削除します。機械的な力が細かい繊毛を混乱させる可能性として、上皮に直接すべてのソリューションを落とさないでください。特に明記していない場合は、RTですべての手順を実行します。 <ol><li>固定液を除去し、PBS + / +で2回洗浄する。 </li><li>ブロッキング溶液を少なくとも1時間、上皮をインキュベートする。 </li><li> （本研究において使用される抗体200 1）及び任意の沈殿物を除去するために、最高速度で5分間遠心分離し、ブロッキング溶液中の抗体を溶解することによって一次抗体溶液を調製する。 </li><li> 4℃CO / Nでの加湿チャンバー内の抗体溶液との上皮をインキュベートする。 </li><li> 5分で少なくとも3回、PBS + / +との上皮を洗ってください。 </li><li>（1：500）をブロッキング溶液中の抗体を溶解することによって二次抗体溶液を調製し、最高速度で5分間遠心する。 </li><li>暗い室内で1時間二次抗体溶液で上皮をインキュベートします。 </li><li> 5分で少なくとも3回、PBS + / +との上皮を洗ってください。 </li><li>かみそりの刃やペーパータオルで液体ブロッカーを削除します。 5秒間蒸留水で上皮を洗ってください。 </li><li>退色防止取り付け試薬で組織を保持します。 注：急速日以内にバックグラウンド蛍光が増加する。マウンティング培地中に埋め込んだ後、遅くとも2日より顕微鏡分析は、したがって、お勧めします。特別な注意は、それが深刻な準備に損傷を与える可能性があるため、正確な焦点面を検索する際に組織を圧迫しないように注意する必要があります。ためピンボケ蛍光に、共焦点顕微鏡とのさらなる調査が推奨されます。 </li></ol>

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The authors have nothing to disclose.

The en face preparation technique described in this protocol provides a powerful tool for the detailed analysis of the olfactory system. So far, most studies characterizing the localization of signaling proteins use immunostainings of cryosections. They present a good overview of the olfactory epithelium, and protein expression in distinct cell types or regions can be easily identified. However, expression in olfactory cilia is sometimes hard to detect. Even if ciliary localization is obvious, cryosections offer a very limited view on the morphology of the ciliary layer. En face preparations provide an additional perspective onto the epithelium. 
The olfactory epithelium preparation described here is especially useful for the investigation of proteins that are expressed in a small subset of olfactory neurons. In this case, single knobs are well identifiable, so that localization in knobs and extending cilia can easily be analyzed. In contrast to staining of signaling proteins in cryosections, it is possible to determine the amount of cilia per knob and the length of the cilia. Moreover, en face preparations allow the exact localization of proteins along the cilia. It is also possible to analyze whether proteins are distributed homogenously along the ciliary membrane. Moreover, changes in protein localization upon application of odorant ligands can be investigated. Another aspect is the investigation of developmental changes in protein localization. We experienced good results not only for en face preparations from adult mice, but also from younger animals, up to E18. 
Limitations for this technique are the analyses of highly abundant signaling proteins that exhibit strong expression in (almost) all neurons of the olfactory epithelium. In this case, distinguishing single cilia is difficult due to the strong labeling of many intermingled cilia. Another aspect that should be considered is the zonal expression of certain proteins in the olfactory system. It is necessary to analyze the distribution of neurons expressing a protein of interest to figure out where in the epithelium cells are located. We describe the preparation of olfactory epithelium from the nasal septum, but if proteins would only be expressed on specific turbinates such as mOR262 (mOR37) family receptors12, the protocol for tissue preparation must be adapted. 
We successfully demonstrate that the en face preparation technique can be used to gain additional insights into the protein organization in cilia of the olfactory system. Using en-face preparations ee uncovered that Kirrel2, a membrane protein which has previously been shown to be important for axon sorting, is also localized to the cilia of sensory neurons. Moreover, ciliary localization was inhomogeneous and not observed in all sensory neurons expressing Kirrel2, indicating that ciliary localization of Kirrel2 is regulated.
Taken together, en face preparations can help to understand the organization of the olfactory system by providing a different view on whether certain signaling proteins are localized to the ciliary compartment of sensory neurons. Many ciliopathies affect the olfactory system13,14, the assessment of the anatomy and the molecular composition of olfactory cilia may therefore be used as a tool for improved analysis of the associated defects.

鼻腔内マウス嗅上皮は6から10000000バイポーラ嗅細胞1を備える。各嗅覚ニューロンは表現のために1200嗅覚受容体遺伝子のいずれかを選択します。臭気物質の検出は、その後4 OLF嗅覚特定のGタンパク質Gαを経由してアデニリルシクラーゼIII型（ACIII）3を活性化する嗅覚受容体2、に結合すること、付臭剤によって開始されます。環状アデノシン一リン酸（cAMP）の結果として生じる上昇は、環状ヌクレオチド依存性（CNG）のCa 2+およびNa +の流入につながる非選択性陽イオンチャネルを開き、続いてのCa 2+流入は、Ca 2+活性化Cl -の開口部につながる-チャネル5,6。外側へのCl結果-フラックスは高い細胞内のClによって促進される-のNa + / K + / CL経由そう取り込み、 - -共輸送体NKCC1、濃度が安定したのClによって維持のCl - / HCO3 -交換SLC4A1、そしておそらく追加のまだ同定されるトランスポーター6-8。 バイポーラ嗅覚ニューロンは嗅球に直接投影する単一の非分枝の軸索を有し、そして上皮の表面まで延びており、特殊なコンパートメント、樹状ノブとして終了デンドライト。このノブから、最大で50〜60程度の長さに到達することができ10-30繊毛は、上皮表面9を覆っている粘液に発する。カノニカルシグナル伝達カスケードのタンパク質は、主にこれらの繊毛の膜に局在している。上皮の増加感覚表面が臭気物質を検出する能力を増幅する。起因する感覚ニューロンの密度に、近隣の樹状ノブから延びる繊毛が混在。この、別のニューロンからの繊毛のランダムな混合をもたらす交絡上皮の表面上の嗅覚受容体の異なるタイプを発現する。検出とセル感覚ニューロンのサブセットにおいてのみ存在する毛様タンパク質のウラル割り当てが凍結切片では困難である。凍結切片は、通常、繊毛の長さの平均よりも薄いので、また、繊毛に沿ってこのようなタンパク質の正確な局在化は、かろうじて可能です。 嗅覚ニューロンにおけるこれまでの特徴付けされていない膜タンパク質の毛様体の局在の調査を可能にするために、我々は繊毛におけるタンパク質の局在の詳細な分析を可能にする専用顔調製技術を最適化した。簡単に言えば、マウスを犠牲にし、頭が正中線付近で分割されます。鼻甲介、鼻、および前頭骨が中隔を露出するために除去される。ライニング上皮の嗅覚部分とセプタムは、鼻腔にすべての接続を切断することによって緩められる。リンゲル液で満たされたペトリ皿にセプタムを入れた後、上皮は、コーティングされたガラススライドに移しウントを剥離する。短いfixatに続いて取り扱いが脆弱な組織の損傷を避けるために、できるだけ穏やかであればイオンステップは、免疫染色の手順を行うことができる。我々は、古典的な凍結切片にし、説明エンフェイス調製における嗅覚繊毛2つの異なる膜タンパク質の染色を比較することによって達成可能な解像度を示す。

<table><tbody><tr><td>Spring scissors</td><td></td><td></td><td>straight tip, multiple suppliers</td></tr><tr><td>Surgical scissors</td><td></td><td></td><td>sharp and blunt end, multiple suppliers</td></tr><tr><td>Fine forceps</td><td></td><td></td><td>curved tips, Dumont #7, multiple suppliers</td></tr><tr><td>Razor blade</td><td></td><td></td><td>extra thin, multiple suppliers</td></tr><tr><td>Binocular with illumination</td><td></td><td></td><td>multiple suppliers, Stemi 2000-C, Zeiss</td></tr><tr><td>Petri dish</td><td></td><td></td><td>multiple suppliers</td></tr><tr><td>Liquid-blocker pen</td><td>Science Services</td><td>N71310</td><td></td></tr><tr><td>Polysine coated slides</td><td>Thermo Scientific</td><td>J2800AMNZ</td><td></td></tr><tr><td>Confocal microscope</td><td>Leica Microsystems</td><td>TCS SPE</td><td></td></tr><tr><td>primary antibody Goat anti-Kirrel2</td><td>R&amp;amp;D Systems</td><td>AF2930</td><td>1:200</td></tr><tr><td>primary antibody Rabbit anti-mOR-EG</td><td>Baumgart et al., 2014</td><td></td><td>1:200</td></tr><tr><td>secondary antibodies</td><td>Life Technologies</td><td>A21206, A11057</td><td>1:500</td></tr><tr><td>Mounting medium, ProLong Gold antifade reagent</td><td>Life Technologies</td><td>P36930</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>441244</td><td>toxic, work under fume hood</td></tr></tbody></table>

whole mount labeling of cilia in the main olfactory system of mice

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.

顔製剤エン嗅上皮に局在凍結切片を分析した後には不明であるタンパク質の詳細な研究を可能にする、感覚ニューロンの繊毛のタンパク質の局在を調べるために使用することができる。この問題は、IRRE様タンパク質2（Kirrel2）の親族のための染色の場合を例示することができる。 （またNeph3呼ばれる）Kirrel2は同種親和性接着タンパク質などの膜タンパク質と機能の免疫グロブリン（Ig）スーパーファミリーのメンバーである。これは、嗅覚系における軸索ガイダンスにおいて役割を果たすこと、および活動依存的に10で嗅覚ニューロンのサブセットにおいて発現されることが示された。 嗅上皮を通して典型的な凍結切片にKirrel2ための免疫染色は軸索、細胞体と樹状突起（ 図2A）でKirrel2の局在を明らかにした。 アン顔調製技術について上記したように、すべての溶液を調製した。いくつかのLもののabelingは、樹状ノブからなる層の上に表示され、毛様体局在は凍結切片の分析の後に不確実なままであった。いくつかのニューロンは、彼らの嗅覚ノブとその周辺染色を示したが、単独の繊毛は特定することが困難であった。対照的に、上述のプロトコルに従って行っ嗅上皮の顔調製専用明白Kirrel2（ 図2B）の繊毛局在化を示した。染色された凍結切片の分析は、ニューロンの非常に大きな割合で発現を示したが、興味深いことに、Kirrel2は、嗅覚ニューロンの比較的小さいサブセットの繊毛で発現させた。また、EN顔の準備は、単に一般的には毛様体染色を実証したが、多くの長い繊毛を持つニューロンからわずか数繊毛を持つ神経細胞に及ぶ毛様体発現パターンの変化を明らかにしませんでした。いくつかの細胞では、Kirrel2はなく、このノブから延びる繊毛で、樹状ノブで検出された。 ℃<html> auseとこの知見の機能的関連性はまだ解明されていないが、それは嗅覚系におけるタンパク質の局在化に新たな洞察を提供するために、専用の顔の準備の可能性を示している。 加えて、我々は強い繊毛表現11を示す嗅覚受容体MOR-EG、のための免疫染色を行った。受容体は、すでに凍結切片における毛様体層（ 図3A）で明確に識別される。それにもかかわらず、そのようなノブあたりの繊毛の数や個々の繊毛の長さと詳細な特性解析は不可能である。嗅覚受容体を発現するニューロンからアン顔製剤 、繊毛の識別を使用する（ 図3B、C）が容易に可能である。 アン顔調製物は、異なる年齢の動物を用いて行うことができる。出生前の動物からのさえ繊毛は正常に染色した（ 図3D）可視化した。 <html> 顔製剤エン内容は&quot;&gt;ので良く毛様体の形態および詳細によく知られている毛様体タンパク質の局在を分析するために適したツールを表す。 <img alt="図1" src="/files/ftp_upload/52299/52299fig1highres.jpg" /> マウスの頭部の調製図。 （A）解剖職場の配置。ペトリ皿は、リンゲル液を充填し、接着剤のガラススライドを保持する。外科用器具は、フェース調製専用に推奨されている。（B）は、マウス頭部の半分を除去した後、セプタムが露出している。セプタムは嗅上皮（OE）及び呼吸上皮（RE）で被覆されている。 OB：嗅球、V：。鋤鼻器官（C）嗅上皮が並ぶ中隔の一部を分離するためには、細かいハサミで実証した線に沿ってセプタムをカット。<html> S：//www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52299/52299fig1large.jpg &quot;ターゲット=&quot; _空白 &quot;&gt;この図の拡大版をご覧になるにはこちらをクリックしてください。 <img alt="図2" src="/files/ftp_upload/52299/52299fig2highres.jpg" /> 凍結切片で、 フェースの準備EN Kirrel2免疫染色の図2.比較。 Kirrel2に対して免疫染色生後60日目（P60）凍結切片の（A）共焦点画像（最大突起）が嗅細胞におけるタンパク質の局在を明らかにしている。シングル嗅覚ノブは繊毛に強い染色およびKirrel2定位（アスタリスク）を想定したが、不確実であることを示している。（B）の共焦点画像（最大突起）P65の嗅覚上皮の顔の準備専用嗅覚感覚ニューロンのサブセットの繊毛に明確なKirrel2染色を示し、 。 （スケールバーは10μm）9 / 52299fig2large.jpg「ターゲット= &quot;_空白&quot;&gt;この図の拡大版をご覧になるにはこちらをクリックしてください。 <img alt="図3" src="/files/ftp_upload/52299/52299fig3highres.jpg" /> 凍結切片で、 顔の準備EN MOR-EGの免疫染色の3の比較図。嗅覚受容体MOR-EGに対して免疫染色P60の凍結切片の（A）共焦点画像（最大投影）。毛様体の局在は既に検出可能である。（B）嗅覚受容体MOR-EGに対して免疫染色顔準備EN P65の共焦点画像。 MOR-EGを強く嗅覚感覚ニューロンのサブセットの繊毛で表されます。凍結切片を、長さに関する繊毛特性とは対照的に、ノブと正確なタンパク質局在当たり繊毛の数を分析することができる。（B）中の四角で囲まれた領域の（C）高倍率。 （D）Confoc嗅覚受容体MOR-EGに対して免疫染色顔準備EN胎生18日（E18）のアル画像。繊毛の染色は、明瞭に見ることができる。 （スケールバー、10μm）を<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52299/52299fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Whole Mount Labeling of Cilia in the Main Olfactory System of Mice at JoVE.com

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

マウスの主嗅覚系における繊毛のホールマウントのラベリング

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...

whole-mount-labeling-of-cilia-in-the-main-olfactory-system-of-mice

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

11.1K ビュー.  Universitaetsklinikum Jena. Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

ビデオ: Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

Simple and Computer-assisted Olfactory Testing for Mice

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected with aging and is a strong predictor of neurodegenerative diseases. Thus, olfaction testing is used as a non-invasive diagnostic method to detect neurological deficits early on. In order to understand the mechanisms underlying olfactory network susceptibility, olfactory research in rodents has gained momentum in the past decade.
Here, we present a very simple, time efficient and reproducible olfactory testing method of innate odor perception and sensitivity in mice without the need of any prior food or water restriction. The tests are performed in a familiar environment to the mice, require only the scents and a 2 min session of odorant exposure. The analysis is performed, post-hoc, using computer-assisted commands on ImageJ and can be, therefore, carried out from start to end by one researcher.
This protocol does not require any special hardware or setup and is indicated for any laboratory interested in testing olfactory perception and sensitivity.

We present a simple and unbiased olfactory test in mice. With this protocol olfactory discrimination, preference, avoidance and sensitivity to a novel odor as compared to water can be assessed in single behavioral sessions. This method is indicated for a single experimenter and analysis is based on computer-assisted video processing.

シンプルでコンピュータ支援嗅覚テストマウス用

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected ...

行動学

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

マウス鋤鼻急性組織スライス標本のイメージングフェロモンセンシング

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

神経科学

Imaging Odor-Evoked Activities in the Mouse Olfactory Bulb using Optical Reflectance and Autofluorescence Signals

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. Functional optical imaging techniques are advantageous to visualize the activation of these modules in sensory cortices with high spatial resolution. In this context, endogenous optical signals that arise from molecular mechanisms linked to neuroenergetics are valuable sources of contrast to record spatial maps of sensory stimuli over wide fields in the rodent brain.
Here, we present two techniques based on changes of endogenous optical properties of the brain tissue during activation. First the intrinsic optical signals (IOS) are produced by a local alteration in red light reflectance due to: (i) absorption by changes in blood oxygenation level and blood volume (ii) photon scattering. The use of in vivo IOS to record spatial maps started in the mid 1980's with the observation of optical maps of whisker barrels in the rat and the orientation columns in the cat visual cortex1. IOS imaging of the surface of the rodent main olfactory bulb (OB) in response to odorants was later demonstrated by Larry Katz's group2. The second approach relies on flavoprotein autofluorescence signals (FAS) due to changes in the redox state of these mitochondrial metabolic intermediates. More precisely, the technique is based on the green fluorescence due to oxidized state of flavoproteins when the tissue is excited with blue light. Although such signals were probably among the first fluorescent molecules recorded for the study of brain activity by the pioneer studies of Britton Chances and colleagues3, it was not until recently that they have been used for mapping of brain activation in vivo. FAS imaging was first applied to the somatosensory cortex in rodents in response to hindpaw stimulation by Katsuei Shibuki's group4.
The olfactory system is of central importance for the survival of the vast majority of living species because it allows efficient detection and identification of chemical substances in the environment (food, predators). The OB is the first relay of olfactory information processing in the brain. It receives afferent projections from the olfactory primary sensory neurons that detect volatile odorant molecules. Each sensory neuron expresses only one type of odorant receptor and neurons carrying the same type of receptor send their nerve processes to the same well-defined microregions of &tilde;100&mu;m3 constituted of discrete neuropil, the olfactory glomerulus (Fig. 1). In the last decade, IOS imaging has fostered the functional exploration of the OB5, 6, 7 which has become one of the most studied sensory structures. The mapping of OB activity with FAS imaging has not been performed yet.
Here, we show the successive steps of an efficient protocol for IOS and FAS imaging to map odor-evoked activities in the mouse OB.

This article presents the protocols of intrinsic optical signals and flavoproteins autofluorescence signals imaging to map odor-evoked activities at the surface of the olfactory bulb in mice.

光反射率と自家蛍光信号を用いたマウス嗅球におけるイメージングの臭い誘発活動

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

における嗅覚受容ニューロンのホールマウント免疫標識<em&gt;ショウジョウバエ</em&gt;アンテナ

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

無傷の神経上皮におけるマウス嗅細胞の穿孔パッチクランプ記録：同定された嗅覚受容体を発現するニューロンの機能解析

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

成体マウス蝸牛のホールマウント解剖および免疫蛍光

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

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 ...

An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and parenting. Importantly, mice can be trained to associate novel odors with specific behavioral responses to provide insight into olfactory circuit function. This protocol details the procedure for training mice on a Go/No-Go operant learning task. In this approach, mice are trained on hundreds of automated trials daily for 2&#8211;4 weeks and can then be tested on novel Go/No-Go odor pairs to assess olfactory discrimination, or be used for studies on how odor learning alters the structure or function of the olfactory circuit. Additionally, the mouse olfactory bulb (OB) features ongoing integration of adult-born neurons. Interestingly, olfactory learning increases both the survival and synaptic connections of these adult-born neurons. Therefore, this protocol can be combined with other biochemical, electrophysiological, and imaging techniques to study learning and activity-dependent factors that mediate neuronal survival and plasticity.

Here, we train mice on an associative learning task to test odor discrimination. This protocol also allows for studies on learning-induced structural changes in the brain.

嗅覚学習とマウスにおける差別の客観的かつ再現性のあるテスト

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and ...

An Effective Manual Deboning Method To Prepare Intact Mouse Nasal Tissue With Preserved Anatomical Organization

The mammalian nose is a multi-functional organ with intricate internal structures. The nasal cavity is lined with various epithelia such as olfactory, respiratory, and squamous epithelia which differ markedly in anatomical locations, morphology, and functions. In adult mice, the nose is covered with various skull bones, limiting experimental access to internal structures, especially those in the posterior such as the main olfactory epithelium (MOE). Here we describe an effective method for obtaining almost the entire and intact nasal tissues with preserved anatomical organization. Using surgical tools under a dissecting microscope, we sequentially remove the skull bones surrounding the nasal tissue. This procedure can be performed on both paraformaldehyde-fixed and freshly dissected, skinned mouse heads. The entire deboning procedure takes about 20-30 min, which is significantly shorter than the experimental time required for conventional chemical-based decalcification. In addition, we present an easy method to remove air bubbles trapped between turbinates, which is critical for obtaining intact thin horizontal or coronal or sagittal sections from the nasal tissue preparation. Nasal tissue prepared using our method can be used for whole mount observation of the entire epithelia, as well as morphological, immunocytochemical, RNA in situ hybridization, and physiological studies, especially in studies where region-specific examination and comparison are of interest.

プリザーブド解剖組織と無傷マウスの鼻腔組織を準備するための効果的なマニュアル脱骨方法

The mammalian nose is a multi-functional organ with intricate internal structures.  The nasal cavity is lined with various epithelia such as olfactory, ...

生物学

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of available mating partners or threats. In mice, this task is executed primarily by the olfactory system and its underlying subsystems, including the main and accessory olfactory systems. Both have peripheral organs populated by sensory neurons expressing G-protein coupled receptors able to bind chemical cues that reach the nasal cavity. Even though the molecular characteristics of these receptors is well understood, little is known about their cognate specific ligands. The method described here combines in situ hybridization detection of olfactory or vomeronasal receptors with immunodetection of phosphorylated ribosomal protein S6 (pS6) - a marker of neuronal activation. This protocol was devised to identify neurons activated after a single event of exposure to purified or complex chemical stimuli detected by the olfactory organs. Importantly, this technique allows the investigation of neurons triggered in biologically relevant contexts. Ideally, this method should be used to probe the molecular biology of the olfactory system and to study olfactory behaviors.

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

This protocol combines in situ hybridization with immunofluorescence to identify olfactory and vomeronasal receptor genes expressed in olfactory sensory neurons after activation by chemical stimuli in the mouse.

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of ...

マウスの主嗅覚系における繊毛のホールマウントのラベリング

要約

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シンプルでコンピュータ支援嗅覚テストマウス用

マウス鋤鼻急性組織スライス標本のイメージングフェロモンセンシング

光反射率と自家蛍光信号を用いたマウス嗅球におけるイメージングの臭い誘発活動

における嗅覚受容ニューロンのホールマウント免疫標識

無傷の神経上皮におけるマウス嗅細胞の穿孔パッチクランプ記録：同定された嗅覚受容体を発現するニューロンの機能解析

成体マウス蝸牛のホールマウント解剖および免疫蛍光

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

嗅覚学習とマウスにおける差別の客観的かつ再現性のあるテスト

プリザーブド解剖組織と無傷マウスの鼻腔組織を準備するための効果的なマニュアル脱骨方法

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization