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>

<ol>
	<li>Firestein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+the+olfactory+system+makes+sense+of+scents.">How the olfactory system makes sense of scents.</a> Nature. 413 (6852), 211-218 (2001).</li><li>Buck, L., Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+multigene+family+may+encode+odorant+receptors:+a+molecular+basis+for+odor+recognition.">A novel multigene family may encode odorant receptors: a molecular basis for odor recognition.</a> Cell. 65 (1), 175-187 (1991).</li><li>Wong, S. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+the+type+III+adenylyl+cyclase+gene+leads+to+peripheral+and+behavioral+anosmia+in+transgenic+mice.">Disruption of the type III adenylyl cyclase gene leads to peripheral and behavioral anosmia in transgenic mice.</a> Neuron. 27 (3), 487-497 (2000).</li><li>Belluscio, L., Gold, G. H., Nemes, A., Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+deficient+in+G(olf)+are+anosmic.">Mice deficient in G(olf) are anosmic.</a> Neuron. 20 (1), 69-81 (1998).</li><li>Brunet, L. J., Gold, G. H., Ngai, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+anosmia+caused+by+a+targeted+disruption+of+the+mouse+olfactory+cyclic+nucleotide-gated+cation+channel.">General anosmia caused by a targeted disruption of the mouse olfactory cyclic nucleotide-gated cation channel.</a> Neuron. 17 (4), 681-693 (1996).</li><li>Reisert, J., Lai, J., Yau, K. W., Bradley, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+the+excitatory+Cl-+response+in+mouse+olfactory+receptor+neurons.">Mechanism of the excitatory Cl- response in mouse olfactory receptor neurons.</a> Neuron. 45 (4), 553-561 (2005).</li><li>Hengl, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+components+of+signal+amplification+in+olfactory+sensory+cilia.">Molecular components of signal amplification in olfactory sensory cilia.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (13), 6052-6057 (2010).</li><li>Smith, D. W., Thach, S., Marshall, E. L., Mendoza, M. G., Kleene, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+lacking+NKCC1+have+normal+olfactory+sensitivity.">Mice lacking NKCC1 have normal olfactory sensitivity.</a> Physiolog., &amp; Behavior. 93 (1-2), 44-49 (2008).</li><li>Menco, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+aspects+of+olfactory+signaling.">Ultrastructural aspects of olfactory signaling.</a> Chemical Senses. 22 (3), 295-311 (1997).</li><li>Serizawa, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neuronal+identity+code+for+the+odorant+receptor-specific+and+activity-dependent+axon+sorting.">A neuronal identity code for the odorant receptor-specific and activity-dependent axon sorting.</a> Cell. 127 (5), 1057-1069 (2006).</li><li>Baumgart, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffolding+by+MUPP1+regulates+odorant-mediated+signaling+in+olfactory+sensory+neurons.">Scaffolding by MUPP1 regulates odorant-mediated signaling in olfactory sensory neurons.</a> Journal Of Cell Science. 127 (11), 2518-2527 (2014).</li><li>Strotmann, J., Wanner, I., Krieger, J., Raming, K., Breer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+odorant+receptors+in+spatially+restricted+subsets+of+chemosensory+neurones.">Expression of odorant receptors in spatially restricted subsets of chemosensory neurones.</a> Neuroreport. 3 (12), 1053-1056 (1992).</li><li>Jenkins, P. M., McEwen, D. P., Martens, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+cilia:+linking+sensory+cilia+function+and+human+disease.">Olfactory cilia: linking sensory cilia function and human disease.</a> Chemical Senses. 34 (5), 451-464 (2009).</li><li>Tadenev, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+Bardet-Biedl+syndrome+protein-8+(BBS8)+perturbs+olfactory+function,+protein+localization,+and+axon+targeting.">Loss of Bardet-Biedl syndrome protein-8 (BBS8) perturbs olfactory function, protein localization, and axon targeting.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (25), 10320-10325 (2011).</li></ol>

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...

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 border="0" cellpadding="0" cellspacing="0" width="629">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:127px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Spring scissors</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">straight tip, multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Surgical scissors</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">sharp and blunt end, multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fine forceps</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">curved tips, Dumont #7, multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Razor blade</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">extra thin, multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Binocular with illumination</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">multiple suppliers, Stemi 2000-C, Zeiss</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri dish</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Liquid-blocker pen</td>
			<td style="width:127px;">Science Services</td>
			<td style="width:119px;">N71310</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polysine coated slides</td>
			<td style="width:127px;">Thermo Scientific</td>
			<td style="width:119px;">J2800AMNZ</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Confocal microscope</td>
			<td style="width:127px;">Leica Microsystems</td>
			<td style="width:119px;">TCS SPE</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">primary antibody Goat anti-Kirrel2</td>
			<td style="width:127px;">R&#38;D Systems</td>
			<td style="width:119px;">AF2930</td>
			<td style="width:167px;">1:200</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">primary antibody Rabbit anti-mOR-EG</td>
			<td style="width:127px;">Baumgart et al., 2014</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">secondary antibodies</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;">A21206, A11057</td>
			<td style="width:167px;">1:500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mounting medium, ProLong Gold antifade reagent</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;">P36930</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Paraformaldehyde</td>
			<td style="width:127px;">Sigma</td>
			<td align="right" style="width:119px;">441244</td>
			<td style="width:167px;">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...

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

Research

JoVE Journal

Neuroscience

11.0K 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

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

ホールマウントの胎児神経系の可視化ショウジョウバエ胚

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

神経科学

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

嗅神経細胞の繊毛のケージド化合物のフラッシュフォトリシス

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1).
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however, other options have been discussed as well5.
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.

マウス胚の脊髄の全載標本での分岐軸索勉強するDRGニューロンのDII -標識

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

の中枢神経系における単一細胞のラベリング<em&gt;キイロショウジョウバエ</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

血管新生を調査するために新生児マウス網膜の全体のマウント免疫蛍光染色<em生体内で&gt;</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

全マウント神経筋接合解析のための トランス対アブドミン筋 の解剖

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

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 ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

マウスの脳の側脳室における上衣繊毛のライブイメージング

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

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 ...

HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2. Consequently, investigation of structural details of the interaction between 5-HT2A and mGlu2 affecting schizophrenia-related behaviors represents a powerful translational tool. As previously shown, the head-twitch response (HTR) in mice is elicited by hallucinogenic drugs and this behavioral response is absent in 5-HT2A knockout (KO) mice3,4. Additionally, by conditionally expressing the 5-HT2A receptor only in cortex, it was demonstrated that 5-HT2A receptor-dependent signaling pathways on cortical pyramidal neurons are sufficient to elicit head-twitch behavior in response to hallucinogenic drugs3. Finally, it has been shown that the head-twitch behavioral response induced by the hallucinogens DOI and lysergic acid diethylamide (LSD) is significantly decreased in mGlu2-KO mice5. These findings suggest that mGlu2 is at least in part necessary for the 5-HT2A receptor-dependent psychosis-like behavioral effects induced by LSD-like drugs. However, this does not provide evidence as to whether the 5-HT2A-mGlu2 receptor complex is necessary for this behavioral phenotype. To address this question, herpes simplex virus (HSV) constructs to express either mGlu2 or mGlu2&#916;TM4N (mGlu2/mGlu3 chimeric construct that does not form the 5-HT2A-mGlu2 receptor complex) in the frontal cortex of mGlu2-KO mice were used to examine whether this GPCR heteromeric complex is needed for the behavioral effects induced by LSD-like drugs6.

This article describes how to inject viral vectors into the mouse frontal cortex to test behavioral assays that require GPCR heteromeric formation.

マウスにおけるGPCRヘテロマーの行動機能を研究するためのキメラ構築物のHSV媒介導入遺伝子発現

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2.  ...