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1。細胞の培養<ol><li>皮質ニューロンを単離し、前述の技法を用いて成長とポリ- D -リジンとラミニン1でコートしたガラスボトム（マテック（株）、アッシュランド、マサチューセッツ州）で培養皿に播種されています。 </li></ol> 2。蛍光プローブTMRMとH 2 DCF - DA用のストック溶液を準備する<ol><li>無水ジメチルスルホキシド1mlにTMRM 5.0 mgを溶解してTMRMの10 mMのストック溶液を調製。 1分間ボルテックスして。その後、アリコートを行い、-20℃に保存する、光から保護し、一ヶ月以内に使用してください。 </li><li>次に、無水DMSO 1ml中にH 2 DCF - DAの4.87 mgを溶解することによってH 2 DCF - DA 10 - mMのストック溶液を調製。同様に、1分間攪拌のためのそれ。その後、アリコートを行い、-20℃に保存する、光から保護し、一週間以内に使用してください。 </li></ol> 3。 TMRMとH 2 DCF - DAでのロードラット皮質ニューロン TMRMは、ミトコンドリアの高度に負に帯電内部に蓄積される電位差、細胞透過性蛍光指示薬です。ミトコンドリアTMRMの自動消光を避けるためにTMRMの低濃度（10〜50 nMの範囲）を使用することが重要です。その後、TMRMの蛍光シグナルはミトコンドリア内膜を通過するΔΨmに直接協力関係することができます。 ΔΨmの消失は、蛍光強度が失われるおそれがミトコンドリアからリークするTMRMなります。 H 2 DCF - DAは細胞透過性の細胞内エステラーゼによってDCF - DAに変換プローブ、および蛍光DCFでの酸化の結果です。高負荷の濃度もH 2の不在O 2におけるDCF蛍光の飽和を引き起こす可能性があるので、DCF - DA 2〜10μMとそれの間の範囲H 2の最終濃度は、異なる脳領域から派生した神経細胞で実験的にテストする必要があります。いかなる内因性または外因性の酸化剤（例えば、一酸化窒素、過酸化水素）の存在は、DFC蛍光強度が増加します。以下、我々はTMRMとH 2 DCF - DAでラット皮質ニューロンをロードするためのプロトコルを提供しています。 <ol><li> TB：：145 mMのNaCl、5mMのKCl、10mMのグルコース、1.5mMのCaCl 2を 、1mMのMgCl 2、および10 TMRMとラット皮質ニューロンをロードするには、まず、タイロードバッファ（テキストオーバーレイで培養神経細胞を3回洗浄mMのHEPES、NaOHで7.4にpHを調整）。その後、結核で1 / 1000倍の10mM TMRMの株式を希釈することによりTMRMの20 nMのを準備して、TBの1mlあたり希薄化後TMRMの2μlを加える。室温、暗所で45分間TMRMと神経細胞をインキュベートします。 45分後、顕微鏡のステージ上に培養皿をマウントし、イメージングを起動します。 </li><li> H 2 DCF - DAとラット皮質ニューロンをロードするには、結核と培養神経細胞を3回洗浄。次に、TBで10mMのH 2 DCF - DA株式1 / 10倍に希釈してH 2 DCF - DAの2μMを準備して、TBの1mlあたり希薄化後のH 2 DCF - DAの2μlを加える。その後、室温で暗所で45分間H 2 DCF - DAを持つニューロンをインキュベートする。 45分後、画像を取得する前に、余分な蛍光指示薬を削除するには、結核と神経細胞を4回洗浄する。 </li></ol> 4。 ΔΨmを決定するためにTMRMとともにインキュベートした神経細胞のイメージングを生きる<ol><li> TMRMとともにインキュベートした神経細胞のライブイメージング、共焦点レーザー走査顕微鏡（テキストオーバーレイ：LSM 510、カールツァイス社）を実行するために、ライブの時系列のプログラムのアプリケーションとを、使用されています。画像を取得し、退色を避けるために必要な時間を最小限に抑えるために：、低解像度と減衰レーザーパワー（1％：低解像度レーザパワー256 × 256テキストオーバーレイ）を適用します。 </li><li> 。次に、反射光を使用してTMRMでロードされたマウントされているニューロンのフォーカスを調整します。 514 nmで照射し、570nmで検出によるTMRM蛍光を調べます。ちょうど飽和レベル以下のカメラの検出のゲインを設定します。 </li><li>一度画像を得るために解像度、レーザーパワー、カメラの検出のゲイン、およびタイムラプス間隔を含むすべてのパラメータが設定されています。実験間でこれらの設定を変更しないでください。次に、フィールドを変更します。画像の収集を開始。 </li><li> ΔΨmの変更をテストするには、そのようなFCCPまたは2μgの/オリゴmlの、1μMのような刺激が著しく、それぞれ、ミトコンドリア膜電位を脱分極や過分極となる、適用することができます。これらの変更は、FCCPの場合にはベースライン蛍光強度と比較してTMRM蛍光強度の減少、またはオリゴマイシンの場合にTMRM蛍光強度の増加が反映されます。 </li></ol> 5。 ROSを決定するためにH 2 DCF - DAとインキュベートした神経細胞のライブイメージング<ol><li> H 2 DCF - DA、最初にインキュベートニューロンのライブイメージングを実行するには、顕微鏡のステージ上に培養皿をマウント。私たちの細胞のフォーカスを調整しますINGは、反射光を。 488 nmで励起と515 nmの放射によるDCFの蛍光を調べる。 </li><li>次に、5-7％、検出器のゲイン、および256 × 256の解像度にレーザーパワーを調整する。実験間でこれらの設定を変更しないでください。その後、時系列のプログラムを使用してライブ映像を得るために周波数を設定します。 </li><li>新しいフィールドを選択して、画像を取得開始します。 ROSレベルの変化を検出するには、H 2 O 2の100から200μMで細胞を扱う。これは、ベースラインレベルと比較してDCF蛍光強度の増加が反映されます。 </li></ol> 6。データの分析<ol><li>エリアを選択するLSMのプログラムからツール：関心領域を（ROIテキストオーバーレイ）を使用してください。その後、TMRMまたはROS蛍光強度を測定する。それぞれ、TMRMまたはROSの蛍光強度を測定するために撮像された細胞の全体の細胞体からミトコンドリアの地域またはROIのからROIを選択してください。 </li><li> TMRM用または各時間ポイントのROSのすべての画像形成されたセルのための全体の細胞体からの各セルのすべてのROIの平均蛍光強度を計算する。バックグラウンドの蛍光強度を計算するセルの隣の領域を選択します。いくつかの測定を行い、平均バックグラウンド強度を計算する。 </li><li> Microsoft Excelを使用して、各時間ポイントの各セル内のROIの平均蛍光強度から平均的なバックグラウンドの蛍光強度を引きます。バックグラウンド強度を差し引いた、この式を用いてベースラインの蛍光（：=ベースライン蛍光の任意の時点でFが=蛍光強度、ΔF= FF O / F O × 100、テキストオーバーレイ）にTMRMまたはDCF蛍光強度を正規化する。その後、時間の経過とともに蛍光強度の変化を示すグラフを生成するシグマプロットプログラムを使用します。 </li></ol> 7。代表的な結果図1Aは、TMRMとインキュベートラット皮質ニューロンの蛍光画像を示しています。 FCCP、ミトコンドリア脱共役剤、の添加は、ミトコンドリアの脱分極とTMRM蛍光強度（図1B）の損失につながる。ベースラインTMRMの蛍光レベルは、FCCPの添加（。;図1C第一350秒）の前に安定している。時間をかけてTMRM蛍光変化の定量分析は、FCCP（図1C）の添加後TMRMの蛍光の有意な減少を示しています。 図1Dは、DCFを搭載したラット皮質ニューロンの蛍光画像を示しています。細胞体の増加DCF蛍光強度（図1E）のH 2 O 2の添加により。ベースラインDCF蛍光レベルは、H 2 O 2のアプリケーションの前に（最初の120秒）変更されていません。 DCF蛍光の経時的測定は、着実なレベルを示し、H 2 O 2処理（図1F）の後にどの増加。 <img src="/files/ftp_upload/2704/2704fig1.jpg" alt="図1"/> 図1。ミトコンドリア膜電位とライブラット皮質ニューロンのROSレベルの評価。 TMRMを搭載した皮質ニューロンの（A）代表蛍光画像。ベースラインTMRM蛍光をスキャンした後、神経細胞はprotonophore FCCP（1μM）で処理した。右側にはそれぞれ、明るい黄色と表す黒の最大値と最小輝度とTMRM蛍光の疑似強度バーです。ミトコンドリアの領域からTMRMの蛍光の消失は、FCCP治療（パネルB）に応じてΔΨmの崩壊を示している。異なる時点でTMRM蛍光強度の変化の定量的表現は、FCCPの治療の前と後のパネルC.（D）でH2DCF - DAを搭載したラット皮質ニューロンの蛍光画像を示しています。ベースラインDCF蛍光を決定した後、細胞を200μMのH 2 O 2で処理し、及びDCF蛍光の変化を評価した。 DCF蛍光の増加は、H 2 O 2処理（E）に応じてROSレベルの増加を反映している。 DCF蛍光の変化の定量分析、H 2 O 2処理の前後には、パネルF.スケールバーに=10μmを示しています Video.7.1 - labmedia 2704_Joshi.avi 40倍対物レンズを用いてFCCPを添加する前と後の皮質ニューロンにおけるTMRMの細胞イメージングを生きる。擬似カラーの強度は、FCCPの添加後（赤色、FCCPの添加後の）最大値（FCCPの添加前に、明るい黄色、）と減少TMRM蛍光強度を示しています。 <a href="http://www.jove.com/files/ftp_upload/2704/supplemental_video1.avi">ビデオを表示するにはここをクリック</a> ビデオ。 7.5 - labmedia 2704_Joshi.avi 40倍対物レンズを用いてH 2 O 2を添加する前と後の皮質ニューロンにおけるDCFの細胞イメージングを生きる。ベースラインDCF蛍光は、細胞体にライトグリーン色をしているとH2O2添加は、DCを増加させる鮮やかな緑色にF蛍光強度は。 <a href="http://www.jove.com/files/ftp_upload/2704/supplemental_video2.avi">ビデオを表示するにはここをクリック</a>

<ol>
	<li>Hilgenberg, L. G., Smith, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+dissociated+mouse+cortical+neuron+cultures.">Preparation of dissociated mouse cortical neuron cultures.</a> J. Vis. Exp. , (2007).</li><li>Scaduto, R. C., Grotyohann, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+mitochondrial+membrane+potential+using+fluorescent+rhodamine+derivatives.">Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives.</a> Biophys. J. 76, 469-477 (1999).</li><li>Ward, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amyloid+precursor+protein+intracellular+domain+(AICD)+disrupts+actin+dynamics+and+mitochondrial+bioenergetics.">The amyloid precursor protein intracellular domain (AICD) disrupts actin dynamics and mitochondrial bioenergetics.</a> J. Neurochem. 113, 275-284 (2010).</li><li>Gunasekar, P. G., Kanthasamy, A. G., Borowitz, J. L., Isom, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDA+receptor+activation+produces+concurrent+generation+of+nitric+oxide+and+reactive+oxygen+species:+implication+for+cell+death.">NMDA receptor activation produces concurrent generation of nitric oxide and reactive oxygen species: implication for cell death.</a> J. Neurochem. 65, 2016-2021 (1995).</li></ol>

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私たちは、それぞれ蛍光指示薬TMRMとH 2 DCF - DAを使用して、ラット皮質ニューロンでΔΨmとROSを決定する方法を説明するステップバイステップの手順を提示している。他の細胞型の場合は、経験的にTMRMまたはH 2 DCF - DAの最終濃度とローディング時間を決定することが重要です。一般的に、TMRM 20から200 nMから濃度範囲、およびTMRMと細胞のインキュベーション時間は20〜60分ごとに異なります。 H 2の最終濃度は、DCF - DAは、2〜10μMの範囲であり、そしてこの指標を含むローディング溶液中の細胞のインキュベーションは、30〜45分ごとに異なります。 どんな刺激がないのに細胞と蛍光強度の変化（TMRM蛍光のちらつきなど）の両方の光毒性を避けるために写真を撮るのレーザーパワーと走査速度を最適化することが重要です。最適化された光の設定は、刺激のない状態で（閾値）飽和上または下にない蛍光シグナルになるはずです。ライブイメージングの10〜15分の任意の刺激の非存在下でのプローブの蛍光強度には変化がないときに、特定のレーザーパワーおよび走査速度で選択したフィールドから画像を収集するために最適な条件が達成されています。 ΔΨmを決定するために他の蛍光プローブはローダミン123およびテトラメチルローダミンエチルエステル（TMRE）が含まれています。しかし、それらは単離されたミトコンドリア2の呼吸のプロセスを阻害することが見出された。重要なのは、TMRMは、低濃度でミトコンドリア呼吸に影響を与えません2と低光毒性と他のプローブに比べて3退色しています。 H 2 DCF - DAは、それがよく細胞内に保持されるROSのための良い指標であり、そのような過酸化物、超酸化物、および一酸化窒素4のようないくつかの酸化剤の種を、認識する。

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th></tr><tr><td>グラスの底の培養皿</td><td>マテック株式会社</td><td> P35G - 1.5 - 14 - C </td></tr><tr><td> NbActive4 </td><td> BrainBits </td><td> NbActive4 </td></tr><tr><td> TMRM </td><td>インビトロジェン</td><td> T668 </td></tr><tr><td> H 2 DCF - DA </td><td>インビトロジェン</td><td> C400 </td></tr><tr><td> NaClの</td><td>シグマ</td><td> S6191 </td></tr><tr><td>塩化カリウム</td><td>シグマ</td><td> P3911 </td></tr><tr><td>のCaCl 2•2H 2 O </td><td>シグマ</td><td> C3306 </td></tr><tr><td>のMgCl 2•6H 2 O </td><td>シグマ</td><td> M2670 </td></tr><tr><td> D -グルコース</td><td>シグマ</td><td> G6152 </td></tr><tr><td> HEPES </td><td>インビトロジェン</td><td> 15630 </td></tr></tbody></table>

determination of mitochondrial membrane potential and reactive oxygen species in live rat cortical neurons

ミトコンドリア膜電位（ΔΨm）は、ATPを生成するための呼吸鎖の生理的機能を維持するために不可欠です。 ΔΨmの大幅な損失は、その後の死でエネルギーが枯渇セルをレンダリングします。活性酸素種（ROS）が重要なシグナル伝達分子であるが、病的状態におけるそれらの蓄積は、酸化ストレスにつながる。細胞内ROSの二つの主要な源は、環境の毒素と酸化的リン酸化のプロセスです。ミトコンドリア機能障害と酸化ストレスは多くの疾患の病態生理に関与しているので、ΔΨmとROSを判断する能力は、細胞とミトコンドリアの機能の生理学的状態に関する重要な手がかりを提供することができます。 いくつかの蛍光プローブ（ローダミン123、TMRM、TMRE、JC - 1）種々の細胞型でΔψmを決定するために使用することができる、と多くの蛍光指標（Dihydroethidium、Dihydrorhodamine 123、H 2 DCF - DA）はROSを決定するために使用することができます。ほぼすべてのΔΨmまたはROSを評価するために使用可能な蛍光プローブのは、単一波長の指標であり、ΔΨmまたはROSのレベルを増加または減少させる刺激に比例した蛍光強度を増加または減少される。従って、それはベースラインレベルと特定の刺激を加えた後にこれらのプローブの蛍光強度を測定することが不可欠です。これは、1つはベースラインレベルと刺激間の蛍光強度の変化の割合を決定することができます。蛍光強度の変化はΔΨmまたはROSの相対的なレベルの変化を反映している。このビデオでは、我々はベースラインレベルとの間およびFCCP、ミトコンドリアの脱共役剤を適用した後TMRM蛍光強度の変化率を決定するためにラット皮質ニューロンの蛍光指示薬、TMRMを、適用する方法を示しています。 FCCPの治療に起因するTMRM蛍光の低いレベルは、ミトコンドリア膜電位の脱分極を反映している。我々はまた、H 2 O 2の適用後、ベースラインの最初の皮質ニューロンにおけるROSのレベルを、評価し、蛍光プローブH 2 DCF - DAを適用する方法を示しています。このプロトコルは、（若干変更して）また、異なる細胞型で、他の脳領域から分離されたニューロンのΔΨmとROSの変化を決定するために使用することができます。

Watch this Scientific Journal Video about Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons at JoVE.com

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

我々は、特定の刺激の適用前と適用後のTMRM蛍光強度の相対的変化を決定するために皮質ニューロンで、蛍光指示薬、TMRMのアプリケーションを実証する。我々はまた、蛍光プローブHのアプリケーションを表示<sub&gt; 2</sub&gt; DCF - DA皮質ニューロンにおける活性酸素種の相対的レベルを評価する。

ライブラット皮質ニューロンのミトコンドリア膜電位と活性酸素種の定量

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

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64.7K ビュー.  Loyola University Chicago. 我々は、特定の刺激の適用前と適用後のTMRM蛍光強度の相対的変化を決定するために皮質ニューロンで、蛍光指示薬、TMRMのアプリケーションを実証する。我々はまた、蛍光プローブHのアプリケーションを表示 2 DCF - DA皮質ニューロンにおける活性酸素種の相対的レベルを評価する。

ビデオ: Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

ニューロモデュレーションとミトコンドリア輸送：長時間にわたって海馬ニューロンのライブイメージング

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

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 ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

初代ラット皮質ニューロンとグリアのBilaminarの共培養

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

皮質ニューロンにおける経験依存性分子の変化の in vivo二光子イメージングにおける

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

グルコース感知を勉強する成体マウス由来の膜視床下部腹内側部のニューロンの電位色素イメージング

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

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

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

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

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.

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

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

生体内で新生仔マウスの大脳皮質ニューロンの 2 光子イメージング

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Stimulus-specific Cortical Visual Evoked Potential Morphological Patterns

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using 128-channel high-density electroencephalography (EEG). The specific aim of the described stimuli and analyses is to examine whether it is feasible to replicate previously reported CVEP morphological patterns elicited by an apparent motion stimulus, designed to simultaneously stimulate both ventral and dorsal central visual networks, using object and motion stimuli designed to separately stimulate ventral and dorsal visual cortical networks.&#160; Four visual paradigms are presented: 1. Randomized visual objects with consistent temporal presentation. 2. Randomized visual objects with inconsistent temporal presentation (or jitter).&#160; 3. Visual motion via a radial field of coherent central dot motion without jitter.&#160; 4. Visual motion via a radial field of coherent central dot motion with jitter.&#160; These four paradigms are presented in a pseudo-randomized order for each participant.&#160; Jitter is introduced in order to view how possible anticipatory-related effects may affect the morphology of the object-onset and motion-onset CVEP response.&#160; EEG data analyses are described in detail, including steps of data exportation from and importation to signal processing platforms, bad channel identification&#160;and removal, artifact rejection, averaging, and categorization of average CVEP morphological pattern type based upon latency ranges of component peaks. Representative data show that the methodological approach is indeed sensitive in eliciting differential object-onset and motion-onset CVEP morphological patterns and may, therefore, be useful in addressing the larger research aim. Given the high temporal resolution of EEG and the possible application of high-density EEG in source localization analyses, this protocol is ideal for the investigation of distinct CVEP morphological patterns and the underlying neural mechanisms which generate these differential responses.&#160; &#160; &#160;&#160;

In this paper, we present a protocol to investigate differential cortical visual evoked potential morphological patterns through stimulation of ventral and dorsal networks using high-density EEG. Visual object and motion stimulus paradigms, with and without temporal jitter, are described. Visual evoked potential morphological analyses are also outlined.&#160;&#160;

刺激特異的皮質視覚誘発潜在的形態パターン

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

E14-E16スプラーグドーリーラット胚から分離された混合原発海馬および皮質ニューロンからの神経球の生成

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. In addition, photoreceptors also exhibit high aerobic glycolysis like cancer cells. Precise measurements of these metabolic activities can provide valuable insights into cellular homeostasis under physiological conditions and in disease states. High throughput microplate-based assays have been developed to measure mitochondrial respiration and various metabolic activities in live cells. However, a vast majority of these are developed for cultured cells and have not been optimized for intact tissue samples and for application ex vivo. Described here is a detailed step-by-step protocol, using microplate-based fluorescence technology, to directly measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration, as well as extracellular acidification rate (ECAR) as an indicator of glycolysis, in intact ex vivo retinal tissue. This method has been used to successfully assess metabolic activities in adult mouse retina and demonstrate its application in investigating cellular mechanisms of aging and disease.

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Described here is a detailed protocol for performing mitochondrial stress assay and glycolytic rate assay in ex vivo retinal tissue samples using a commercial bioanalyzer.

Ex Vivoのレティナル組織サンプルにおけるミトコンドリア呼吸と解糖の測定

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. ...