この作品は、研究開発、医学研究サービス、退役軍人省のOfficeでサポートされています。この研究は、VAメリットレビュー賞（MCL）とテネシー大学ヘルスサイエンスセンター多発性硬化症研究基金によって賄われていた。

1。抗体標識（図1） <ol><li> 、0.1M NaHCO 3緩衝液を加えます。 <ul type="circle"><li> 8.4グラムのNaHCO 3 </li><li> 29.2グラムのNaCl </li><li> 1リットル、H 2 O </li></ul></li><li>この溶液を用いて8.4のpH（10.6グラムのNa 2 CO 3、29.2グラムのNaCl、1リットル、H 2 0）にバッファを立ち上げます。 </li><li> 70μlの抗hnRNPA1と500μlのNaHCO 3緩衝液に35μlのAtto550 NHSを加え、室温で1時間回転させます。時間の回転した後、透析に混合物を注入し、一晩のPBSで2リットルで透析する。 </li><li>翌日、スピンカラム（アミコンウルトラ0.5）を用いた抗hnRNPA1透析しAtto550 NHSを集中する。 </li><li> Atto550 NHSは抗hnRNPA1抗体が濃縮された標識の後、ナノドロップサンプル濃度を決定する。 </li></ol> 2。抗体のトランスフェクション（図2） <ol><li>シード10 5細胞/ウェルDulbecc500μlの中へトランスフェクションの8ウェルスライド24時間におけるoダルベッコ改変イーグル培地（DMEM/F12 +10％FBS +1％抗生物質）。コンフルエントの細胞の少なくとも70％であるべきである。 </li><li>二十四時間播種後、エッペンドルフチュー​​ブにAB-DeliverIN試薬の2μlを添加する。 </li><li>次に、同じエッペンドルフチュー​​ブに抗hnRNPA1抗体（0.5μg/μLの）標識Atto550 NHSの2μgを追加し、室温で10〜15分間インキュベートする。 </li><li>インキュベーションの間、培地を吸引し、細胞に新鮮なDMEM培地の394μlを添加する。 </li></ol> 注：コントロールとして動作するように手つかずの細胞の1室に500μlのDMEMを追加します。 <ol start="5"><li>インキュベーション後、上下にピペッティングして混合、抗体液に100μlのDMEMを追加し、500μlの合計ボリュームに対して、DMEM中のセルに追加します。 </li><li>抗体配信の48時間後にメディアを変更してください。 </li></ol> 注：プロトコルは、FITCはポジティブコントロールとしてRIGGラベル付きで繰り返した。 3。効率を決定するライブと固定撮像<ol><li>画像の細胞は、トランスフェクト画像Atto550 NHSの標識抗体をに蛍光顕微鏡でCy3のフィルタを使用して48時間で生活しています。 </li><li>ライブイメージングが完了した後、メディアやフィックス細胞を吸引除去する。 </li><li>メディアを吸引した後、室温で15分間、0.4％パラホルムアルデヒドで細胞を治療。細胞をPBSで4×5分間ずつを洗ってください。その後、DAPI試薬およびフィックスカバースリップを含むマウントメディアを持つ細胞をマウントします。 AxioVisionのソフトウェアを使用して固定された細胞のトランスフェクション効率を測定します。 AxioVisionのソフトが手元にない場合は、単に3.7の計算に手やプラグ結果によってあなたのイメージからのイベントをカウントします。あるいは、ImageJのソフトウェアは、これらの計算に使用することができます。 </li><li>まず、20倍の倍率でお好みの画像内のセルの総数をカウントする &quot;イベント&quot;の測定を使用しています。 </li><li>次に、なかった細胞をカウントする &quot;イベント&quot;の測定値を使用正常に同じ20倍倍率画像で抗体でトランスフェクションした。 </li><li> Excelのスタイル形式で測定された &quot;イベント&quot;にアクセスするには、 &quot;プロパティ&quot;をクリックして、 &quot;計測&quot;タブをクリックし、表示をクリックし、 &quot;テーブルとして。&quot; </li><li>最後に、2つの以前に測定されたイベントを比較することによって、あなたのトランスフェクション効率を決定するには、次の計算式を使用します（（総細胞 - 非トランスフェクト細胞）/総細胞数）×100 =トランスフェクション効率。 </li></ol> 4。代表的な結果トランスフェクトされた（ 図3B）ニューロンに比べてトランスフェクトしていないのライブ映像（ 図3A）は 、抗体の標識は（各図中の赤または緑）が明確に（ 図3B、C、D）を細胞内に観察されることが明らかになった。吸引後、細胞の表面に付着した間、または抗体を除去するために、メディアで洗浄し、標識された抗体は、まだ神経細胞（ 図3C、D）内に存在する。<html>トランスフェクション効率を計算するには、正常に導入しなかった細胞と比較して、合計のセルを計算するAxioVisionのソフトウェアを使用しています。これらの値は、全細胞の割合（ 図4）のようにトランスフェクション効率を獲得するため、トランスフェクション効率の計算式に入力した。 <img alt="図1" src="/files/ftp_upload/4154/4154fig1.jpg" /> 図標識抗体を濃縮するために必要な手順が続くアト550 NHSエステルを有する抗体を標識するために使用される手順の1。フロー図は<a href="http://www.jove.com/files/ftp_upload/4154/4154fig1large.jpg" target="_blank">拡大図を表示するには、ここをクリックしてください</a> 。 <img alt="図2" src="/files/ftp_upload/4154/4154fig2.jpg" /> 図2培養中の生細胞に標識された抗体をトランスフェクトするために使用される手続きのフロー図。arge.jpg &quot;ターゲット=&quot; _blank &quot;&gt;拡大図を表示するには、ここをクリックしてください。 <img alt="図3" src="/files/ftp_upload/4154/4154fig3.jpg" /> 図3：トランスフェクトされたニューロンは48時間トランスフェクション後の画像をライブ。 <img alt="図4" src="/files/ftp_upload/4154/4154fig4.jpg" /> 図4は、トランスフェクション効率を決定するためにカウントされたイベントを使用して画像を修正してすすいだ。

<ol>
	<li>Burkhardt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epitope-specific+recognition+of+type+II+collagen+by+rheumatoid+arthritis+antibodies+is+shared+with+recognition+by+antibodies+that+are+arthritogenic+in+collagen-induced+arthritis+in+the+mouse.">Epitope-specific recognition of type II collagen by rheumatoid arthritis antibodies is shared with recognition by antibodies that are arthritogenic in collagen-induced arthritis in the mouse.</a> Arthritis Rheum. 46, 2339-2348 (2002).</li><li>Chester, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lambert-Eaton+syndrome+antibodies:+reaction+with+membranes+from+a+small+cell+lung+cancer+xenograft.">Lambert-Eaton syndrome antibodies: reaction with membranes from a small cell lung cancer xenograft.</a> J. Neuroimmunol. 18, 97-104 (1988).</li><li>Lee, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HTLV-1+induced+molecular+mimicry+in+neurological+disease.">HTLV-1 induced molecular mimicry in neurological disease.</a> Curr. Top Microbiol. Immunol. 29, 125-136 (2005).</li><li>Reeves, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+lupus+erythematosus.+Antibodies+to+DNA,+DNA-binding+proteins,+and+histones.">Systemic lupus erythematosus. Antibodies to DNA, DNA-binding proteins, and histones.</a> Rheum. Dis. Clin. North Am. 20, 1-28 (1994).</li><li>Levin, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmunity+due+to+molecular+mimicry+as+a+cause+of+neurological+disease.">Autoimmunity due to molecular mimicry as a cause of neurological disease.</a> Nat. Med. 8, 509-513 (2002).</li><li>Lee, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoantibodies+that+recognize+functional+domains+of+hnRNPA1+implicate+molecular+mimicry+in+the+pathogenesis+of+neurological+disease.">Autoantibodies that recognize functional domains of hnRNPA1 implicate molecular mimicry in the pathogenesis of neurological disease.</a> Neurosci. Lett. 401, 188-193 (2006).</li><li>Kalume, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mimicry:+cross-reactive+antibodies+from+patients+with+immune-mediated+neurologic+disease+inhibit+neuronal+firing.">Molecular mimicry: cross-reactive antibodies from patients with immune-mediated neurologic disease inhibit neuronal firing.</a> J. Neurosci. Res. 77, 82-89 (2004).</li><li>Levin, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-reactivity+between+immunodominant+human+T+lymphotropic+virus+type+I+tax+and+neurons:+implications+for+molecular+mimicry.">Cross-reactivity between immunodominant human T lymphotropic virus type I tax and neurons: implications for molecular mimicry.</a> J. Infect. Dis. 186, 1514-1517 (2002).</li><li>Lee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+potential+link+between+autoimmunity+and+neurodegeneration+in+immune-mediated+neurological+disease.">A potential link between autoimmunity and neurodegeneration in immune-mediated neurological disease.</a> J. Neuroimmunol. 235, 56-69 (2011).</li><li>Smeenk, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antinuclear+antibodies:+cause+of+disease+or+caused+by+disease.">Antinuclear antibodies: cause of disease or caused by disease.</a> Rheumatology (Oxford). 39, 581-584 (2000).</li><li>Lee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-translational+glycosylation+of+target+proteins+implicate+molecular+mimicry+in+the+pathogenesis+of+HTLV-1+associated+neurological+disease.">Post-translational glycosylation of target proteins implicate molecular mimicry in the pathogenesis of HTLV-1 associated neurological disease.</a> J. Neuroimmunol. 204, 140-148 (2008).</li><li>Reisinger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-free+transfection+of+CHO+cells+with+chemically+defined+transfection+systems+and+investigation+of+their+potential+for+transient+and+stable+transfection.">Serum-free transfection of CHO cells with chemically defined transfection systems and investigation of their potential for transient and stable transfection.</a> Cytotechnology. 60, 115-123 (2009).</li><li>Weill, C. O., Biri, S., Erbacher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cationic+lipid-mediated+intracellular+delivery+of+antibodies+into+live+cells.">Cationic lipid-mediated intracellular delivery of antibodies into live cells.</a> Biotechniques. 44, Pvii-Pxi (2008).</li><li>Radic, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+nuclear+ribonucleoprotein+P2+is+an+autoantibody+target+in+mice+deficient+for+Mer,+Axl,+and+Tyro3+receptor+tyrosine+kinases.">Heterogeneous nuclear ribonucleoprotein P2 is an autoantibody target in mice deficient for Mer, Axl, and Tyro3 receptor tyrosine kinases.</a> J. Immunol. 176, 68-74 (2006).</li><li>Vincent, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+'passive+transfer'+of+paraneoplastic+stiff+person+syndrome+with+antibodies+to+an+intracellular+antigen.">Successful 'passive transfer' of paraneoplastic stiff person syndrome with antibodies to an intracellular antigen.</a> Brain. 133, 3164-3165 (2010).</li><li>Vincent, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stiff,+twitchy+or+wobbly:+are+GAD+antibodies+pathogenic.">Stiff, twitchy or wobbly: are GAD antibodies pathogenic.</a> Brain. 131 (Pt 10), 2536-2537 (2008).</li><li>Magrys, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+anti-alpha-enolase+autoantibodies+in+pathogenicity+of+autoimmune-mediated+retinopathy.">The role of anti-alpha-enolase autoantibodies in pathogenicity of autoimmune-mediated retinopathy.</a> J. Clin. Immunol. 27, 181-192 (2007).</li><li>Geis, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stiff+person+syndrome-associated+autoantibodies+to+amphiphysin+mediate+reduced+GABAergic+inhibition.">Stiff person syndrome-associated autoantibodies to amphiphysin mediate reduced GABAergic inhibition.</a> Brain. 133, 3166-3180 (2010).</li></ol>

特別な利害関係は宣言されません。

特定の分子とその発現についての仮説をテストするためには、遺伝子および他のベクターは、一般的に細胞にトランスフェクトされる。このプロシージャのユニークな点は、容易に標的細胞に抗体をトランスフェクトする能力である。我々は、重複での5つの別々の実験ごとにこの方法を行った。トランスフェクション効率の結果は、すべての実験間で同様であった。重要なのは、方法は、抗...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>蛍光顕微鏡AxioObserver A1 </td><td>ツァイス</td><td></td></tr><tr><td> AxioVisionのバージョン4.2 </td><td>ツァイス</td><td></td></tr><tr><td>アンチrhnRNPA1 </td><td>アブカム</td><td> Ab4791 </td></tr><tr><td> FITCは抗RIGGラベル付き</td><td> OZ Biosciences社</td><td> AI20100 </td></tr><tr><td> Atto550 NHSエステル</td><td>シグマ</td><td> 92835 </td></tr><tr><td> AB-DeliverIN </td><td> OZ Biosciences社</td><td> AI20100 </td></tr><tr><td> DMEM/F12 +10％FBS +1％抗生物質</td><td>ギブコ</td><td> 11320-033 </td></tr><tr><td>ポリ-D-リジン8ウェル培養スライド</td><td> BD </td><td> 354632 </td></tr><tr><td>多目的ローテータ</td><td>科学的なインダスES株式会社</td><td>モデル151 </td></tr><tr><td> DAPIマウントメディア</td><td>ミリポア</td><td> S7113 </td></tr><tr><td> PBS </td><td>アンビオン</td><td> 9626 </td></tr><tr><td>ダイアライザー</td><td>サーモフィッシャーサイエンティフィック</td><td> 66380 </td></tr><tr><td>アミコンウルトラ0.5 </td><td>ミリポア</td><td> UFC501096 </td></tr><tr><td>パラホルムアルデヒド（4％溶液） </td><td>電子顕微鏡学</td><td></td></tr><tr><td>ナノドロップ</td><td>サーモフィッシャーサイエンティフィック</td><td></td></tr></tbody></table>

antibody transfection into neurons as a tool to study disease pathogenesis

抗体は、生体のシステムで行われている様々なイベントに新たな洞察を得るための機能を提供します。標的タンパク質に特異性の高い抗体を産生する能力が対処しなければ、非常に正確な生物学的な質問のために許可されています。重要なのは、抗体は全身性エリテマトーデス（SLE）、関節リウマチ（RA）、腫瘍随伴症候群、多発性硬化症（MS）とヒトTリンパ球向性ウイルス1型（HTLV-1）を含むヒトの多くの疾患の病因に関与している脊髄症/熱帯性痙性対麻痺（HAM / TSP）1-9関連付けられています。抗体は、病気が進行中の調査の領域であり、データは抗体および炎症の様々な細胞内分子の結果の間の相互作用は、携帯メッセージング、およびアポトーシス10を変更していることを示唆している原因になる方法。これは、MSとHAM / TSP患者は細胞内のRNA結合タンパク質異種リボ核タンパク質に対する自己抗体を産生することが示されている5月7日 1（のhnRNP A1）は3、9、11。最近のデータは、イントラ神経および表面抗原の両方に対する抗体が病原3、5月9日、11であることを示している。したがって、細胞内抗体の研究を可能にする手順：タンパク質相互作用は、疾患の病因に偉大な洞察力を貸すだろう。 遺伝子は一般的に初代培養細胞や培養細胞株にトランスフェクトされ、細胞内への抗体のトランスフェクションは、しかし抗体構造または貧弱トランスフェクション効率を12の改変によって妨げられている。トランスフェクションの他の方法としては、カチオン性リポソーム（DOTAP / DOPEから成る）とポリエチレンイミン（PEI）に基づく抗体のトランスフェクションを含む;コントロール12と比較して、抗体のトランスフェクションの10倍に減少したどちらも。私たちの研究で行った方法では、陽イオン脂質媒介方法に類似しており、静とhydrを通じて抗体と非共有結合複合体を形成するために、脂質ベースのメ​​カニズムを使用しophobic相互作用13。我々は、非共有結合、静電性および疎水性相互作用を介して抗体を捕捉し、細胞内でそれらを提供することができる脂質製剤であるAB-DeliverIN試薬を利用した。したがって、化学的および遺伝的カップリングは生細胞に機能的な抗体の送達のためには必要ありません。この方法では、さまざまなトレース抗体やタンパク質の局在化実験と同様に、細胞内抗体の分子の結果の分析を行うことができるようになりました：タンパク質相互作用9。 このプロトコルでは、再現性とトランスフェクション効率の高い、急速に神経細胞に抗体をトランスフェクトする方法を示します。一例として、我々はアンチのhnRNP A1および抗IgG抗体を使用します。トランスフェクション効率を簡単に定量化のために、我々はアト-550-NHSとFITC標識IgGで標識した抗のhnRNP A1抗体を使用していました。 Atto550 NHSは高分子吸収と量子yiを持つ新しいラベルですELD。励起源とAtto550用蛍光フィルターはCy3（例：556エム。578）に似ています。また、Atto550は高い光安定性を持っています。 FITC標識IgGは、このメソッドは汎用性であることを示していると依存染めないようにコントロールとして用いた。生成されたこのアプローチは、データは細胞内標的抗原に対する抗体は、ヒト疾患の病因に果たしていることが役割の理解に役立ちます。

Watch this Scientific Journal Video about Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis at JoVE.com

Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis

相互作用や細胞内環境における抗体の存在に関連する分子メカニズムに及ぼす影響を調査するための迅速なアプローチが説明されています。この方法は、脂質製剤に基づく非共有結合複合体形成を利用した生細胞への抗体のトランスフェクションが含まれます。技術は不死化細胞株および初代培養細胞に適応可能です。

疾患の病因を研究するツールとしてのニューロンへの抗体のトランスフェクション

Antibodies provide the ability to gain novel insight into various events taking place in living systems.  The ability to produce highly specific ...

antibody-transfection-into-neurons-as-tool-to-study-disease

Research

JoVE Journal

Neuroscience

10.7K ビュー.  Veterans Administration Medical Center, Memphis, TN. 相互作用や細胞内環境における抗体の存在に関連する分子メカニズムに及ぼす影響を調査するための迅速なアプローチが説明されています。この方法は、脂質製剤に基づく非共有結合複合体形成を利用した生細胞への抗体のトランスフェクションが含まれます。技術は不死化細胞株および初代培養細胞に適応可能です。

ビデオ: Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

のレーザーキャプチャーマイクロダイセクションショウジョウバエ末梢ニューロン

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

神経科学

Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic transmission. Increasing evidence indicates that gap junctions not only permit electrical current flow and synchronous activity between interconnected or coupled cells, but that the strength or effectiveness of electrical communication between coupled cells can be modulated to a great extent1,2. In addition, the large internal diameter (~1.2 nm) of many gap junction channels permits not only electric current flow, but also the diffusion of intracellular signaling molecules and small metabolites between interconnected cells, so that gap junctions may also mediate metabolic and chemical communication. The strength of gap junctional communication between neurons and its modulation by neurotransmitters and other factors can be studied by simultaneously electrically recording from coupled cells and by determining the extent of diffusion of tracer molecules, which are gap junction permeable, but not membrane permeable, following iontophoretic injection into single cells. However, these procedures can be extremely difficult to perform on neurons with small somata in intact neural tissue.
Numerous studies on electrical synapses and the modulation of electrical communication have been conducted in the vertebrate retina, since each of the five retinal neuron types is electrically connected by gap junctions3,4. Increasing evidence has shown that the circadian (24-hour) clock in the retina and changes in light stimulation regulate gap junction coupling3-8. For example, recent work has demonstrated that the retinal circadian clock decreases gap junction coupling between rod and cone photoreceptor cells during the day by increasing dopamine D2 receptor activation, and dramatically increases rod-cone coupling at night by reducing D2 receptor activation7,8. However, not only are these studies extremely difficult to perform on neurons with small somata in intact neural retinal tissue, but it can be difficult to adequately control the illumination conditions during the electrophysiological study of single retinal neurons to avoid light-induced changes in gap junction conductance.
Here, we present a straightforward method of determining the extent of gap junction tracer coupling between retinal neurons under different illumination conditions and at different times of the day and night. This cut-loading technique is a modification of scrape loading9-12, which is based on dye loading and diffusion through open gap junction channels. Scrape loading works well in cultured cells, but not in thick slices such as intact retinas. The cut-loading technique has been used to study photoreceptor coupling in intact fish and mammalian retinas7, 8,13, and can be used to study coupling between other retinal neurons, as described here.

An easy and convenient method to determine the extent of gap junction tracer coupling between retinal neurons is described. This technique enables one to investigate the function of the electrical synapses between neurons in the intact retina under different illumination conditions and at different times of the day and night.

カットローディング：網膜ニューロン間ギャップ結合トレーサーカップリングの程度を調べるための便利なツール

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

初代海馬ニューロンのリン酸カルシウムトランスフェクション

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations &#8211; most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

リアルタイム神経細胞株に神経毒性および神経保護に関与する分子経路を描写するツールとしてのインピーダンスベースの細胞分析装置

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

モデルとしての嗅覚系は、軸索の成長パターンと形態を研究する<em&gt;インビボ</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the development of several animal models of these neuromuscular disorders has provided the scientific community with different therapeutic scenarios aimed at delaying or reversing the progression of these conditions. By taking advantage of the retrograde machinery of neurons, one of these approaches has been to target skeletal muscles in order to shuttle therapeutic genes into corresponding spinal cord motor neurons. Although once promising, the success of such gene delivery approach has been hampered by the sub-optimal number of transduced motor neurons it has so far shown to yield. Motor end plates (MEPs) are highly specialized regions on the skeletal musculature that are in direct synaptic contact to the spinal cord &#945; motor neurons. In this regard, it is important to note that, so far, the efforts to retrogradely transfer genes into motor neurons were made without reference to the location of the MEP region in the targeted muscles. Here, we describe a simple protocol 1) to reveal the exact location of the MEPs on the surface of skeletal muscles and 2) to use this information to guide the intramuscular delivery and subsequent optimal retrograde transport of retrograde tracers into motor neurons. We hope to utilize the results from these tracing experiments in further studies into investigating retrograde transport of therapeutic genes to spinal cord motor neurons through the targeting of MEPs.

The efficacy of intramuscular uptake and retrograde transport of molecules to corresponding motor neurons depends on the location of the injection sites with respect to the motor end plates (MEPs). Here, we describe how to locate MEPs on skeletal muscles to optimise retrograde transport of tracers into motor neurons.

モーターエンドプレートに沿って筋肉内注射：直接運動ニューロンへのシャトルトレーサーへの低侵襲アプローチ

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the ...

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.

ストライプアッセイは、解離海馬ニューロンを用いたタンパク質基質の魅力や反発作用を検討します

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

研究保護および腫瘍細胞の侵襲性をモデルとして脊髄海馬スライス培養

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

線維形成の強力な細胞モデルとして α-シヌクレインのミクロソーム関連付けられた集計一次ニューロンの外因性投与

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing neuronal dysfunction which eventually leads to cognitive decline. Hyperphosphorylated tau aggregates in the neuron are believed to cause most of the pathology associated with AD. These aggregates are assumed to be released into the extracellular compartment and taken up by adjacent healthy neurons where they induce further tau aggregation. This &#34;prion-like&#34; spreading can be interrupted by antibodies capable of binding and &#34;neutralizing&#34; extracellular tau aggregates as shown in preclinical mouse models of AD. One of the proposed mechanisms by which therapeutic antibodies reduce pathology is antibody-mediated uptake and clearance of pathological aggregated forms of tau by microglia. Here, we describe a quantitative cell-based assay to assess tau uptake by microglia. This assay uses the mouse microglial cell line BV-2, allows for high specificity, low variability and medium throughput. Data generated with this assay can contribute to a better characterization of anti-tau antibody effector functions.

Here we describe a cell-based assay to quantitatively assess tau uptake by microglia with the aim of creating an investigational tool to better characterize the mechanisms of action of anti-tau antibodies.

タウの抗体を介したミクログリアによってクリアランスを勉強するセルベースのアッセイ

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing ...