Name: quantification of filamentous actin (f-actin) puncta in rat cortical neurons
Uploaded: 2016-02-10T14:00:00.000+00:00
Duration: 10 min 13 s
Description: Watch this Scientific Journal Video about Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons at JoVE.com

This work was funded by NIH grants DA013137, DA031604, and HD043680. Partial support was provided by a NIH T32 training grant in Biomedical-Behavioral science.

全ての動物プロトコルを見直し、サウスカロライナ大学の動物実験委員会（保証番号：A3049-01）によって承認されました。 1.低密度胚性神経文化<ol><li> 初代皮質神経細胞培養のための準備 <ol><li>ソリューション： <ol><li> 10 mlのホウ酸緩衝液中のポリ-L-リジン5mgを溶解することによりポリ-L-リシンストック溶液を調製します。 </li><li> 49ミリリットルホウ酸緩衝液中で1ミリリットルポリ-L-リジンストックを希釈して使用液を調製します。 </li><li> 395ミリリットルのdH 2 Oで開始し、その後、全ての成分（ホウ砂の1.24グラムのホウ酸+ 1.9グラム）を加えることによりホウ酸緩衝液、pH 8.4を準備します。 1MのNaOHにより8.4にpHを調整します。 400ミリリットルに調整し、ボンネットの下に0.2μmのナイロン膜フィルターでろ過します。 </li><li> 445ミリリットルのdH 2 Oで始まるとすべての他の成分（50ミリリットル10X HBSS証券+ 1.2グラムHEPES）を追加することで、HBSS溶液、pHが7.2を準備します。 1NのHCl、BRIにより7.2にpHを調整しますNGに500ミリリットルとは、ボンネットの下に0.2μmのナイロン膜フィルターでろ過します。 </li><li> 10％ウシ胎児血清を含むDMEM / F12：メッキ培地を準備します。ボンネットの下に0.2μmのナイロン膜フィルターでフィルタリングします。 </li><li>完全増殖培地を準備します50ミリリットルNeurobasal培地に、サプリメントを追加する（500μlのグルタマックス（100X）+ 500μlのグルコース抗生物質 - 抗真菌溶液（100Xの+ 1ミリリットルのB-27（50X）+500μlの）+100μlの7.5％炭酸水素ナトリウムはボンネットの下に0.2μmのナイロン膜フィルターでフィルタリングします。 </li></ol></li><li>文化の前に二日： <ol><li>ポリ-L-リジンの2ミリリットルワーキング溶液でコート12 35ミリメートルのガラスボトムディッシュを、フードのO / Nの下におきます。 </li></ol></li><li>文化の一日前： <ol><li>皿から空のコーティング剤とのddH 2 Oですすぎ料理はフードの下で時間乾燥することができます。 </li><li> 10％ウシ胎児血清（全溶液の10％）でメッキ培地（DMEM / F12を準備します。ピペット2ミリリットル培地に各皿、および37°Cの 、5％CO 2でO / Nインキュベートします。 注：培養する場合には、プラスチック培養皿を回避することが重要です。 （直立顕微鏡用）（倒立顕微鏡用）ガラスボトムディッシュの使用またはカバーガラスは、シャープでクリアな画像が得られます。プラスチックボトムディッシュに比べて、ガラスボトムディッシュは、蛍光顕微鏡検査の際に、不要な色合いやまぶしさを避けることができます。 </li></ol></li></ol></li><li> 細胞培養プロトコル： <ol><li> HBSSバッファー（100〜150ミリリットル）を、冷蔵置く100ミリメートルペトリ皿、鉗子、はさみ、50ミリリットルチューブ、およびフードでUV光の下で15 mlチューブ。 </li><li>培養室の外でフードの下、5％セボフルランの致死吸入で妊娠ラットを安楽死させます。 </li><li> 70％エタノール、10トン胎盤を露出するようにハサミを使用して腹部cavity.Openの途中で尾から子宮をカットアップしながら、鉗子を使用して、下腹部の皮膚に滅菌します。 </li><li> 8-10 E18の胎児を削除します。プラHBSS溶液を含むペトリ皿中のCe 8-10胎児を。 </li><li>鉗子で胎児の頭の後ろを持ち、その後、身体から頭部を切断するはさみを使用しています。 HBSS 5-7ミリリットルで満たされた別のペトリ皿に入れてください。フードに皿を転送します。 </li><li>冷HBSSで2つの追加の100ミリメートルのペトリ皿を埋めます。 </li><li>ピールさておき頭蓋骨は、そしてHBSSで満たされた新しいペトリ皿に脳をスクープ。 </li><li>それに冷HBSSで新しいペトリ皿に脳を転送し、脳から鋭い湾曲鉗子、別々の小脳および脳幹を使用してください。 </li><li>湾曲した鉗子で半球を分離、確保するためにピンセットを使用してください。髄膜を削除します。マークされた15ミリリットルの遠心管に前頭皮質および転送の部分を分離します。 </li><li>新鮮な2ミリリットルHBSSで15ミリリットルチューブを記入し、各15ミリリットルチューブに20μlのトリプシンEDTA（0.5％トリプシンおよび5.3 mMのEDTAで10倍濃縮混合物を。）を追加します。 </li><li> RTで10〜15分間インキュベートします。ゆっくり、数分おきにチューブを旋回セトリングことはできません。 </li><li> Afター10-15分は、古いHBSSを削除し、新しいHBSSで二回リンスするためにガラスピペットを使用しています。 </li><li> （2ミリリットルHBSS中1mg / mlの、2mgのトリプシンインヒビター）トリプシン阻害剤に浸し、よく混ぜ、それは5分放置します。新鮮なHBSSで二回洗浄します。 </li><li>均質になるまで非常にゆっくりと減少し、先端径較正ピペット及び滅菌チップを用いて再度bubbles.Triturateを回避するように非常にゆっくりとゴム球、磨砕脳片で10~15回ガラスピペットを使用して。 </li><li>転送は、事前に準備コーティングした培養皿（50細胞/ mm2の）に所望の密度で細胞を解離しました。 37°Cの 、5％CO 2でO / Nインキュベートします。 </li><li> 24時間後に、新たに作られた完全増殖無血清Neurobasal培地を含むDMEM / F12培地を交換してください。 </li><li>実験前の少なくとも12から14日間、5％CO 2/95％室内空気の加湿インキュベーター中ですべての回で細胞を維持します。新鮮なNeurobasal培地は、古い培地の約50％を交換しておき5-6日細胞を補います。 </li></ol></li></ol> 2.蛍光標識および免疫細胞化学注：初代皮質細胞培養物の免疫蛍光標識を1 mlと作業容積を有するガラス底面35 mmの細胞培養皿で行いました。 <ol><li>リン酸緩衝生理食塩水のpHが7.4（PBS）で2回ガラスボトムディッシュを洗ってください。 </li><li> RTで15分間、4％パラホルムアルデヒドで細胞を固定してください。 </li><li> PBSで細胞を2回洗浄し、5分間、PBS中の0.1％トリトンX-100で透過性。 </li><li> F-アクチン特異的な染色で、室温で20分間細胞を処理するため、のAlexaFluor 488ファロイジン（1時40分、1ミリリットルPBS中ファロイジン25μlの）。 </li><li> PBSで2回細胞を洗浄し、1〜2時間、室温で10％正常ヤギ血清でブロックします。 </li><li> 2％正常ヤギ血清を含むPBSで4 O℃でO / Nインキュベートする：ニワトリポリクローナル抗MAP2抗体（2,500 1）希釈</li><li>インキュベーション後、PBSで2回すすぎます。 </li><li>二次抗体（AlexaのRを希釈ED 594結合ヤギ抗ニワトリIgG抗体（1：500）2％正常ヤギ血清で、室温で2時間インキュベートします。 </li><li> PBSですすぎ、ヘキスト染料を10μl/皿を追加します。 </li><li>室温で3分間インキュベートし、PBSで二回洗浄します。 注：ステップ3（F-アクチン涙点カウント）のための標識された細胞は、準備が整いました。 </li><li>封入剤として退色防止試薬100μlの細胞サンプルを維持し、長期保存のため、暗所で4°Cのでおきます。 注：ファロイジンは退色防止封入試薬で保存することができる1週間と蛍光まで暗所で4℃でPBS中で比較的安定しています。ファロイジンを長期間にわたって保管して外に拡散を開始することができるので、最適な画像が染色後1-3日から取得されます。 </li></ol> 3. F-アクチン斑点カウント<ol><li>蛍光顕微鏡をオンにして、20倍の対物レンズに切り替えます。オープン顕微鏡ソフトウェア、および1280×960ピクセルの画像サイズでプログラムを設定し、0.11倍ズームで7ミクロン/ピクセル画像の解像度。 </li><li>赤、緑（495nmで）/（613 nm）の蛍光チャネルの下で共同標識F-アクチン/ MAP2ニューロンの画像を取得します。 </li><li> 5グリーン（F-アクチン）/レッド（MAP2）免疫標識/ブルー（ヘキスト）明確に定義された樹状アーバーと個々のニューロンの蛍光画像を選択してください。 </li><li>連続MAP2免疫蛍光と二次樹枝状のセグメント（長さの範囲25から75ミクロン）でF-アクチン豊富な構造を特定します。 </li><li>水平レベルに画像の選択した領域を回転させます。新しいイメージとしてコピーして貼り付けます。 </li><li>各皿のための一貫性の調整を使用して、画像の背景を引きます。 </li><li>明るい緑色のF-アクチン涙点を数え、訓練を受けた独立した観察者によって手動で選択された樹状セグメントの長さをトレースします。スプレッドシートファイルにデータをエクスポートします。 </li><li> MAP2の長さ（L）総F-アクチン標識された涙点（N）を割ることで密度を計算する樹状突起を標識しました。 F-交流の数などのデータを表現しますデンドライトの10ミクロンあたり錫涙点 注：涙点の樹状シャフト内染色の平均強度よりも少なくとも50％のピーク強度とF-アクチンの蛍光（サ ​​イズ≤1.5μm）をそれぞれ選択した樹状セグメントに含まれていました。 </li></ol>

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None of the authors have conflicts of interest to declare.

このプロトコルでは、我々は、私たちは、個々の神経細胞の樹状突起を識別することを可能にする35ミリメートルのガラスボトムディッシュで低密度で培養ラット皮質ニューロンを記述する。次に、我々は、樹状変化を検出するファロイジンとMAP2染色を使用します。その後、我々はF-アクチン斑点の変化を定量化するために特別なソフトウェアを使用していました。 F-ア...

この研究の主な目的は、ニューロンの樹状突起ネットワークのシナプス完全性の測定（推定）の信頼性の高い方法を開発することです。ここでは、ファロイジン染色および特殊な（NIS-要素）ソフトウェアを使用して、その後の分析と樹状突起の免疫細胞化学（ICC）の検出の組み合わせを用いて、ラット初代培養神経細胞におけるF-アクチン涙点の定量化について説明します。 標識ファロトキシンは、大小両方のフィラメント（F-アクチン）に対して同様の親和性を有するが、いくつかのアクチン抗体1とは異なり、単量体の球状アクチン（G-アクチン）に結合しません。ファロイジンの非特異的結合は、このように細胞イメージングの間に最小限の背景を提供し、ごくわずかです。ファロイジンは、典型的には、ファロイジンによるF-アクチンのはるかに強い標識化を可能にする蛍光顕微鏡用の細胞タンパク質を標識するために使用される抗体よりもはるかに小さいです。このように、神経細胞におけるF-アクチンの局在の詳細な画像をすることができます標識されたファロイジンを使用することによって得られます。 神経細胞の樹状突起のファロイジン（F-アクチン）染色は、成熟した棘、非とげシナプス2と未熟棘を含む樹枝状構造の多様性を表す離散的「ホットスポット」や明るい「斑点」を生成します。未熟棘薄い糸状仮足及びパッチの形態のいくつかの形態を含み、そしてspinogenesis 3の開始を表すことができます。未熟棘と非とげ状のパッチがPSD95 4を欠いています 。のみならず、棘におけるその後の変化だけでなく、追加の樹枝状構造へのF-アクチンの鉛の生産の変化は、このようにsynaptodendritic整合性5-7を調査するための重要なツールファロイジンを作ります。一般的には、ファロイジン陽性（F-アクチン）涙点の数は、アクティブなシナプス（興奮性および抑制）、アクチンダイナミクスとシナプスの安定性8のバランスを反映しています。 特定のトンを研究することは重要であるが、シナプスのYPES（ すなわち 、興奮棘）、治療の標的が未知であるとき、最初の樹枝状構造体の種々の一般的な整合性を推定する必要があります。 F-アクチンは、抑制性シナプスを含む樹状突起棘および他の構造の主要な構成要素であるので、F-アクチン涙点の変更された数は、シナプス変性症を示すことができます。このシナプス変性症は、より具体的な変化についてさらに調査することができます。複数のシナプスのタイプ/構造を検出するための当社の定量方法は、様々な実験処置後の樹状シナプスの変化（増減）の全体​​的な推定値が得られます。

<table><tbody><tr><td>35 mm Glass Bottom Dishes No. 1.5 coverglass</td><td>MatTek Corporation</td><td>P35G-1.5-20-C</td><td></td></tr><tr><td>DMEM/F12 medium</td><td>Life Technologies</td><td>10565-018</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Life Technologies</td><td>15400-054</td><td></td></tr><tr><td>Poly-L-Lysine</td><td>Sigma</td><td>P9155</td><td></td></tr><tr><td>Boric acid</td><td>Sigma</td><td>B0252</td><td></td></tr><tr><td>Borax</td><td>Sigma</td><td>B9876</td><td></td></tr><tr><td>GlutaMax</td><td>Life Technologies</td><td>35050-061</td><td>100X</td></tr><tr><td>Glucose</td><td>VWR</td><td>101174Y</td><td></td></tr><tr><td>HBSS</td><td>Sigma</td><td>H4641</td><td>10X</td></tr><tr><td>Neurobasal medium</td><td>Life Technologies</td><td>21103-049</td><td></td></tr><tr><td>B-27 supplement</td><td>Life Technologies</td><td>17504-044</td><td>50X</td></tr><tr><td>Antibiotic-Antimycotic solution</td><td>Cellgro</td><td>30004CI</td><td>100X</td></tr><tr><td>Sodium Bicarbonate</td><td>Life Technologies</td><td>25080</td><td></td></tr><tr><td>Vannas Scissors</td><td>World Precision Instruments</td><td>500086</td><td></td></tr><tr><td>Iris Scissors</td><td>World Precision Instruments</td><td>500216</td><td></td></tr><tr><td>Iris Forceps</td><td>World Precision Instruments</td><td>15914</td><td></td></tr><tr><td>Dumont #7 Forceps</td><td>World Precision Instruments</td><td>14097</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>World Precision Instruments</td><td>14095</td><td></td></tr><tr><td>ProLong Gold</td><td>Life Technologies</td><td>P36930</td><td></td></tr><tr><td>Paraformaldehyde&amp;nbsp;</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Cover glass</td><td>VWR</td><td>631-0137</td><td></td></tr><tr><td>AlexaFluor 488 Phalloidin</td><td>Life Technologies</td><td>A12379</td><td></td></tr><tr><td>Normal horse serum</td><td>Life Technologies</td><td>26050-070</td><td></td></tr><tr><td>Chicken polyclonal anti-MAP2</td><td>abcam</td><td>Ab92434</td><td></td></tr><tr><td>Alexa Red 594-conjugated goat anti-chicken IgG</td><td>Life Technologies</td><td>A11042</td><td></td></tr><tr><td>NucBlue Live cell stain ReadyProbes Reagent Hoescht 33342</td><td>Life Technologies</td><td>R37605</td><td></td></tr><tr><td>NIS-Elements software package</td><td>Nikon Instruments</td><td></td><td></td></tr><tr><td>Nikon Eclipse TE2000-E inverted fluorescent computer-controlled microscope&amp;nbsp;</td><td>Nikon Instruments</td><td></td><td></td></tr><tr><td>0.2 &amp;mu;m Nalgene nylon membrane filter</td><td>Fishersci</td><td>151-4020</td><td></td></tr></tbody></table>

quantification of filamentous actin (f-actin) puncta in rat cortical neurons

（F-アクチン）繊維状アクチンタンパク質はspinogenesis、シナプス可塑性、およびシナプスの安定性に大きな役割を果たしています。樹状F-アクチン豊富な構造の変化は、シナプスの完全性と接続性の変化を示唆しています。ここでは、初代ラット皮質ニューロン、F-アクチン斑点のためのファロイジン染色、およびその後の定量化手法を培養するための詳細なプロトコルを提供します。まず、E18ラット胚の前頭皮質は、その後、低密度の細胞培養物中に少なくとも12〜14日間、in vitroで増殖させた神経細胞を解離します。実験的治療に続いて、皮質ニューロンがのAlexaFluor 488ファロイジンで染色され、微小管結合タンパク質2（樹状F-アクチン斑点標識する）（MAP2を、神経細胞や樹状突起の整合性を検証するために）。最後に、特別なソフトウェアがランダムに選択された神経細胞の樹状突起を分析し、定量化するために使用されます。 F-アクチン豊富な構造は、二次樹枝状の枝に識別されます（長さの範囲25-75＆＃181; m）を連続MAP2免疫蛍光を持ちます。ここで紹介するプロトコルは、実験処理に続いて、樹状シナプス構造の変化を調査するための有用な方法となります。

私たちは、個々の神経細胞の樹状突起を識別することを可能にする35ミリメートルのガラスボトムディッシュで低密度で存在する方法で、私たち第1の培養ラット皮質ニューロン、。 図1では 、微分干渉コントラスト（DIC）画像は、日4、6、10、14、21、in vitroで 27に胎児ラット皮質ニューロンの開発における形態学的変化を示しています。樹状突起の?...

Watch this Scientific Journal Video about Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons at JoVE.com

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

ラット皮質ニューロンにおける繊維状アクチン（F-アクチン）涙点の定量化

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

quantification-filamentous-actin-f-actin-puncta-rat-cortical

Research

JoVE Journal

Neuroscience

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

12.0K ビュー.  University of South Carolina. Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

ビデオ: Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become imperative to develop objective approaches to quantify changes in synaptic connectivity. Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers. The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, c.eroglu@cellbio.duke.edu) under the ImageJ analysis software platform. The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers. This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development.

This protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers.

定量シナプス：シナプス数を定量する免疫細胞ベースのアッセイ

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become ...

神経科学

In Vitro Polymerization of F-actin on Early Endosomes

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto the endosomal membrane. We have established a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to genetic and biochemical manipulations. Endosomal fractions are prepared by floatation in sucrose gradients from cells expressing the early endosomal protein GFP-RAB5. Cytosolic fractions are prepared from separate batches of cells. Both endosomal and cytosolic fractions can be stored frozen in liquid nitrogen, if needed. In the assay, the endosomal and cytosolic fractions are mixed, and the mixture is incubated at 37 &#176;C under appropriate conditions (e.g., ionic strength, reducing environment). At the desired time, the reaction mixture is fixed, and the F-actin is revealed with phalloidin. Actin nucleation and polymerization are then analyzed by fluorescence microscopy. Here, we report that this assay can be used to investigate the role of factors that are involved either in actin nucleation on the membrane, or in the subsequent elongation, branching, or crosslinking of F-actin filaments.

In Vitro Polymerization of F-actin on Early Endosomes

Early endosome functions depend on F-actin polymerization. Here, we describe a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to biochemical and genetic manipulations.

体外初期エンドソームの F-アクチンの重合

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto ...

生化学

Labeling F-actin Barbed Ends with Rhodamine-actin in Permeabilized Neuronal Growth Cones

The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally expressed molecular guidance cues that bind growth cone receptors and regulate the dynamics and organization of the growth cone cytoskeleton3-6. The main target of these navigational signals is the actin filament meshwork that fills the growth cone periphery and that drives growth cone motility through continual actin polymerization and dynamic remodeling7. Positive or attractive guidance cues induce growth cone turning by stimulating actin filament (F-actin) polymerization in the region of the growth cone periphery that is nearer the source of the attractant cue. This actin polymerization drives local growth cone protrusion, adhesion of the leading margin and axonal elongation toward the attractant.
Actin filament polymerization depends on the availability of sufficient actin monomer and on polymerization nuclei or actin filament barbed ends for the addition of monomer. Actin monomer is abundantly available in chick retinal and dorsal root ganglion (DRG) growth cones. Consequently, polymerization increases rapidly when free F-actin barbed ends become available for monomer addition. This occurs in chick DRG and retinal growth cones via the local activation of the F-actin severing protein actin depolymerizing factor (ADF/cofilin) in the growth cone region closer to an attractant8-10. This heightened ADF/cofilin activity severs actin filaments to create new F-actin barbed ends for polymerization. The following method demonstrates this mechanism. Total content of F-actin is visualized by staining with fluorescent phalloidin. F-actin barbed ends are visualized by the incorporation of rhodamine-actin within growth cones that are permeabilized with the procedure described in the following, which is adapted from previous studies of other motile cells11, 12. When rhodamine-actin is added at a concentration above the critical concentration for actin monomer addition to barbed ends, rhodamine-actin assembles onto free barbed ends. If the attractive cue is presented in a gradient, such as being released from a micropipette positioned to one side of a growth cone, the incorporation of rhodamine-actin onto F-actin barbed ends will be greater in the growth cone side toward the micropipette10.
Growth cones are small and delicate cell structures. The procedures of permeabilization, rhodamine-actin incorporation, fixation and fluorescence visualization are all carefully done and can be conducted on the stage of an inverted microscope. These methods can be applied to studying local actin polymerization in migrating neurons, other primary tissue cells or cell lines.

A method to visualize and quantify F-actin barbed ends in neuronal growth cones is described. After culturing neurons on glass coverslips, cells are permeabilized with a saponin-containing solution. Then, a short incubation with the saponin buffer containing rhodamine-actin incorporates fluorescent actin onto free actin barbed ends.

透過処理神経成長円錐におけるローダミン-アクチンとF -アクチンばら両端にラベルを付ける

The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally ...

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

皮質ネットワークの開発で機能的カルシウムイメージング

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

DetectSyn: A Rapid, Unbiased Fluorescent Method to Detect Changes in Synapse Density

Synapses are the site of communication between neurons. Neuronal circuit strength is related to synaptic density, and the breakdown of synapses is characteristic of disease states like major depressive disorder (MDD) and Alzheimer's disease. Traditional techniques to investigate synapse numbers include genetic expression of fluorescent markers (e.g., green fluorescent protein (GFP)), dyes that fill a neuron (e.g., carbocyanine dye, DiI), and immunofluorescent detection of spine markers (e.g., postsynaptic density 95 (PSD95)). A major caveat to these proxy techniques is that they only identify postsynaptic changes. Yet, a synapse is a connection between a presynaptic terminal and a postsynaptic spine. The gold standard for measuring synapse formation/elimination requires time-consuming electron microscopy or array tomography techniques. These techniques require specialized training and costly equipment. Further, only a limited number of neurons can be assessed and are used to represent changes to an entire brain region. DetectSyn is a rapid fluorescent technique that identifies changes to synapse formation or elimination due to a disease state or drug activity. DetectSyn utilizes a rapid proximity ligation assay to detect juxtaposed pre- and postsynaptic proteins and standard fluorescent microscopy, a technique readily available to most laboratories. Fluorescent detection of the resulting puncta allows for quick and unbiased analysis of experiments. DetectSyn provides more representative results than electron microscopy because larger areas can be analyzed than a limited number of fluorescent neurons. Moreover, DetectSyn works for in vitro cultured neurons and fixed tissue slices. Finally, a method is provided to analyze the data collected from this technique. Overall, DetectSyn offers a procedure for detecting relative changes in synapse density across treatments or disease states and is more accessible than traditional techniques.

DetectSyn is an unbiased, rapid fluorescent assay that measures changes in relative synapse (pre- and postsynaptic engagement) number across treatments or disease states. This technique utilizes a proximity ligation technique that can be used both in cultured neurons and fixed tissue.

DetectSyn:シナプス密度の変化を検出するための迅速で偏りのない蛍光法

Synapses are the site of communication between neurons. Neuronal circuit strength is related to synaptic density, and the breakdown of synapses is ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

海馬の齧歯動物の脳スライスにおけるシナプス密度の評価

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

脳スライスと一次細胞培養における錐体ニューロンの弾道標識

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin occurs in equilibrium in its two forms: monomeric globular (G-actin) and polymerized filamentous (F- actin). At the synaptic terminals, actin cytoskeleton forms the basis for critical pre- and post-synaptic functions. Moreover, dynamic changes in the actin polymerization status (interconversion between globular and filamentous forms of actin) are closely linked to plasticity-related alterations in synaptic structure and function. We report here a modified fluorescence-based methodology to assess polymerization status of actin in ex vivo conditions. The assay employs fluorescently labelled phalloidin, a phallotoxin that specifically binds to actin filaments (F-actin), providing a direct measure of polymerized filamentous actin. As a proof of principle, we provide evidence for the suitability of the assay both in rodent and post-mortem human brain tissue homogenates. Using latrunculin A (a drug that depolymerizes actin filaments), we confirm the utility of the assay in monitoring alterations in F-actin levels. Further, we extend the assay to biochemical fractions of isolated synaptic terminals wherein we confirm increased actin polymerization upon stimulation by depolarization with high extracellular K+.

We report a simple, time-efficient and high-throughput fluorescence spectroscopy-based assay for the quantification of actin filaments in ex vivo biological samples from brain tissues of rodents and human subjects.

げっ歯類とヒト脳組織におけるアクチン重合状態評価のための時間効率の高い蛍光分光法ベースアッセイ

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin ...

Measuring Properties of the Membrane Periodic Skeleton of the Axon Initial Segment using 3D-Structured Illumination Microscopy (3D-SIM)

The axon initial segment (AIS) is the site at which action potentials initiate and constitutes a transport filter and diffusion barrier that contribute to the maintenance of neuronal polarity by sorting somato-dendritic cargo. A membrane periodic skeleton (MPS) comprising periodic actin rings provides a scaffold for anchoring various AIS proteins, including structural proteins and different ion channels. Although recent proteomic approaches have identified a considerable number of novel AIS components, details of the structure of the MPS and the roles of its individual components are lacking. The distance between individual actin rings in the MPS (~190 nm) necessitates the employment of super-resolution microscopy techniques to resolve the structural details of the MPS. This protocol describes a method for using cultured rat hippocampal neurons to examine the precise localization of an AIS protein in the MPS relative to sub-membranous actin rings using 3D-structured illumination microscopy (3D-SIM). In addition, an analytical approach to quantitively assess the periodicity of individual components and their position relative to actin rings is also described.

Measuring Properties of the Membrane Periodic Skeleton of the Axon Initial Segment using 3D-Structured Illumination Microscopy 3D-SIM

The present protocol describes a method to visualize and measure actin rings and other components of the membrane periodic skeleton of the axon initial segment using cultured rat hippocampal neurons and 3D-structured illumination microscopy (3D-SIM).

3D構造化照明顕微鏡(3D-SIM)を用いた軸索初期セグメントの膜周期骨格の特性の測定

The axon initial segment (AIS) is the site at which action potentials initiate and constitutes a transport filter and diffusion barrier that contribute to ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

動的細胞突起におけるソフトウェア支援トラッキングのためのグラフィカルユーザインタフェース

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...