キイロショウジョウバエ神経筋接合部の1）準備（NMJ） <ol><li>標準ショウジョウバエ生理食塩水を（130 mMのNaCl、36 mMスクロース、5mMのKClを、2mMのCaCl 2を 、2mMのMgCl 2、5mMのHepes緩衝液、pH 7.3 4準備。 </li><li>生理食塩水（1.1）の準備を細かく分析。ショウジョウバエの幼虫は、Sylgard皿に背側を上向きにしてpinedされ、背側には長手方向の切片であり、そして内臓は削除されます。準備は、引き伸ばされ、pinedれる。いくつかの腹側の筋肉は、使用することができます。 </li></ol> 2）刺激とFM染色<ol><li>漂白からFM染料を保護するために、暗い場所でもすべて、次の手順を実行することをお勧めします。 </li><li>神経の化学的刺激の場合は、高カリウムバッファーが使用されます。塩化カリウムの90 mMのとFM1 -​​ 43色素の10μMを含む（1.1参照）ショウジョウバエ生理食塩水を準備します。光から保護ソリューションをキープ。 </li><li>バス、室温で1分間、事前に準備されたバッファと準備。 </li><li>細胞外FM1 -​​ 43色素を除去するために標準のショウジョウバエ生理食塩水（1.1）で洗浄する。固定するために急速に進んでください。 </li></ol> 3）固定<ol><li>室温で45分間リン酸緩衝生理食塩水（PBS）で2.5％グルタルアルデヒドを有する製剤バス。形態学的特徴の良い保全のために、グルタルアルデヒドは、ホルムアルデヒドが好ましい。 </li><li> PBSで1回洗浄し、その後NH 4 Clの100mMのに15分間水没の準備を残す。このステップは、残りのグルタルアルデヒド固定液の遊離アルデヒド基をクエンチするために行われます。 </li><li>通常のPBSでNH 4 Cl溶液を洗い流す。 </li></ol> 4）光変換<ol><li> 4℃で30分間NMJの準備をインキュベートしますPBSでCは、ジアミノベンジジン（DAB）の1.5mgのML - 1を含む。 </li><li>蛍光顕微鏡下でサンプルを置きます。比較的低倍率の水浸対物レンズ（20倍0.5 NA）を用いて蛍光シグナルを検出し、フォーカス。 </li><li> FMの染料は、（図1C）、完全に漂白されるまでの最大ランプ強度とサンプルを照らす。光変換が発生したかどうかを制御するために、FMの染料漂白段階の前後に透過光の下にサンプルで確認することをお勧めします。光変換が行われる場合、暗褐色の沈殿物が（図1C）が期待される。照明時間は、準備にDABの浸透時にも、照明の強さとに応じて、変数です。ほとんどの準備のために、我々は20倍の対物レンズを用いて時〜30〜45分の照射時間が最適であることがわかった。短いイルミネーション期間は、より高い倍率の目標（60倍）に使用されます。 </li><li>照明のために我々は、後方散乱光を収集し、そのため全体の強度を増加させるバックミラーを、含んでいるランプハウジングと、水銀ランプを使用することを好む。 </li></ol> 5）EMのサンプルを処理<ol><li> Osmication <ol class="lalpha"><li>水の木の量のオスミウム四酸化（サンプルあたり約300μL）の一冊で解決策を準備。四酸化オスミウムを使用して作業は手袋とアイアイの保護装置を使用して、フードの下で行う必要があります。 </li><li>室温で1時間四酸化オスミウム溶液（フードの下で）の準備をインキュベートする。 </li><li> PBSで洗う（4-5回5分）と清浄なガラスのフラスコに各サンプルを移す。 </li></ol></li><li>脱水<ol class="lalpha"><li> 30、50、70 90、95およびエタノールの100％を含む別の溶液を調製。 </li><li> 5分間、各サンプルに30％エタノール1 mlを追加します。 </li><li> 5分間の各サンプルに50％エタノール1 mlを追加します。 </li><li> 5分間、各サンプルに70％エタノール1 mlを追加します。 </li><li> 5分間、各サンプルに90％エタノール1 mlを追加します。 </li><li> （3倍を繰り返す）5分間、各サンプルに95％エタノール1 mlを追加します。 </li><li> （3倍を繰り返す）5分間、各サンプルに100％エタノール1 mlを追加します。 </li></ol></li><li>埋め込みとその後の処理<ol class="lalpha"><li>エタノールの一冊とエポン樹脂の1ボリュームを混在させる。連続回転の下準備（2-4時間）に追加します。 </li><li>オープンフラスコで100％エポンで準備を置き、残りのエタノールを蒸発させる（4-6時間）。 </li><li> 36時間60℃で金型の準備を置きます。 </li><li>電子顕微鏡の処理。標準ultramicrotomyの手順を使用して、50〜80 nmの薄いセクションで準備を処理します。 </li></ol></li><li>電子顕微鏡イメージング<ol class="lalpha"><li>製剤は、従来のEMの手順を用いて結像​​される。 </li></ol></li></ol> 6）代表結果期待した結果が図1Cと図2に概説されています。仲間の形成における照明の手続の結果蛍光と透過イメージングの両方で表示されるWNのDABの沈殿物、。照明時の最初のオカレンスは、FM色素の染色に関連付けられている蛍光の消失です。アルデヒド系固定液により誘発されるバックグラウンドの蛍光は、対照的に、実験を通して目に見えるようになります。漂白は、照明の開始後〜10〜20分で一般的に行われます。一般的には光電製品は、この時点で観察することはできません。 照明は継続すべきである、と〜10分後の製剤は、それは同時には行われませんなぜDAB降水量とFM色素が漂白不明であるDAB蓄積による茶色の色調（図1C IV）に変わります、それは、その比較的大きな可能性があります酸化DABの量は、凝集し沈殿する前に蓄積する必要があるため、この反応は、漂白工程よりも遅いということ。 DABの降水量はおそらく不完全であるとして、この段階では、しかし、準備には、電子顕微鏡の処理のための準備ができていません。我々は準備（シナプス前神経終末が）完全な変換を示す暗褐色（または黒色）の色を、想定している間に、5〜10分長く待つことを好む。準備の一般的な表面のわずかな変換（例えば、神経筋接合部における筋線維のは）この段階で観察することができます。それはおそらく自家蛍光（および/または固定液蛍光）により誘発されるDABの変換に関連して、そしてそれは、プロシージャに有害ではない。 準備をして電子顕微鏡用に処理され、そしてダーク（ラベル）小胞の有無をチェックする必要があります。我々は5,6の前に説明したように、これらの小胞は、非標識のものよりもはるかに高密度であるため、容易に（図2B）区別されます。よく小胞の異なる種類を区別する機会を増やすために、セクションのないコントラスト増強後の染色は、（無酢酸ウラニルまたはクエン酸鉛で染色）実行されるべきである。オスミウム染色は、ほとんどの携帯電話の要素を観察するのに十分である、ととしてではないDABの沈殿物として暗い。 <img src="/files/ftp_upload/1790/1790fig1.jpg" alt="図1" /> 図1：FM染料、それは光変換のために使用します 。 （A）刺激下でFM色素を使用して小胞をロードするとdistainingのための古典的な実験の手順を表します。 （ 私は ）バッファが含まれているFM色素でサンプルをインキュベートする。 （ⅱ）FM色素の存在下で小胞融合を誘導するために（電気的または化学的）刺激。 （ⅲ）刺激後のサブシーケンスのエンドサイトーシスは細胞外のバッファに残っているFM色素で、いくつかの小胞をロードします。 （ⅳ）バッファーで洗浄することにより細胞外FM色素を交換してください。 （V）新しい刺激がロードされた小胞は、エキソサイトーシスの際、FM色素を解放するために誘発される。 （B）回路図は、FM1 - 43色素のヘッド、ブリッジとテールを示している。 ショウジョウバエ NMJの光電変換実験の（C）の例。 （ⅰ）外部色素分子と固定し、洗浄、FM1 - 43でNMJをロードした後、神経末端の蛍光は、従来の落射蛍光顕微鏡（63x）で観察することができます。 （II - III）の連続照明下では色素が完全に漂白です。 （ⅳ）照明が続くと、黒い色は、ジアミノベンジジンの析出による表示されます。スケールバーは10μmを表す。 <img src="/files/ftp_upload/1790/1790fig2.jpg" alt="図2" /> 図2：photoconvertedサンプルのEMの例（A）画像はシナプス小胞を含む神経端末を示していますが、それらのphotoconvertedであり、これは組織に十分な照明や ​​貧しいジアミノベンジジンの浸透を持っていないことが原因である可能性があります。 （B）FM光変換を経ていくつかの暗い（充填）小胞とシナプスブートン。は小胞やオルガネラは区別がない全体的な暗いターミナルの照明の結果の（C）超過。スケールバーは200nmを表す。

<ol>
	<li>Betz, W. J., Bewick, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+analysis+of+synaptic+vesicle+recycling+at+the+frog+neuromuscular+junction.">Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction.</a> Science. 255, 200-203 (1992).</li><li>Henkel, A. W., L&#252;bke, J., Betz, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FM1-43+dye+ultrastructural+localization+in+and+release+from+frog+motor+nerve+terminals.">FM1-43 dye ultrastructural localization in and release from frog motor nerve terminals.</a> Proc Natl Acad Sci USA. 93, 1918-1923 (1996).</li><li>Sandell, J. H., Masland, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoconversion+of+some+fluorescent+markers+to+a+diaminobenzidine+product.">Photoconversion of some fluorescent markers to a diaminobenzidine product.</a> J Histochem Cytochem. 36, 555-559 (1988).</li><li>Kuromi, H., Kidokoro, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+optically+determined+size+of+exo/endo+cycling+vesicle+pool+correlates+with+the+quantal+content+at+the+neuromuscular+junction+of+Drosophila+larvae.">The optically determined size of exo/endo cycling vesicle pool correlates with the quantal content at the neuromuscular junction of Drosophila larvae.</a> J Neurosci. 19, 1557-1565 (1999).</li><li>Denker, A., Kr&#246;hnert, K., Rizzoli, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revisiting+synaptic+vesicle+pool+localization+in+the+Drosophila+neuromuscular+junction.">Revisiting synaptic vesicle pool localization in the Drosophila neuromuscular junction.</a> J Physiol (Lond). 587, 2919-2926 (2009).</li><li>Rizzoli, S. O., Betz, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structural+organization+of+the+readily+releasable+pool+of+synaptic+vesicles.">The structural organization of the readily releasable pool of synaptic vesicles.</a> Science. 303, 2037-2039 (2004).</li><li>Darcy, K. J., Staras, K., Collinson, L. M., Goda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutive+sharing+of+recycling+synaptic+vesicles+between+presynaptic+boutons.">Constitutive sharing of recycling synaptic vesicles between presynaptic boutons.</a> Nat Neurosci. 9, 315-321 (2006).</li><li>Harata, N., Ryan, T. A., Smith, S. J., Buchanan, J., Tsien, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+recycling+synaptic+vesicles+in+hippocampal+neurons+by+FM1-43+photoconversion.">Visualizing recycling synaptic vesicles in hippocampal neurons by FM1-43 photoconversion.</a> Proc Natl Acad Sci USA. 98, 12748-12753 (2001).</li><li>Lange, R. P. J. d. e., de Roos, A. D. G., Borst, J. G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+modes+of+vesicle+recycling+in+the+rat+calyx+of+Held.">Two modes of vesicle recycling in the rat calyx of Held.</a> J Neurosci. 23, 10164-10173 (2003).</li><li>Richards, D. A., Guatimosim, C., Rizzoli, S. O., Betz, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+vesicle+pools+at+the+frog+neuromuscular+junction.">Synaptic vesicle pools at the frog neuromuscular junction.</a> Neuron. 39, 529-541 (2003).</li></ol>

いくつかの重要なステップを考慮する必要があります。 <ul><li> DABのインキュベーションは、準備の徹底した洗浄と急冷後に行ってください。そうでなければ非反応グルタルアルデヒドは、DABと相互作用し、（通常は高密度電子ではないフラットな結晶の形で）その降水量が発生します。この降水量が豊富に行われる準備は、電子顕微鏡のためまれに使用できます。 </li><li>照明時間は、異?...

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		<thead>
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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>FM 1-43</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>F10317</td>
<td>&nbsp;</td>
<tr>
<td>Epon resin</td>
<td>&nbsp;</td>
<td>Plano</td>
<td>R1030</td>
<td>&nbsp;</td>
<tr>
<td>di-aminobenzidine hydrochloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D5905</td>
<td>&nbsp;</td>
<tr>
<td>50% Glutaraldehyde</td>
<td>&nbsp;</td>
<td>AppliChem</td>
<td>A3166</td>
<td>EM grade</td>
</tr>
<tr>
<td>Sylgard</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>104186298</td>
<td>&nbsp;</td>
<tr>
<td>Axioskop 2 FS plus</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Objective 20x 0.5 NA</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>Dry objective</td>
</tr>
<tr>
<td>100W Hg Lamp</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Lamp housing with back mirror</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>1007-980</td>
<td>&nbsp;</td>
<tr>
<td>MRm camera</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>0445-554</td>
<td>Image acquisition</td>
</tr>
<tr>
<td>Ex. Filter (HQ 470/40)</td>
<td>&nbsp;</td>
<td>AHF</td>
<td>F49-671</td>
<td>&nbsp;</td>
<tr>
<td>Dichroic (495 DCLP)</td>
<td>&nbsp;</td>
<td>AHF</td>
<td>F33-100</td>
<td>&nbsp;</td>
<tr>
<td>Em. Filter (HQ 500 LP)</td>
<td>&nbsp;</td>
<td>AHF</td>
<td>F42-018</td>
<td>&nbsp;</td>
<tr>
<td>EM</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Proscan CCD HSS</td>
<td>&nbsp;</td>
<td>Proscan Electronic Sys.</td>
<td>&nbsp;</td>
<td>1024 x 1024</td>
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studying synaptic vesicle pools using photoconversion of styryl dyes

形質膜（エキソサイトーシス）とシナプス小胞の融合は、神経伝達物質の放出と神経細胞のコミュニケーションに必要なステップです。彼らは新しいエキソおよびエンドサイトーシスのサイクル（小胞のリサイクル）を受けるまで、小胞はその後、形質膜（エンドサイトーシス）から取得し、神経終末内の小胞の一般的なプールと一緒にグループ化されます。これらのプロセスは、電子顕微鏡、電気生理学的記録、アンペロメトリーとキャパシタンスの測定など、さまざまな手法を用いて研究されている。重要なのは、最後の二十年の間に蛍光標識したマーカーの数は、光学技術は、そのリサイクルのダイナミクスに小胞を追跡できるように、浮上した。最も一般的に使用されるマーカーの一つは、スチリルまたはFM色素1であり、構造的に、すべてのFM色素は親水性の頭部と芳香環と1つまたは複数の二重結合（図1B）を介して接続親油性尾が含まれています。小胞のプールをラベル付けするために古典的なFM色素の実験は、神経の刺激（電気的または高K +の）中に色素と（図1Ai）準備の入浴で構成されています。これは最近、エンドサイトーシス小胞（図1A I - III）に小胞のリサイクルと染料のその後のロードを誘発する。染料、色素フリーバスの刺激の第二ラウンドで小胞をロードした後にエキソサイトーシスを介してFMのリリース（図1A IV - V）、蛍光強度の減少（脱色）を監視し続けることができますプロセスの引き金となるでしょう。 FM色素が小胞のリサイクルの分野に大きく貢献しているが、それは従来の蛍光顕微鏡を使用して、個々の小胞の正確な局在や形態を決定することはできません。また、光変換を介して、電子顕微鏡を用いてエンドサイトーシスのマーカーとして使用する方法をFM色素そのため、我々はここで説明する。光電変換技術は、強烈な照明下での活性酸素種を生成する蛍光色素のプロパティを利用している。蛍光標識された調製物は、ジアミノベンジジン（DAB）を含む溶液に浸漬し、点灯されています。色素分子によって生成される反応種は暗い外観を持っているし、簡単に電子顕微鏡2,3で区別することができる安定した、不溶性の沈殿物を形成するDABを、酸化する。 DABにのみ蛍光分子（活性酸素種は短命であるため）のすぐ近くに酸化されるように、テクニックだけ蛍光標識構造は電子密度の高い沈殿物を含むとしていることを保証します。テクニックは、このように積極的に細胞内小器官のリサイクルの正確な位置と形態の研究をすることができます。

Watch this Scientific Journal Video about Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes at JoVE.com

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

FM色素は、シナプスのダイナミクスの理解に非常に貴重な助けとなった。 FMSは、通常、異なる刺激条件の中に蛍光顕微鏡下で続いている。しかし、電子顕微鏡と組み合わせてFM色素の光変換は、シナプスboutonsで、他の超微細構造のコンポーネント間で、明確なシナプス小胞プールの可視化が可能になります。

スチリル染料の光変換を使用して、シナプス小胞プールの勉強

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The ...

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JoVE Journal

Neuroscience

11.6K ビュー.  European Neuroscience Institute Göttingen. FM色素は、シナプスのダイナミクスの理解に非常に貴重な助けとなった。 FMSは、通常、異なる刺激条件の中に蛍光顕微鏡下で続いている。しかし、電子顕微鏡と組み合わせてFM色素の光変換は、シナプスboutonsで、他の超微細構造のコンポーネント間で、明確なシナプス小胞プールの可視化が可能になります。

ビデオ: Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

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 ...

神経科学

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 ...

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 ...

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

アストロサイトトランスポーター電流のデコンボリューション解析グルタミン酸クリアランスの時間経過の導出

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

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 ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

生体内培養-高速、シンプルで、安価な AWESAM プロトコルを使用してマウスのアストロ サイトのように

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...

Pentylenetetrazole-Induced Kindling Mouse Model

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high dose, whereas sequential injections of a subconvulsive dose have been used for the development of chemical kindling, an epilepsy model. A single low-dose injection of PTZ induces a mild seizure without convulsion. However, repetitive low-dose injections of PTZ decrease the threshold to evoke a convulsive seizure. Finally, continuous low-dose administration of PTZ induces a severe tonic-clonic seizure. This method is simple and widely applicable to investigate the pathophysiology of epilepsy, which is defined as a chronic disease that involves repetitive seizures. This chemical kindling protocol causes repetitive seizures in animals. With this method, vulnerability to PTZ-mediated seizures or the degree of aggravation of epileptic seizures was estimated. These advantages have led to the use of this method for screening anti-epileptic drugs and epilepsy-related genes. In addition, this method has been used to investigate neuronal damage after epileptic seizures because the histological changes observed in the brains of epileptic patients also appear in the brains of chemical-kindled animals. Thus, this protocol is useful for conveniently producing animal models of epilepsy.

This protocol describes a method of chemical kindling with pentylenetetrazole and provides a mouse model of epilepsy. This protocol can also be used to investigate vulnerability to seizure induction and pathogenesis after epileptic seizures in mice.

Pentylenetetrazole 誘起キンドリング マウス モデル

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

オプトジェネティクスを組み合わせてそのまま神経回路内でのシナプス前機能に関する遺伝子ノックダウンの効果を特徴付ける人工マイクロ Rna

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

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 ...

Evaluation of Synaptic Multiplicity Using Whole-cell Patch-clamp Electrophysiology

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic multiplicity). Synaptic multiplicity is plastic and changes throughout development and in different physiological conditions, being an important determinant for the efficacy of synaptic transmission. Here, we outline experiments for estimating the degree of multiplicity of synapses terminating onto a given postsynaptic neuron using whole-cell patch clamp electrophysiology in acute brain slices. Specifically, voltage-clamp recording is used to compare the difference between the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs). The theory behind this method is that afferent inputs that exhibit multiplicity will show large, action potential-dependent sEPSCs due to the synchronous release that occurs at each synaptic contact. In contrast, action potential-independent release (which is asynchronous) will generate smaller amplitude mEPSCs. This article outlines a set of experiments and analyses to characterize the existence of synaptic multiplicity and discusses the requirements and limitations of the technique. This technique can be applied to investigate how different behavioral, pharmacological or environmental interventions in vivo affect the organization of synaptic contacts in different brain areas.

Here, we present a protocol for evaluating the functional synaptic multiplicity using whole-cell patch clamp electrophysiology in acute brain slices.

全細胞パッチク電気生理学を用いたシナプスの多様性の評価

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic ...