Name: measuring synaptic vesicle endocytosis in cultured hippocampal neurons
Uploaded: 2017-09-04T12:30:00.000+00:00
Duration: 7 min 30 s
Description: Watch this Scientific Journal Video about Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons at JoVE.com

朱永陵先生にありがとう VAMP2 phluorin を提供するため、シナプトフィジン pHluorin2x 構造と博士ジェームズ e. ロスマンを提供します。彼らのテクニカル サポートおよびヘルプ、博士スーザン ・ チェンと組織プラスミノーゲンアクテベータ電子顕微鏡施設のバージニア クロッカーに感謝します。この作品は国立研究所の神経疾患と脳卒中学内研究プログラム米国の生命研究イニシアティブ プログラム (韓国生物医学科学者フェローシップ プログラム)、韓国研究所からの助成金によって支持されました。バイオ サイエンスとバイオ テクノロジー、大韓民国。

注: 次のプロトコルは、pHluorin イメージング法と培養海馬ニューロン pHluorin モニター シナプス小胞タンパク質吸収細胞内で使用されている EM 方法を説明しますと、EM はシナプス小胞の取り込みを検出し、。微細構造変化します。
 動物のケアとプロシージャの NIH ガイドラインに従った NIH 動物ケアおよび使用委員会によって承認されました。
 1 です。 pHluorin イメージング <ol><li>海馬ニューロンの文化 <ol><li>4 mM NaHCO 3 と 5 mM HEPES を組み合わせることによって準備海馬バッファー (HB) 5 M NaOH で pH 7.3 に調整。Neurobasal、中 2 %b27、0.5 mM L グルタミンと 1% ペニシリン-ストレプトマイシンを混合することによって培養液を作る。さらに、HB バッファーと 20% の混合物を準備ウシ胎児血清 (FBS HB/20%). 
		注: この養殖のプロトコルは、Sankaranarayanan、ら に基づいてください。 29 日 呉 、ら 24</li> <li>培養液に 2 日目に生後 0 日間マウスの子犬の首をはねるし、4 ° C HB/20% FBS に脳を抽出します。脳幹、視床、なったを公開するを削除します。脳幹と視床の除去によってそれを公開した後、なったを分析し、新鮮な 4 ° C HB/20% FBS に転送します 。
		注: 通常、利回りは約 4 x 10 5 セル/mL 1 匹の子犬 (2 つなった) のです。ピンセットやはさみを使用して付着膜をきれいにし、新鮮な 4 ° C HB/20% FBS に転送します。海馬歯状回を引っ張り出すと、はさみで切断することによって、subiculum を分離し、新鮮な 4 ° C HB/20% FBS に転送します</li>。
		<li>約 10 スライスに端から端までを切断することによって各海馬を分割し、15 mL のポリプロピレンの円錐管にそれらを転送します</li>。
		<li>を解決する組織を可能にした後 HB/20% FBS の 10 mL で洗浄し、HB の 10 mL で 3 回洗う</li>。
		<li>は 137 mM の NaCl、KCl 5 mM、7 mM Na2HPO 4 25 mM HEPES、消化ソリューションを準備し、5 M NaOH で pH 7.2 に調整します。なったから上澄みを削除します。トリプシンの 10 mg と 1 mg DNase の消化液 2 mL を加えるし、サンプル ペレットに直接滅菌 0.22 μ m 膜を介してフィルターします</li>。
		<li>なった 37 ° C で 5 分間インキュベートし、HB/20% FBS の 10 mL で 2 回洗浄し、HB の 10 mL で 1 回洗浄.</li>
		<li>解離 MgSO 4 mg を 6 セル &#8729; 7 H 2 O と DNase の 1 mg 2 mL HB と、なった上に滅菌フィルター滅菌 0.22 μ m のフィルターを通してペレットします。軽く気泡を導入することを避けるために世話をしながらの慎重な triturating によって細胞を切り離します。2 分のために解決する組織微粒子を許可し、別のチューブに上清をゆっくり転送します</li>。
		<li>追加 3 mL HB/20% FBS に細胞懸濁液、および遠心分離、または 1,000 rpm、4 ° C で 10 分。上澄みを廃棄し、培地の再懸濁します</li>。
		<li>プレート 60,000 セル、coverslip オフ流出なしのポリ-D-リジン コーティングされた直径 25 mm coverslip の 150 μ L の培地の中断します。めっき後に予め温めておいた培 2 h の 2 mL を追加します。Coverslips 6 ウェル培養プレートまたは滅菌シャーレに保持されます</li>。
		<li>37 ° c 5% で維持する細胞の CO 2 は、14-21 日録音前の培地のインキュベーターを加湿しました。培地の上半分を週に 2 回変更文化成長中です</li>。
	</ol></li> <li>トランスフェクション <ol><li>メッキ後、6-7 日 transfect シナプトフィジン pHluorin 2 X (SpH) または海馬ニューロンに VAMP2 pHluorin。SpH、サイトメガロ ウイルス (CMV) のプロモーターは、pcDNA3 ベクトル 30 に挿入を使用します。VAMP2-pHluorin を使用して pCI ベクトル 31 挿入 CMV プロモーター</li>。
		<li>は、1 μ g のプラスミドを用いた標的細胞に脂質キャリアを使用していずれかのベクトルを transfect します。Transfection のため血清を欠いている、プロトコル手順 1.1.1 から培を使用します。毒性を軽減するトランスフェクション後中 2 h を変更します 。
		注: ボタンで、SpH の低式の場合細胞が健康でない限り、DNA 濃度を脂質キャリアと 2 μ g または培養時間を増加します。通常、4-10 セル (0.006 0.008%) 神経細胞のトランスフェクションした</li>。
	</ol></li> <li>光顕微鏡 <ol><li>119 mM 2 mM の CaCl 2、2.5 mM KCl、25 mM HEPES (pH 7.4) 30 mM グルコース 2 mM MgCl 2、0.01 mM 6-cyano-7-nitroquinoxaline-2,3-dione (塩化ナトリウムから成る生理食塩水の準備CNQX) は、0.05 mM DL-2-アミノ-5-phosphonovaleric 酸 (AP5)。刺激、潤滑剤、シール剤を使用してリークを回避することができるイメージング室培養培地プレートと場所から観察を取る。ガラス、カバーガラスのプレートから商工会議所への転送中にすぐに食塩通常 750 μ L を追加して乾燥させない 。
		注: CNQX と AP5 再発活動の可能性のあるシナプスの活動をブロックするために使用されました。配置する前に倒立蛍光顕微鏡の商工会議所は商工会議所が漏れていないことを確認するのに洗浄ティッシュを使用します。屈折率の変化の結果通常生理食塩水および水浸オイルの混合によって記録中にフォーカスの変更を引き起こす遅い漏れ</li>。
		<li>メタルハライド ランプとレンズの刺激と <ol><li>画像 60 X で広視野倒立顕微鏡上 (1.4 開口) オイルの液浸を記録します。480 の励起ピーク設定フィルター pHluorin を視覚化するフリップ マニュアル 500 550 nm 発光フィルター、490 nm ロングパス鏡 nm。電子乗算電荷結合デバイス (EMCCD) カメラを使用して 2 x 2 のビニングと 100 ms 毎の画像をキャプチャします 。
			注: いくつかの機能は、退色、画像キャプチャ間隔を含むを避けるために適切な条件を達成してフィルター処理に考慮すべき、ビニング、機器に依存しています。共焦点レーザー顕微鏡の場合、レーザー パワーと暴露時間退色を避けるために見なす必要があります。刺激がなければ、少なくとも 3 分を記録した後セットアップを決定し、少なくとも 10 の記録を行った退色をチェックするための刺激がなければ s.</li>
			<li>は、各実験の研究分析の容易さのための高密度エリアを選択します。ブートン、ボタン間の連続式の円形または楕円形の表現パターンに重点を置いて、緑の蛍光性信号によって transfected セルを識別します</li>。
			<li>を使用して 1 刺激による誘発エンドサイトーシス ms パルス、20 mA 活動電位 (AP) パルス刺激装置から供給および白金電極刺激・ アイソレーション ・ ユニット内で配信。刺激と細胞回復中にわたって蛍光活性をイメージします 。
			注: 死んでいるセルの場合式生活研究より強かったし、ボタン間斑点状のパターンを示した</li>。
		</ol></li></ol></li> <li>画像解析 <ol><li>単一のブートンで蛍光強度を分析するため設定を地域の関心 (ROI) 1.5 倍の 1.5 μ m 正方形;ブートンのサイズは約 1.5 μ m 使用退色を確認し、背景として減算への刺激の前にトレース内です</li>。
		<li>正規化蛍光式 (ΔF) を変更: <img alt="Equation"height="11"src="/files/ftp_upload/55862/55862eq1.jpg"width="172"/> 、F 最大 F 0 を参照してください最大増加刺激後・ ベースライン蛍光、それぞれ。として測定するエンドサイトーシス速度減衰率最初 4-10 s pHluorin 蛍光の最大のポイントの後。エンドサイトーシスの継手ベースライン モノラル指数関数にするピーク増加から pHluorin 蛍光減衰時定数 (τ) を取得します</li>。
	</ol></li></ol> 2。電子顕微鏡 <ol><li>準備ポリ-D-リジン コーティング 6 ウェル プレート、常温で 1 h の各ウェルに 0.01% 無菌ろ過ポリ-D-リジン溶液 1.5 mL を適用し、滅菌水で 3 回を洗浄します。分析、文化、1.1.1、1.1.2、1.1.3 の手順のように海馬ニューロンをそれぞれ保持します</li>。
	<li>準備 31.5 mM の NaCl、2 mM CaCl 2、90 mM KCl、25 mM HEPES (pH 7.4) 30 mM グルコースとして HRP に高 K + 刺激ソリューション pH 5 m 7.4 に調整する 2 mM MgCl 2、CNQX 0.01 mM、0.05 mM AP5、5 mg/mL HRP、NaOH。
	<ol><li>1.5 ml の高い海馬ニューロンの文化を刺激するため K + 刺激ソリューション室温 (呼ばれる K +) 各ウェルに 1.5 mL の添加により 90 で s。90 の HRP を 5 mg/mL の同じ濃度を適用 (R と呼ばれる) 静止状態で s、生理食塩水で。回収サンプルの K + サンプル高 K + 刺激ソリューションを適用し、急速に洗って食塩に置き換えるし、10 分間インキュベート</li>
	</ol></li> <li>固定および汚損 <ol><li>pH 7.4 で 21.4 グラム/L の Na cacodylate を使用して 0.1 M Na cacodylate バッファーを準備します。室温で少なくとも 1 時間 0.1 M Na cacodylate バッファーの 4% グルタルアルデヒドでセルを修復します。7 分の 0.1 M Na cacodylate バッファーで 3 回洗って</li>。
		<li>準備ジアミノベンジジン (軽打) ソリューションでは、0.5 mg/mL から成る軽く H 2 O 2 ddH 2 O、0.22 μ m フィルターでフィルターで 0.3%。3 回 7 分の 0.1 M Na cacodylate バッファーを 37 ° c. の洗浄で 30 分の 1.5 mL DAB ソリューションを適用します 。
		注意: DAB は有害と疑われる発がん性物質です。手袋、白衣を使用してください 。
		注: 軽打とラベリング H 2 O 2、その酸化による HRP により触媒とに発生します。サンプルでは、増加する信号のコンポーネントの結果に小さな増加。HRP の濃度を増加させる、触媒の効果がスピードアップします。H 2 O 2 の十分に大きい濃度 32 をラベリングの効果を阻害すると HRP、副反応を衰弱させるを可能にします。今回、このラベリング システムの濃度は現在利用可能な研究 33 , 34 に基づいて選ばれた</li>。
		<li>では、0.1 M Na cacodylate 緩衝固定の記事、4 ° C で 1 時間に 1% OsO 4 の 1.5 mL でニューロンを孵化させなさい。7 分の 0.1 M Na cacodylate バッファーの 1.5 mL で 3 回洗って 。
		注意: 毒性と OsO 4 の反応性のため氷のサンプルをおいて化学フードは孵化に冷蔵庫を使用する多くの場合望ましい</li>。
		<li>準備 0.1 M 酢酸緩衝液 13.61 グラム/L のナトリウム酢酸と 11.43 mL/L 氷酢酸で ph 5.0。1.5 mL の 0.1 M 酢酸緩衝液 pH 5.0 7 分間で 3 回洗浄し、pH 5.0 4 で 1 時間で 0.1 M 酢酸緩衝液で 1.5 mL 1 %005. incubate ° C. 洗浄 3 回 7 分の 1.5 mL の 0.1 M 酢酸緩衝液と</li>。
	</ol></li> <li>エポキシを埋め込む <ol><li>シングル 1.5 mL 各 7 分、50%、70%、90% のエタノールの洗浄とニューロンの文化を脱水し、3 は発煙のフード 7 分 1.5 mL 100% エタノールの洗浄です</li>。
		<li>ミックス 485 mL/L bisphenol-A-(epichlorhydrin) エポキシ樹脂、160 mL/L ドデセニル無水コハク酸 (性)、340 mL/L メチル-5-ノルボルネン-2, 3-ジカルボン酸 (NMA) と 15 mL/L 2,4,6-トリス (dimethylaminomethyl) フェノール (DMP 30) エポキシを作成するには樹脂です。ミックスが徹底的に、空気の泡を削除する真空下で格納します 。
		注: それは特にそれらが肉眼に目に見えるよりも小さい樹脂に空気の泡を削除する重要なので断面中に樹脂のキャビティを引き起こす可能性が</li>。
		<li>50% エポキシとエタノールを置き換えることによってサンプル樹脂、シェーカーで室温で 30 分間エタノールを 70% エタノール シェーカーで室温で 30 分間のエポキシ樹脂そして潜入</li>。
		<li>スイッチ エポキシ 100% エポキシ樹脂でソリューションを樹脂し、50 ° C 追加新鮮な 100% エポキシで 1 時間孵化と新鮮な 100% エポキシ樹脂の 50 ° C の実行の 2 つの取引所で 10 分間インキュベート樹脂し、50 ° c 夜間、60 ・ #17 でのセキュリティを強化することができます。6;36 h 以上を強化するための C.</li>
	</ol></li> <li>ムーティ ウェル プレートから宝石商の各サンプルを削除 &#39; s ハンドソー。密な濃度は、倒立顕微鏡を使用し、ミクロトームによる区分のための 70 に 80 nm ブロックのセルの関心の領域を選択します。ミクロトーム チャック内カットの領域をマウントし、ミクロトームをロードします。ミクロトームでチャックを置き、エッジ ブロックの表面に平行にダイヤモンド ナイフをマウントします。個々 のグリッドに直接 70 に 80 nm 厚のセクションを収集します</li>。
	<li>は、重量、1% 溶液を水に酢酸ウランを溶解し、個別に重量によって 3% の解決のため水にクエン酸鉛を溶解します。15 分間の 1% 水溶液 005. とし、試料のコントラストを改善するために 5 分間 3% 水溶液鉛クエン酸によるセクションを counterstain</li>。
	<li>EM イメージング <ol><li>電子顕微鏡セクションと 10,000-20, 000 × 3 の主な倍率で CCD のデジタル カメラ画像を記録確認します</li>。
	</ol></li> <li>統計 <ol><li>t を実行-制御と実験サンプル平均と標準誤差測定 (s.e.m.) を比較することによって大きな違いを識別するためにテストします</li>。
	</ol>
	</li>
</ol>

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ここでは、シナプス小胞エンドサイトーシスを監視するための 2 つの方法を示します。最初のメソッドでは、pHluorin transfected ニューロンのシナプス小胞タンパク質と融合し、その後電気刺激を監視しました。第二に、kcl と HRP 取り込みの EM イメージングを使用しました。2 つの理由のためにさまざまな刺激を使いました。まず、高カリウム施肥は文化のすべてのニューロンの脱分極を誘導し?...

神経伝達物質は、シナプス小胞に格納され、エキソサイトーシスによってリリースされました。シナプス小胞膜および蛋白質は、エンドサイトーシス、によって、開口放出の次のラウンドで再利用します。シナプス小胞エンドサイトーシスは、細胞膜から突き出た小胞を削除しますシナプス小胞のプールを維持するため重要です。酸性情況で急冷、中性 pH で蛍光 pH 敏感な緑色蛍光タンパク質 pHluorin は、エンドサイトーシス時間コース ライブ セル1,2,3を測定する使用されています。PHluorin 蛋白質は通常シナプトフィジンや VAMP2/シナプトブレビンなどのシナプス小胞タンパク質の内腔側に接続されています。でも、pHluorin はシナプス小胞の 5.5 の pH ルーメンで急冷され。細胞膜に小胞の融合を公開、pH が 7.3 〜 細胞外の溶液に小胞の内腔 pHluorin 蛍光性の増加の結果します。エキソサイトーシス後、シナプス小胞蛋白質それら回復小胞内小胞再酸性化に続いてのエンドサイトーシスによる増加の蛍光が崩壊します。崩壊は、エンドサイトーシスと小胞の再酸性化を反映しているが、再酸性化がほとんど条件1,4エンドサイトーシスよりも速いのでそれ主のエンドサイトーシスを反映します。再酸性の時定数は 3-4 秒または 10 よりも一般に高速は5,6に少ない s 以上の小胞エンドサイトーシス4,5に必要な。シナプス小胞タンパク質を取得するかどうかを決定する 5.5 の pH の 4 Morpholineethanesulfonic 酸 (MES) 溶液 (25 mM) を用いた酸焼入れ実験を使用ことができます実験が再酸性からエンドサイトーシスを区別するために必要な場合エンドサイトーシス1,3,4を介して原形質膜。したがって、pHluorin 蛍光強度増加エキソおよびエンドサイトーシス、残高反映して反映されます神経刺激後減少特にエンドサイトーシス。
確率誘発リリースと自発的な解放9および pHluorin イメージングがエンドサイトーシスの時間のコースもシナプス小胞プール7、8サイズを測定するだけではなく使えます。多くの要因とエンドサイトーシスなどカルシウム、水溶性 NSF 添付ファイル蛋白質受容体 (スネア) 蛋白質、脳由来神経栄養 factor(BDNF)、カルシニューリンの調節に関与するタンパク質は、pHluorin イメージング1を使用して識別されています。,2,10,11,12,13,14,15,16. 唯一の第一次ニューロンではなく観察17神経芽細胞腫、さらに、神経伝達物質の放出が検出できます。最近では、pHluorin 亜種、下流、mOrange、pHTomato は、単一シナプス18,19で複数の要因を同時記録を監視するため開発されました。たとえば、pHTomato シナプトフィジンと融合され遺伝的コード化カルシウム インジケーター (GCaMP5K) とシナプス小胞の融合およびシナプス コンパートメント20の Ca2 +流入監視に使用します。そのため、シナプス蛋白質に接続されている pHluorin は、エンドサイトーシスとエキソサイトーシスとの関係を分析する有用な方法を提供します。
EM は一般的エンドサイトーシス中の微細構造の変化を示す高空間分解能のためのエンドサイトーシスを研究するために使用する別の方法です。2 つの一般的な領域は、神経細胞21内の病理学的変化を視覚化し、小胞蛋白質22を追跡する機能です。特に、シナプス小胞の取り込み、膜の曲率の観察 periactive ゾーンにクラスリン被覆し、エンドソーム構造が EM3,23,24,25 で可能 ,26,,2728。EM で定着剤による奇形などの潜在的なアーティファクトは、エンドサイトーシス、影響を与える可能性があります、データ分析は労働集約型、解像度は細胞構造を視覚化するための魅力的な機会を提供します。潜在的な定着性の問題と EM の時間分解能の限界は、凍結、エンドサイトーシス27の間に現在の繊細な構造の安定化の高速で非化学メソッドを提供する高圧で克服できます。

<table><tbody><tr><td>Lipofectamine LTX with Plus</td><td>Thermo Fisher</td><td>15338-100</td><td>Transfection of plasmid DNA including synaptophysin or VAMP2-pHluorin</td></tr><tr><td>neurobasal medium</td><td>Thermo Fisher</td><td>21103-049</td><td>Growth medium for neuron, Warm up to 37&amp;deg;C before use</td></tr><tr><td>B27</td><td>Thermo Fisher</td><td>17504-044</td><td>Gradient for neuronal differentiation</td></tr><tr><td>Glutamax</td><td>Thermo Fisher</td><td>35050-061</td><td>Gradient for neuronal culture</td></tr><tr><td>Poly-D-Lysine coated coverslip</td><td>Neuvitro</td><td>GG-25-pdl</td><td>Substrate for neuronal growth and imaging of pHluorin</td></tr><tr><td>Trypsin XI from bovine pancrease</td><td>Sigma</td><td>T1005</td><td>Neuronal culture-digest hippocampal tissues</td></tr><tr><td>Deoxyribonuclease I from bovine pancreas</td><td>Sigma</td><td>D5025</td><td>Neuronal culture-inhibits viscous cell suspension</td></tr><tr><td>pulse stimulator</td><td>A-M systems</td><td>model 2100</td><td>Apply electrical stimulation</td></tr><tr><td>Slotted bath with field stimulation</td><td>Warner Instruments</td><td>RC-21BRFS</td><td>Apply electrical stimulation</td></tr><tr><td>stimulus isolation unit</td><td>Warner Instruments</td><td>SIU102</td><td>Apply electrical stimulation</td></tr><tr><td>lubricant</td><td>Dow corning</td><td>111</td><td>pHluorin imaging-seal with coverslip and imaging chamber, avoid leak from chamber</td></tr><tr><td>AP5</td><td>Tocris</td><td>3693</td><td>Gradient for normal saline, selective NMDA receptor antagonist, inhibit postsynaptic activity which have potential for recurrent activity</td></tr><tr><td>CNQX</td><td>Tocris</td><td>190</td><td>Gradient for normal saline, competitive AMPA/kainate receptor antagonist, inhibit postsynaptic activity which have potential for recurrent activity</td></tr><tr><td>Illuminator</td><td>Nikon</td><td>C-HGFI</td><td>Metal halide light source for pHluorin</td></tr><tr><td>EMCCD camera</td><td>Andor</td><td>iXon3</td><td>pHluorin imaging, detect pHluorin fluorescence intensity</td></tr><tr><td>Inverted microscopy</td><td>Nikon</td><td>Ti-E</td><td>Imaging for synaptophysin or VAMP2 pHluorin transfected cells</td></tr><tr><td>NIS-Elements AR</td><td>Nikon</td><td>NIS-Elements Advanced Research</td><td>Software for imaging acquisition and analysis</td></tr><tr><td>Igor Pro</td><td>WaveMetrics</td><td>Igor pro</td><td>Software for imaging analysis and data presentation</td></tr><tr><td>imaging chamber</td><td>Warner Instruments</td><td>RC21B</td><td>pHluorin imaging, apply field stimulation on living cells</td></tr><tr><td>poly-l-lysine</td><td>Sigma</td><td>P4832</td><td>Electron microscopy, substrate for neuronal growth, apply on multiwell plate for 1 h at room temperature then wash with sterilized water 3 times</td></tr><tr><td>Horseradish peroxidase(HRP)</td><td>Sigma</td><td>P6782</td><td>Electron microscopy, labeling of endocytosed synaptic vesicles by catalyzing DAB in presence hydrogen peroxide, final concentration is 5 mg/mL in normal saline, make fresh before use</td></tr><tr><td>Na cacodylate</td><td>Electron Microscopy Sciences</td><td>12300</td><td>Electron microscopy, buffer for fixatives and washing, final concentration is 0.1 N</td></tr><tr><td>3,3&amp;prime;-Diaminobenzidine(DAB)</td><td>Sigma</td><td>D8001</td><td>Electron microscopy, labeling of endocytosed synaptic vesicles, substrate for HRP, final concentration is 0.5 mg/mL in DDW and filtered, make fresh before use</td></tr><tr><td>Hydrogen peroxide solution</td><td>Sigma</td><td>H1009</td><td>Electron microscopy, labeling of endocytosed synaptic vesicles by inducing HRP-DAB reaction, final concentration is 0.3% in DDW, make fresh before use</td></tr><tr><td>glutaraldehyde</td><td>Electron Microscopy Sciences</td><td>16365</td><td>Electron microscopy, fixatives, final concentration is 4% in Na-cacodylate buffer, make fresh before use, shake well before to use</td></tr><tr><td>TEM</td><td>JEOL</td><td>200CX</td><td>Electron microscopy, imaging of endocytosed vesicles and ultrastructural changes</td></tr><tr><td>CCD digital camera</td><td>AMT</td><td>XR-100</td><td>Electron microscopy, capturing images</td></tr><tr><td>Lead citrate</td><td>Leica microsystems</td><td>16707235</td><td>Electron microscopy, grid staining</td></tr></tbody></table>

measuring synaptic vesicle endocytosis in cultured hippocampal neurons

エンドサイトーシス、時にシナプス小胞の融合は小胞リサイクルそしてこうして神経反復焼成時におけるシナプス伝達の維持を可能にする神経終末で取得されます。病理学の条件に障害エンドサイトーシスは、シナプスの強さと脳の機能の低下につながります。ここでは、我々 は哺乳類神経培養海馬シナプスにおけるシナプス小胞エンドサイトーシスを測定するために使用方法を説明します。我々 は pHluorin、増加 pH 敏感な緑色蛍光タンパク質を小胞の内腔側でシナプトフィジンと VAMP2/シナプトブレビンを含むシナプス小胞膜タンパク質を融合することでシナプス小胞タンパク質エンドサイトーシスを監視、pH として蛍光強度が増加します。エキソサイトーシス、時小胞内腔 pH が増加し、pH は酸性再エンドサイトーシス小胞内腔中。したがって、pHluorin の蛍光強度の増加は減少を示す標識のシナプス小胞タンパク質のエンドサイトーシスに対し融合を示します。PHluorin イメージング法を使用して、エンドサイトーシスを記録するに加え、膜小胞エンドサイトーシスの監視西洋わさびペルオキシダーゼ (HRP) 摂取量の電子顕微鏡検査 (EM) 測定による小胞によって。最後に、我々 は神経末端の穴を形成する高カリウム誘起脱分極後様々 な時を監視しました。HRP 吸収と膜ピット形成の時間的経過は、エンドサイトーシスの時間コースを示します。

脂質キャリア メソッドを使用して、SpH に表現された海馬ニューロン ボタン (図 1 a) の同定を可能にします。電気刺激誘発細胞エキソサイトーシスと蛍光強度の増加に対応。蛍光 (ΔF) の増加は、刺激 (図 1 b) を終了することによって停止されました。増加の蛍光は、エンドサイトーシスにより、低速の減少が続いた。VAMP2-pH...

Watch this Scientific Journal Video about Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons at JoVE.com

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

光顕微鏡によるシナプス小胞タンパク質と融合した 1p-169 と吸収小胞の電子顕微鏡によるシナプス小胞エンドサイトーシスが検出されました。

培養海馬神経細胞のシナプス小胞エンドサイトーシスを測定

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

measuring-synaptic-vesicle-endocytosis-in-cultured-hippocampal-neurons

Research

JoVE Journal

Neuroscience

9.8K ビュー.  National Institute of Neurological Disorders and Stroke. 光顕微鏡によるシナプス小胞タンパク質と融合した 1p-169 と吸収小胞の電子顕微鏡によるシナプス小胞エンドサイトーシスが検出されました。

ビデオ: Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

Measuring Exocytosis in Neurons Using FM Labeling

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time imaging of vesicles labeled with FM dye to monitor the rate of presynaptic vesicle release.  FM4-64 is a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated.  Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles and a nominal one when in the extracellular fluid. After a wash step is used to help remove external dye within the plasma membrane, the remaining FM is concentrated within the vesicles and is then expelled when exocytosis is induced by another round of electrical stimulation. The rate of vesicles release is measured from the resulting decrease in fluorescence.  Since FM dye can be applied external and transiently, it is a useful tool for determining rates of exocytosis in neuronal cultures, especially when comparing the rates between transfected synapses and neighboring control boutons.



The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures.

FMラベリングを用いた神経細胞の測定エキソサイトーシス

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

生物学

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

シナプス伝達とイメージシナプス前終末を研究するために齧歯類の脳スライスからの神経細胞のVibrodissociation

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

神経科学

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

FM染料を用いて培養小脳顆粒神経細胞のシナプス小胞プールの補充の定量分析

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

培養神経細胞における膜タンパク質の側方拡散とエキソサイトーシスは、蛍光回復と蛍光損失光退色を用いて評価

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

初代培養海馬ニューロンにおける高密度コア小胞のライブイメージング

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

海馬スライス培養におけるシナプス蛋白のポストエンベディング免疫金ラベリング

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using styryl FM dyes, which reveal membrane turnover. Conventional protocols for the use of FM dyes were designed for analyzing neurons following stimulated (evoked) synaptic activity. Recently, protocols have become available for analyzing the FM signals that accompany weaker synaptic activities, such as spontaneous or miniature synaptic events. Analysis of these small changes in FM signals requires that the imaging system is sufficiently sensitive to detect small changes in intensity, yet that artifactual changes of large amplitude are suppressed. Here we describe a protocol that can be applied to evoked, spontaneous, and miniature synaptic activities, and use cultured hippocampal neurons as an example. This protocol also incorporates a means of assessing the rate of photobleaching of FM dyes, as this is a significant source of artifacts when imaging small changes in intensity.

We describe the use of styryl FM dyes to image synaptic vesicle recycling in functional nerve terminals. This protocol can be applied not only to evoked, but also spontaneous and miniature synaptic activities. The protocol expands the variety of synaptic events that can be effectively evaluated.

誘発、自発的、およびミニチュアシナプス活動中に、FM色素を用いたシナプス小胞のリサイクルの検討

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

蛍光プローブを用いた培養ニューロンにおけるエンドソームおよびリソソームの動力学の定量

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane proteins become exposed at the cell surface. One of these proteins is Synaptotagmin-1 (Syt1). An antibody directed against the luminal domain of Syt1, once added to the culture medium, is taken up during the exo-endocytotic cycle. This uptake is proportional to the amount of SV recycling and can be quantified through immunofluorescence. Here, we combine Syt1 antibody uptake with double transfection of cultured hippocampal neurons. This allows us to (1) localize presynaptic sites based on expression of recombinant presynaptic marker Synaptophysin, (2) determine their functionality using Syt1 uptake, and (3) characterize the targeting and effects of a protein of interest, GFP-Rogdi.

We describe an optical assay for synaptic vesicle (SV) recycling in cultured neurons. Combining this protocol with double transfection to express a presynaptic marker and protein of interest allows us to locate presynaptic sites, their synaptic vesicle recycling capacity, and determine the role of the protein of interest.

シナプス小胞の培養神経細胞シナプス蛋白質の過剰発現でリサイクルのため光アッセイ

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane ...

Transmission Electron Microscopic Imaging of Synaptic Vesicle Endocytosis in Mouse Hippocampal Neurons 

<a href='https://app.jove.com/v/55862/measuring-synaptic-vesicle-endocytosis-in-cultured-hippocampal-neurons'>Source: Villarreal, S., et. al., Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons. J. Vis. Exp. (2017)</a>This video demonstrates the analysis of synaptic vesicle endocytosis in cultured hippocampal neurons through horseradish peroxidase labeling and electron microscopy.

Source: Villarreal, S., et. al., Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons. J. Vis. Exp. (2017)This video demonstrates the ...