ホマイラ・ナワビ、フレデリック・モレット、イザベル・サニャスの助けに感謝します。この研究はCNRS、アソシエーション・フランセーズ・コントル・レ・ミオパシー(AFM)、ANRヤドル、ラベックス・デヴェーカン、ラベック・コルテックス、ERC YODAからV.C.;C.D-BとA.Jは、それぞれラ・リーグ・コントル・ル・ガンとラベックス・DevWeCanフェローシップによってサポートされています。

1. プキシンA1受容体をpHluorinでタグ付けするクローニング戦略
<ol><li>適切な発現ベクターを骨格として選択する(例えばマウス受容体プレキシンA1発現ベクター、アンドレアス・プッシェル博士11の贈り物)。 注:このplexinA1ベクターは、効率的なHAタグ付き受容体挿入を実現するように設計されました。</li>
	<li>適切なプラスミドをテンプレートとして用いたエクリプティックpHluorinコード配列をPCRにより増幅する(例えば、pHluorinタグ付きGABA A受容体、ヤコブ2博士の贈り物)。必要に応じて、バックボーンのクローニングステップを容易にするために、プライマーの5'末端に制限部位を追加します。</li>
	<li>制限部位を用いてシグナルペプチドと受容体コード配列の間のフレーム内にpHluorin配列を挿入する(例えば図1Bに記載されているNruI/Kpn2I制限部位)。 注:受容体の正しい標的を保証するシグナルペプチドが切断されるため、pHluorinはその後に配置する必要があります。これはシグナルペプチドの認識を保証し、関心のある受容体からのpHluorinの切断を防ぐ。</li>
	<li>PCRによって突然変異が導入されないように得られた構成体を配列する。</li>
</ol>2. COS7細胞におけるpHluorinタグ付き受容体 インビトロ の特徴
融合タンパク質がpHが低下するほどの蛍光の血漿膜に到達する能力および蛍光の可逆的損失が低下する能力は、以下の手順を用いて確認できる。
<ol><li>1日目。完全なダルベッコの修正されたイーグルミディアムの2mlのガラス底35mm皿の皿1.5 x 105 COS7細胞(DMEM - 10%胎児の牛血清 - 1 mMナトリウムピルビン酸 - 25 U /mlペニシリン/ストレトマイシン - 2.5 μg/mlアンホテリシンB - pH 7.4)。</li>
	<li>2日目。トランスフェクト細胞: 注: セルは 70-80% コンフルエントである必要があります。<ol>
<li>200 μl NaCl 150 mMを準備し、3 μgのDNA すなわちpHluorinタグ付き受容体をコードするベクターを加えます。穏やかに渦を回転させ、短時間回転します。 注: これらの実験で使用した pHluorin-plexinA1 ベクターのマップを 図 1Bに示します。</li>
		<li>10 μlのトランスフェクション試薬(または使用する試薬の適切な量)を加えます。すぐに渦。</li>
		<li>RTで10分インキュベート。</li>
		<li>細胞に200μlのトランスフェクション試薬/DNAミックスを加えます。</li>
		<li>プレートを穏やかに移動して、ミックスの再パーティションを作成し、細胞を37°Cインキュベーターに戻します。</li>
	</ol>
</li>
	<li>3日目。トランスフェクション培地を取り除き、2mlの新鮮な完全なDMEMで交換してください。</li>
	<li>4日目。COS7 pHluorin-plexinA1 トランスフェクト細胞の生細胞イメージングを行う:<ol>
<li>DMEM完全培地の2つのアリコートを準備し、それぞれ3.5と9.5にpHを調整します。 注:1つの35 mmプレートでは、実験を行うために各溶液の1.5mlが必要です。</li>
		<li>細胞培地を取り出し、1mlのDMEM完全培地pH 7.4で交換してください。</li>
		<li>顕微鏡室を開かなくても細胞培養培地に直接様々な成分を注入するために、適切なタイプのチューブで5mlシリンジを調製します。</li>
		<li>37°C、5%CO2湿気の多い作業雰囲気のメンテナンスが可能なモジュールを使用してください。 注:CO2 チャンバの使用に対する別のアプローチは、HEPES緩衝媒体(通常、細胞タイプに応じて10〜25 mMの範囲)を使用することです。</li>
		<li>チャンバーにセルを置き、チューブと注射器を調整します。 注:記録中の機械的なドリフトを避けるために、顕微鏡は開始する前に平衡化する必要があります。</li>
		<li>イメージングソフトウェアを開き、多次元取得プログラムを選択します。</li>
		<li>40Xの目的を持つトランスフェクトされたCOS7細胞を見つけ、それぞれのソフトウェアで位置をマークします。</li>
		<li>Z スタックを設定して、15 μm の深度取得を行います(プレートにメディアを追加するとフォーカスが変わる可能性があります)。</li>
		<li>GFP フィルタとフェーズの露出を設定します。</li>
		<li>取得タイミングを設定します。 注: 5 つの対象分野を含む実験全体では、10 分間 20 秒ごとに取得すれば十分です。</li>
		<li>取得を開始し、DMEM pH 7.4培地で5つの制御画像を取ります。</li>
		<li>一時停止取得、pH 3.5の1.25 mlを注入し、培地中で5.5のpHを達成し、ソフトウェアでイベントをマークし、さらに5つの時間ポイントの取得を再開する。 注:緑色蛍光は徐々に消えるはずです。</li>
		<li>一時停止取得、pH 9.5完全DMEMの1.2 mlを注入して、培養培地中でpH 7.4を達成し、ソフトウェアでイベントをマークし、さらに5つの時間ポイントの取得を再開する。 注:緑色蛍光は、形質膜に再び現れるはずです。</li>
		<li>画像を分析します。 図2 は、pHluorin-plexinA1コンストラクトを用いてこのようなプロトコルで得られた代表的な画像を示す。
</li>
	</ol>
</li>
</ol>3. ovoでpHluorin-プレキジナ1コンストラクトのエレクトロポレーション
<ol><li>エレクトロポレーション前の卵の取り扱い:<ol>
<li>受精卵は、インキュベーションの1週間前まで14°Cの冷蔵庫に保管してください。</li>
		<li>卵を38.5°C(101.3°F)で、胚がステージHH1412に達するまで50〜52時間飽和湿度のインキュベーターでインキュベートする。 注:卵は卵の上部に浮かぶエレクトロポレーションのために胚が適切に配置されるように、インキュベーション中に水平に配置する必要があります。HH14段階は、適切な生存率を有する脊髄および後根神経節における分化ニューロンにおけるプラスミドの発現を得るのに適している。</li>
	</ol>
</li>
	<li>エレクトロポレート胚8-10:<ol>
<li>エレクトロポレーションを準備する:<ol>
<li>2 μg/μlより優れた濃度の内毒素を含まないDNAプラスミドを調製し、所望の濃度として希釈できるようにします。</li>
			<li>異なるDNA溶液を注入するのに十分なガラス毛細血管を引っ張ります。</li>
			<li>滅菌PBS(-Ca2+;-Mg2+)-100 U/mlペニシリン/ストレプトマイシンを準備し、38.5°Cで平衡化する。</li>
			<li>フード、湾曲したはさみ、細かい鉗子を殺菌する。</li>
			<li>電極間隔を制御します。 注:電極間の4mmのスペースは、一般的に使用されます。</li>
		</ol>
</li>
		<li>卵13(図3A)を窓ます):<ol>
<li>卵の鈍い側にシェルを突き刺すために湾曲したはさみを使用してください。</li>
			<li>0.9 mm x 25 mm の針と 5 ml のシリンジを使用して 2 ml のアルブミンを取り除きます。卵黄嚢を傷つけないように、針を縦方向に向けます。</li>
			<li>殻の完全性を維持するために、卵の上部をテープで覆います。</li>
			<li>湾曲したはさみを使用して、卵から2mlの卵子を取り除くときの圧力を均等にするためにテープの中央にシェルを突き刺す。その後、胚を視覚化するのに十分な大きさの窓を切り、それに取り組むことができる。</li>
			<li>胚の脱水を避け、マニピュレータにアクセスしやすくするために、〜2 mlの無菌温かいPBS(-Ca2+;-Mg2+)-100 U/mlペニシリン/ストレプトマイシンを加えます。</li>
		</ol>
</li>
		<li>DNAを注入し、胚を電気ポレートする<ol>
<li>PBSでプラスミドを希釈し(-Ca2+;-Mg2+)0.5~2 μg/μlの濃度にし、速い緑色の色素を加えて最終的な0.025%濃度に達します。DNAミックスを毛細血管にロードします。インジェクターの使用をお勧めします。 注: 毛細血管抵抗が大きすぎ(つまり、胚を注入するときに困難が発生する可能性があります)、または小さすぎる(毛細血管が大きすぎて胚に損傷を与える可能性があることを意味します)ことを確認してください。また、2μg/μlより高い核酸の濃度は、非特異的な効果を引き起こす可能性があり、制御する必要があります。</li>
			<li>卵黄嚢と神経管を尾頭側に穿刺し、毛細管を積んだ状態で穿刺する。神経管を浅い角度で入れ、尾から頭部までの内腔をDNAミックスで満たす(図3B)。 注: [高速緑色]では、射出精度を制御できます。</li>
			<li>4mmの白金電極を、電気ポレートしたいレベルで神経管の両側に素早く配置し、500ミリ秒間隔で31Vの3パルスを50msecに3パルスで3回塗布します(図3C)。 注:現像胚を損傷しないように、電極を心臓や大きな余分な胚容器に置かないようにしてください。電極に泡が形成されるはずです。</li>
			<li>針で、シェル内のレベルを減少させるためにアルブミンの2 mlを除去します。</li>
			<li>窓と鈍い側をテープで密閉して密封します。</li>
			<li>卵が所望の段階になるまで、インキュベーターで38.5°Cに戻します。</li>
		</ol>
</li>
	</ol>
</li>
</ol>4. 胚埋め込みと凍結切断
<ol><li>エレクトロポレーション後48時間、エレクトロポレート胚を慎重に収穫する(HH24ステージ)。絨毛管内膜のテープと半分を切ります。胚が黄身に沈むのを防ぐために、胚の下にザルを置き、絨毛球膜の後半を切断する。</li>
	<li>胚を氷冷PBSで満たされた解剖皿に移す。</li>
	<li>蛍光ステレオ顕微鏡で神経管内の蛍光を探すことでエレクトロポレーション効率を確認します。 注: RFPをコードするコントロールプラスミドの共エレクトロポレーションは、電気ポポレート領域を可視化するのに役立つ場合があります。</li>
	<li>マイクロスカルプテルを使用して胚を解剖し、脊髄の電気泳動領域を選択する。</li>
	<li>解剖した胚を24ウェルプレートに移し、pH 7.4 4%パラホルムアルデヒド(PFA)-リン酸緩衝液生理塩基(PBS)、O/Nを4°Cで固定する。 注: 固定ステップは、その「生きている」立体構造におけるpHluorinの安定化を可能にし、したがって、固定/透過組織でpHluorinを使用できるようにするために重要です。固定はpH依存的な蛍光変化を劇的に遅くするが、固定後も立体構造/プロトネーションの変化が起こり得ることを考慮する必要がある。したがって、以下のプロトコル(埋め込み、凍結切片および観察)は、固定ステップの3日以内に、すべてのバッファをpH7で行う必要がある。固定が必要ない場合は、生きた組織切片に対する観察を行うことを推奨する。</li>
	<li>4%PFAを取り除き、pH 7.4 PBSで胚を洗浄します。</li>
	<li>PBS-15%スクロースで胚をインキュベートし、胚が沈むまで4°Cに保ちます。</li>
	<li>固定胚をpH 7.4 7.5%ゼラチン-15%スクロースを37°Cで45分間インキュベートし、胚が完全に埋め込まれるようにする。</li>
	<li>氷の上に埋め込み金型を配置し、固体2ミリメートルベースを達成するためにpH 7.4 7.5%ゼラチン- 15%スクロースの400 μlを追加します。</li>
	<li>埋め込まれた胚を切り取り先端で吸引し、固体ゼラチンベースに胚を置く。</li>
	<li>pH 7.4 7.5%ゼラチン- 15%スクロースで覆い、ゼラチンが固まる前に鉗子で胚を配置する。</li>
	<li>ゼラチンが固まったら、-40°Cのイソプロパノール浴(ドライアイスまたは液体窒素を使用)を準備し、ゼラチンブロックを5分間凍結します。</li>
	<li>冷凍ブロックは-80°Cに保ちます。</li>
	<li>冷凍ブロックを-20°Cに1時間置きます。</li>
	<li>モールドを取り外し、ブロックをポリエチレングリコール媒体でチャックに固定します。</li>
	<li>ブロックがしっかりと固定されたら、チャックをクライオスタットシステムに入れます。 注:染色中の組織の損失を避けるためにコーティングされたスライドを使用してください。</li>
	<li>シリアルクライオセクションを実行します(通常は20μmのクライオセクションが行われます)。</li>
	<li>泣き切りはRTで15分間乾燥させます。 注: GFP 蛍光の漂白を避けるために、クライオセクションは不必要な光暴露から保護する必要があります。</li>
</ol>5. クライオセクションの顕微鏡解析
<ol><li>10分間RTでpH 7.4 PBSで凍結切片を水分補給する。 注: 必要に応じて、核はHoechstで染色することができます。</li>
	<li>PBSで0.5 μg/mlのHoechst溶液を使用し、15分間凍結液をインキュベートします。</li>
	<li>スライド3xをpH 7.4 PBSで5分間リンスします。</li>
	<li>スライド取付けに進みます。O/Nを硬化させるpH 7.4(またはより基本的な)ポリビニルアルコール取り付け溶液を使用することができます:慎重にスライドとカバースリップの間の気泡の形成を避けるためにカバースリップを配置します。</li>
	<li>取り付け媒体は、暗闇の中で4°CでO /Nを硬化させます。</li>
	<li>反転共焦点顕微鏡を使用して、pHluorin-plexinA1融合タンパク質を正確に視覚化:最適なピンホールと光学分解能でZスタックを実行し、20X(NA 0.75)または40X(NA 1.3)レンズを使用します。 注: pHluorin は、GFP の検出に使用される同じパラメータ (つまり、509 nm の放出ピーク) で検出可能です 。波長励起および検出フィルタの設定は、イメージングソフトウェアによって最適に定義されます。Hoechstは425-460 nm(励起は405 nm)の間で検出され、GFPまたはpHluorinは485-545 nm(励起は473nm)の間で検出され、RFPは575-675 nm(励起は559nm)の間で検出される。</li>
</ol>チクチの脊髄のpHluorin-plexinA1およびeGFP発現の代表的な画像を 図4に示す。

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著者らは開示するものは何もない。

このプロトコルは、細胞培養およびひよこ胚脊髄の発達コンテキストの両方において軸索誘導受容体のダイナミクスに従うステップバイステップの手順を提供する。
デノボpHluorin タグ付きタンパク質を設計するには、クローニング戦略に関して2つの点を考慮する必要があります。まず、pHluorinタグは、酸性のエンドーソームの内腔に曝され、その結果、細胞膜受?...

彼らのナビゲーション中に、軸索は彼らの目標に向かってそれらを導く複数の環境の手がかりを統合する。これらの手がかりは、軸索末端の表面で誘導受容体を活性化し、成長コーンは、今度は適切なシグナル伝達経路を開始する。したがって、受容体の細胞表面分布の時間的および空間的調節は、成長コーン1の感度を設定するために重要である。この文脈では、コミシャル軸索による正中線交差は、受容体細胞表面レベルの調節を調査するための優れたモデルである。発達中の脊髄では、コミシャル軸索は当初、正中線を横切る腹側床板に向かって引き付けられる。交差した後、床板の誘引物質への応答性を失い、床板を出て神経系2,3の反対側の最終目的地に向かってナビゲートできるように、床板忌避剤に対する応答を得る。成長コーン表面における受容体の利用可能性の調節は、正中のキューへの応答性の切り替えの基礎となるメカニズムの1つである 4,5.したがって、成長コーンの原形質膜に存在する受容体の選択的モニタリングが最も重要である。ここでは、現像するひよこ脊髄において、 生体外 および 生体内で血漿膜に対処される軸索誘導受容体を特異的に可視化する緑色蛍光タンパク質(GFP)変異体のpH依存性蛍光性に基づく方法について説明する。
Rothmanたちの研究グループは、ecliptic pHluorin6を含むGFPのpH感受性変異をポイント変異によって設計した。エクリプティックpHluorinは、中性pHで蛍光されながら酸性pH(<6)に曝露されると非蛍光であるという性質を有する。これにより、細胞内酸性コンパートメントに局在する非蛍光受容体(すなわち、内生小胞)を、細胞膜に取り込まれ、細胞外中性pH7に曝露される蛍光受容体から区別することができる。これを利用して、プレキシンA1の細胞膜局在を監視し、正中忌避セマフォリン3B5 に対する成長コーン応答を媒介する軸索誘導受容体である(図1A)。ここでは、pHluorin-plexinA1コンストラクトの インビトロ 特徴付けについて、発達中のひよこ脊髄におけるこの構造の ovo エレクトロポレーション8-10 と共に、空間および時間的解像度 の両方で生体内 の軸索誘導受容体ダイナミクスに従うことを可能にする極低温分析を行う。

<table border="0" cellpadding="0" cellspacing="0" width="1007">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">COS7 cells</td>
			<td style="width:147px;">ATCC</td>
			<td style="width:219px;">CRL-1651</td>
			<td style="width:379px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">DMEM GlutaMAX</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">61965-026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Sodium pyruvate</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">11360-039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Amphotericin B</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:219px;">A2942</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Fetal bovine serum</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">10270-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Penicillin/Streptomycin</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:264px;">Exgen500 reagent</td>
			<td style="width:147px;">Euromedex Fermentas</td>
			<td style="width:219px;">ET0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">PBS -Ca2+ -Mg2+</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Fast green dye</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:219px;">F7252</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:264px;">32% Paraformaldehyde aqueous solution</td>
			<td style="width:147px;">Electron Microscopy</td>
			<td style="width:219px;">15714-S</td>
			<td>Dilute extemporaneously in PBS to achieve a 4% solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Gelatin from cold water fish skin</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:219px;">G7041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Sucrose</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:219px;">S0389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Cryomount</td>
			<td style="width:147px;">Histolab</td>
			<td style="width:219px;">00890</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Hoechst 34580</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:219px;">H21486</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Mowiol 4-88</td>
			<td style="width:147px;">Fluka</td>
			<td style="width:219px;">81381</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:1008px;">Consumables</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Bottom-glass 35 mm dish</td>
			<td style="width:147px;">MatTek</td>
			<td style="width:219px;">P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">5 ml Syringe</td>
			<td style="width:147px;">Terumo</td>
			<td style="width:219px;">SS-05S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Needles 0.9 mm x 25 mm</td>
			<td style="width:147px;">Terumo</td>
			<td style="width:219px;">NN-2025R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Capillaries</td>
			<td style="width:147px;">CML</td>
			<td style="width:219px;">PP230PO</td>
			<td>capillaries are stretched manually in the flame</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Superfrost Plus Slides</td>
			<td style="width:147px;">Thermo Scientific</td>
			<td>4951PLUS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:1008px;">Material</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Curved scissors</td>
			<td style="width:147px;">FST</td>
			<td style="width:219px;">129-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Microscalpel</td>
			<td style="width:147px;">FST</td>
			<td style="width:219px;">10316-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Forceps</td>
			<td style="width:147px;">FST</td>
			<td style="width:219px;">Dumont #5 REF#11254</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:1008px;">Equipment/software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Time lapse microscope</td>
			<td style="width:147px;">Zeiss</td>
			<td style="width:219px;">Observer 1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:264px;">Temp module S</td>
			<td style="width:147px;">PECON for Zeiss</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">CO2 module S</td>
			<td style="width:147px;">PECON for Zeiss</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Metamorph software</td>
			<td style="width:147px;">Metamorph</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:264px;">Eggs incubator</td>
			<td style="width:147px;">Sanyo</td>
			<td style="width:219px;">MIR154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Electroporator apparatus</td>
			<td style="width:147px;">Nepa Gene CO., LTD</td>
			<td style="width:219px;">CUY21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Electrodes</td>
			<td style="width:147px;">Nepa Gene CO., LTD</td>
			<td style="width:219px;">CUY611P7-4</td>
			<td>4 mm platinum electrodes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Fluorescence stereomicroscope</td>
			<td style="width:147px;">LEICA</td>
			<td style="width:219px;">MZ10F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Cryostat</td>
			<td style="width:147px;">MICROM</td>
			<td style="width:219px;">HM550</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Confocal microscope</td>
			<td style="width:147px;">Olympus</td>
			<td style="width:219px;">FV1000, X81</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Fluoview software</td>
			<td style="width:147px;">Olympus</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CLC Main Workbench software</td>
			<td style="width:147px;">CLC Bio</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

use of phluorin to assess the dynamics of axon guidance receptors in cell culture and in the chick embryo

開発中、軸索誘導受容体は、魅力的で反発的な手がかりの両方に対する軸索感度を調節する上で重要な役割を果たす。実際に、ガイダンス受容体の活性化は、軸索先端、成長コーン、リガンドに応答することを可能にするシグナル伝達機構の第一段階である。したがって、細胞表面でのそれらの可用性の変調は、成長コーン感度の設定に関与するメカニズムの一つである。ここでは、発達中のひよこ脊髄におけるインビトロおよびインビボの両方の軸索誘導受容体の時空間的細胞表面ダイナミクスを正確に可視化する方法について述べている。緑色蛍光タンパク質(GFP)変異体のpH依存性蛍光性を利用して、細胞膜に取り組む軸索誘導受容体の割合を特異的に検出しました。我々は、まず、このようなpH依存性構造のインビトロ検証を記述し、さらに、関心のある軸索誘導受容体の時空間的ダイナミクスを評価するために、ひよこ脊髄における生体内でのそれらの使用をさらに詳述する。

<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50883/50883fig1highres.jpg" src="/files/ftp_upload/50883/50883fig1.jpg"> 図 1.A. 細胞内におけるpHluorin-plexinA1蛍光特性のスキーム。PHluorinは、pHが酸性(<6)である細胞内区画では、小胞や内生体の中などで酸性であり、pHが中性である細胞外媒体に曝露されると蛍光性を有する。これにより、pHluorin-plexinA1受?...

Watch this Scientific Journal Video about Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo at JoVE.com

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

ここでは、pH感受性緑色蛍光タンパク質変異体pHluorinを用いて、細胞表面における軸索誘導受容体の密売の時空間的ダイナミクスを研究する。pHluorinタグ付き受容体は、細胞培養と 生体内の両方で発現し、ひよこ胚のエレクトロポレーションを用いる。

細胞培養およびひよこ胚における軸索誘導受容体のダイナミクス評価におけるpHluorinの使用

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

use-phluorin-to-assess-dynamics-axon-guidance-receptors-cell-culture

Research

JoVE Journal

Neuroscience

8.2K ビュー.  University of Lyon. ここでは、pH感受性緑色蛍光タンパク質変異体pHluorinを用いて、細胞表面における軸索誘導受容体の密売の時空間的ダイナミクスを研究する。pHluorinタグ付き受容体は、細胞培養と 生体内の両方で発現し、ひよこ胚のエレクトロポレーションを用いる。

ビデオ: Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

でProprioceptorsの可視化<em&gt;ショウジョウバエ</em&gt;幼虫および蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

神経科学

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

イメージング初代培養マウス神経細胞における形質膜に受容体の挿入をpHluorinタグ付き

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

ニワトリ胚における軸索軌道とシナプスターゲットを追跡する後脳のエレクトロ

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

神経発火率を操作することによってアストロサイト受容体の可塑性を誘導すること

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

マウスの胚感覚軸索投影のホールマウントイメージング

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

イメージングカルシウム<sup&gt; 2+</supマウス網膜の光受容コーン軸索末端で&gt;ダイナミクス

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

軸索分岐と神経形態形成中に形質膜配信の役割を定義するために組み合わせる方法論を活用

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

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

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

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

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

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

Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, assembly and activity-dependent plasticity. Giant ankyrin-G, the master organizer of AIS, directly associates with membrane-spanning voltage gated sodium (VSVG) and potassium channels (KCNQ2/3), as well as 186 kDa neurofascin, a L1CAM cell adhesion molecule. Giant ankyrin-G also binds to and recruits cytoplasmic AIS molecules including beta-4-spectrin, and the microtubule-binding proteins, EB1/EB3 and Ndel1. Giant ankyrin-G is sufficient to rescue AIS formation in ankyrin-G deficient neurons. Ankyrin-G also includes a smaller 190 kDa isoform located at dendritic spines instead of the AIS, which is incapable of targeting to the AIS or rescuing the AIS in ankyrin-G-deficient neurons. Here, we described a protocol using cultured hippocampal neurons from ANK3-E22/23-flox mice, which, when transfected with Cre-BFP exhibit loss of all isoform of ankyrin-G and impair the formation of AIS. Combined a modified Banker glia/neuron co-culture system, we developed a method to transfect ankyrin-G null neurons with a 480 kDa ankyrin-G-GFP plasmid, which is sufficient to rescue the formation of AIS. We further employ a quantification method, developed by Salzer and colleagues to deal with variation in AIS distance from the neuronal cell bodies that occurs in hippocampal neuron cultures. This protocol allows quantitative studies of the de novo assembly and dynamic behavior of AIS.

Here, we described a protocol to quantitatively study the assembly and structure of the axon initial segments (AIS) of hippocampal neurons that lack pre-assembled AIS due to the absence of a giant ankyrin-G.

主培養海馬ニューロンを用いた軸索初期セグメントの組み立て研究

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, ...

Isolation and Culture of Chick Ciliary Ganglion Neurons

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system and are responsible for the innervation of the muscle tissues present in the eye. This ganglion is constituted by a homogenous population of ciliary and choroidal neurons that innervate striated and smooth muscle fibers, respectively. Each of these neuronal types regulate specific eye structures and functions. Over the years, neuronal cultures of the chick ciliary ganglia were shown to be effective cell models in the study of muscle-nervous system interactions, which communicate through cholinergic synapses. Ciliary ganglion neurons are, in its majority, cholinergic. This cell model has been shown to be useful comparatively to previously used heterogeneous cell models that comprise several neuronal types, besides cholinergic. Anatomically, the ciliary ganglion is localized between the optic nerve (ON) and the choroid fissure (CF). Here, we describe a detailed procedure for the dissection, dissociation and in vitro culture of ciliary ganglia neurons from chick embryos. We provide a step-by-step protocol in order to obtain highly pure and stable cellular cultures of CG neurons, highlighting key steps of the process. These cultures can be maintained in vitro for 15 days and, hereby, we show the normal development of CG cultures. The results also show that these neurons can interact with muscle fibers through neuro-muscular cholinergic synapses.

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system. Neuronal cultures of chick CG neurons were shown to be effective cell models in the study of nerve muscle interactions. We describe a detailed protocol for the dissection, dissociation and in vitro culture of CG neurons from chick embryos.

チラリア性神経節ニューロンの分離と培養

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system and are responsible for the innervation of the muscle tissues present in the ...