私たちは、彼らの技術支援のための越（ポーリン）胡、レイチェルSINITに感謝したいと思います。研究は、NIHの助成金（PN2 EY016525）によって、及びダウン症の研究と治療財団とラリーL. Hillblom財団からの資金によってサポートされています。

外科および動物の手順は厳密に実験動物の管理と使用に関するNI​​Hガイドに従って行われる。動物の使用を含む全ての実験は、UCSDの施設内動物管理使用委員会によって承認されています。 1プラスミドのクローニング、モノビオチン化BDNFの発現および精製（mBtBDNF） 注：HEK293FTセル10におけるpcDNA3.1ベクターと共発現に予めproBDNFaviとBirAをするcDNAを構築します。 mBtBDNFは、成熟生物学的に活性モノビオチン神経成長因子（mBtNGF）10を製造する以前に公開された方法に従ってのNi-NTAビーズを用いて精製する。 マイクロ流体チャンバーの調製マイクロ流体ニューロン培養装置は、流体ニューロン細胞体から軸索を単離することができる。右の各解剖前にコーティングされたばかりカバースリップでチャンバーを組み立てます。使用マイクロ流体室このプロトコルで、商業的に（資機材の表を参照）を購入している。手洗いや5-6xまでの再利用、商業的に購入室。 <ol><li>手洗い1％Alconoxマイクロ流体室。 30分ごとにミリQ水で3回すすいでください。手を70％エタノールで再びチャンバを洗浄する。 </li><li>層流フード中でパラフィルム上で乾燥さ室をレイアウト。 UV下で20分間チャンバーの両側を照射し、滅菌する。ストアは、室温でパラフィルムで密封し、滅菌15cmの皿にチャンバを滅菌した。 </li><li>ガラス容器に24×40ミリメートル1号カバーガラスを配置します。一晩回転装置に35％の塩酸でカバースリップを浸す。翌日、30分水でそれぞれにカバースリップを3回すすいでください。 </li><li>カバースリップを100％エタノールに各カバースリップを浸漬し、ブンゼンバーナーの上に炎によって一枚ずつ滅菌する。無菌ペトリ皿に乾燥カバースリップを保存し、コーティングまで室温で保管してください。 </li><li> OUを置き15cmの培養皿にカバースリップはt。コー​​ト0.01％ポリ-L-リジン（PLL）0.7mlを各カバースリップとは、室温でフード内でインキュベートする。 </li><li> 1時間後、カバースリップを滅菌水で3回リンス。ドライカバーガラス、真空とし、6cmの培養皿に各カバースリップを配置。 </li><li>マイクロ流体チャンバーを組み立てるために、マイクログルーブに触れないように注意して、PLLコーティングしたカバーガラス上に下部にマイクログルーブ側でチャンバーを配置。優しく室が密閉されたことを確認するためにピペットチップでチャンバーを押し下げる。 </li></ol>チェンバースでの神経文化、プレートの3解剖<ol><li> 1％のペニシリン/ストレプトマイシンおよび10mMのHEPESと2ミリリットル解剖バッファー（HBSS、無カルシウム、ないマグネシウムを含む15ミリリットルコニカルチューブ中で解剖2 E17-E18ラット海馬（1脳）を配置します。組織をすすぎ5mlで3倍解剖できる限り解剖バッファを削除してください。たびにバッファリングし、新鮮なディ900μlのを追加ssectionバッファ。 </li><li>組織を消化するために、1×作業濃度を作るために解剖バッファーに10倍のトリプシン（2.5％）の100μlを添加する。 37℃の水浴中で円錐管を置き。消化の10分後、1 mg / mlの周りの最終濃度に10 mg / mlのDNase Iを100μlのを追加します。 </li><li>ファイアーポリッシュパスツールガラスピペットを使用して、静かにピペッティング5〜10倍によって組織を粉砕する。右粉砕後、（10％FBS、2mMのグルタマックス、2％のB27とのNeurobasal）培地めっき2mlのトリプシンをクエンチする。 </li><li>組織のいずれ破片が底に落ち着くことができるように5分間フード内のサンプルのままにしておきます。慎重に細胞をペレットに5分間200×gで清潔な無菌の15ミリリットルコニカルチューブと遠心分離機に上清の2ミリリットルを削除します。 50μlのメッキメディアでペレットを再懸濁</li><li>血球計算板を用いて細胞を数える。負荷のマイクロ流体チャンバーの1区画への15〜20μlの細胞懸濁液（〜40,000細胞）。チャンバーを配置細胞がカバーグラスに付着することを可能にする10分間インキュベーター中。 10分後、チャンバーの両方の区画を埋めるために、よりメッキメディアを追加。 </li><li>解剖の二日目に、完全に細胞体と軸索コンパートメントの両方で維持培地（2mMのグルタマックス、2％のB27と神経基本）でメッキのメディアを交換してください。海馬ニューロンからの軸索は、3日目に微小溝を渡り、一日5-7間の軸索コンパートメントに到達するために開始します。この期間中、新鮮な維持培地ごとに24〜48時間で培養培地の半分を交換してください。 </li></ol> QD-BDNFの4軸索輸送<ol><li>徹底的に2時間BDNFフリー、無血清神経基本培地で両方の区画ごとに、30分をすすぐことにより、マイクロ流体チャンバーの細胞体と軸索のコンパートメントの両方からのBDNFを枯渇、QD-BDNF軸索輸送のイメージングを生きる前に。 </li><li> BDNFの枯渇時には、QD-BDNF結合体を準備します。モノビオチン化BDの50 nMのミックスNFの神経基礎培地中の50 nMのQD655-ストレプトアビジン抱合体と二量体と60分間氷上でインキュベートする。 </li><li>軸索コンパートメントにメディアを取り出し、37℃で4時間、0.25 nMでの最終濃度で300μlのQD-BDNFを追加します。細胞体区画にQD-BDNFの拡散を最小限に抑えるためには、常に軸索区画におけるよりも細胞体コンパートメント内のメディアのより高いレベルを維持することは非常に重要である。ライブイメージングの前インキュベーション後に、結合していないQD-BDNFを洗い流す。 </li><li> 100X油対物レンズを備えた倒立顕微鏡を用いてQD-BDNF輸送のライブイメージングを実施する。範囲および一定温度（37℃）とCO 2（5％）、それに取り付けられた環境室を温める。 QD655信号を可視化するためにテキサスレッド励起/発光キューブのセットを使用してください。 </li><li>取得しCCDカメラを用いて2分間、合計1フレーム/秒の速度で中間軸索内のタイムラプス画像を取り込む。股関節のない軸索とマイクログルーブを使用してくださいtは浸潤のコントロールとして何QDので、無信号がありません。 </li><li>任意の画像解析ソフトウェアまたはNIHのImageJを使用して、BDNF輸送を分析します。 </li></ol>

<ol>
	<li>Wu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+dynein-microtubule+network+is+required+for+NGF+signaling+through+the+Rap1/MAPK+pathway.">A functional dynein-microtubule network is required for NGF signaling through the Rap1/MAPK pathway.</a> Traffic. 8, 1503-1520 (2007).</li><li>Wortzel, I., Seger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ERK+Cascade:+Distinct+Functions+within+Various+Subcellular+Organelles.">The ERK Cascade: Distinct Functions within Various Subcellular Organelles.</a> Genes &amp; cancer. 2, 195-209 (2011).</li><li>Huang, E. J., Reichardt, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trk+receptors:+roles+in+neuronal+signal+transduction.">Trk receptors: roles in neuronal signal transduction.</a> Annual review of biochemistry. 72, 609-642 (2003).</li><li>Nonomura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+pathways+and+survival+effects+of+BDNF+and+NT-3+on+cultured+cerebellar+granule+cells.">Signaling pathways and survival effects of BDNF and NT-3 on cultured cerebellar granule cells.</a> Brain research. Developmental brain research. 97, 42-50 (1996).</li><li>Weissmiller, A. M., Wu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+advances+in+using+neurotrophic+factors+to+treat+neurodegenerative+disorders.">Current advances in using neurotrophic factors to treat neurodegenerative disorders.</a> Translational neurodegeneration. 1, 14 (2012).</li><li>Zhang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+axonal+transport+of+Rab7+GTPase+results+in+dysregulated+trophic+signaling.">Defective axonal transport of Rab7 GTPase results in dysregulated trophic signaling.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 33, 7451-7462 (2013).</li><li>Taylor, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+culture+platform+for+CNS+axonal+injury,+regeneration+and+transport.">A microfluidic culture platform for CNS axonal injury, regeneration and transport.</a> Nature methods. 2, 599-605 (2005).</li><li>Cui, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+at+a+time,+live+tracking+of+NGF+axonal+transport+using+quantum+dots.">One at a time, live tracking of NGF axonal transport using quantum dots.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 13666-13671 (2007).</li><li>Xie, W., Zhang, K., Cui, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterization+and+axonal+transport+of+quantum+dot+labeled+BDNF.">Functional characterization and axonal transport of quantum dot labeled BDNF.</a> Integrative biology : quantitative biosciences from nano to macro. 4, 953-960 (2012).</li><li>Sung, K., Maloney, M. T., Yang, J., Wu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+producing+mono-biotinylated,+biologically+active+neurotrophic+factors:+an+essential+reagent+for+single+molecule+study+of+axonal+transport.">A novel method for producing mono-biotinylated, biologically active neurotrophic factors: an essential reagent for single molecule study of axonal transport.</a> Journal of neuroscience methods. 200, 121-128 (2011).</li><li>Tani, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trafficking+of+a+ligand-receptor+complex+on+the+growth+cones+as+an+essential+step+for+the+uptake+of+nerve+growth+factor+at+the+distal+end+of+the+axon:+a+single-molecule+analysis.">Trafficking of a ligand-receptor complex on the growth cones as an essential step for the uptake of nerve growth factor at the distal end of the axon: a single-molecule analysis.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 25, 2181-2191 (2005).</li><li>Bronfman, F. C., Tcherpakov, M., Jovin, T. M., Fainzilber, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand-induced+internalization+of+the+p75+neurotrophin+receptor:+a+slow+route+to+the+signaling+endosome.">Ligand-induced internalization of the p75 neurotrophin receptor: a slow route to the signaling endosome.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 23, 3209-3220 (2003).</li><li>Kruttgen, A., Heymach, J. V., Kahle, P. J., Shooter, E. M. <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+the+nerve+growth+factor+carboxyl+terminus+in+receptor+binding+and+conformational+stability.">The role of the nerve growth factor carboxyl terminus in receptor binding and conformational stability.</a> The Journal of biological chemistry. 272, 29222-29228 (1997).</li><li>Zuccato, C., Cattaneo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain-derived+neurotrophic+factor+in+neurodegenerative+diseases.">Brain-derived neurotrophic factor in neurodegenerative diseases.</a> Nature reviews. Neurology. 5, 311-322 (2009).</li><li>Gharami, K., Xie, Y., An, J. J., Tonegawa, S., Xu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain-derived+neurotrophic+factor+over-expression+in+the+forebrain+ameliorates+Huntington's+disease+phenotypes+in+mice.">Brain-derived neurotrophic factor over-expression in the forebrain ameliorates Huntington's disease phenotypes in mice.</a> Journal of neurochemistry. 105, 369-379 (2008).</li><li>Gauthier, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Huntingtin+controls+neurotrophic+support+and+survival+of+neurons+by+enhancing+BDNF+vesicular+transport+along+microtubules.">Huntingtin controls neurotrophic support and survival of neurons by enhancing BDNF vesicular transport along microtubules.</a> Cell. 118, 127-138 (2004).</li><li>Her, L. S., Goldstein, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+sensitivity+of+striatal+neurons+to+axonal+transport+defects+induced+by+mutant+huntingtin.">Enhanced sensitivity of striatal neurons to axonal transport defects induced by mutant huntingtin.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 28, 13662-13672 (2008).</li><li>Rong, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+intracellular+trafficking+of+huntingtin-associated+protein-1+is+critical+for+TrkA+protein+levels+and+neurite+outgrowth.">Regulation of intracellular trafficking of huntingtin-associated protein-1 is critical for TrkA protein levels and neurite outgrowth.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 26, 6019-6030 (2006).</li></ol>

利害関係が宣言されていない。

本研究では、生物学的に活性な生成するための新しい技術の開発を報告し、BDNFの軸索輸送を追跡するために使用することができ、BDNF（mBtBDNF）がモノビオチン化。量子ドットストレプトアビジンにタンパク質をコンジュゲート、およびマイクロ流体チャンバを使用することにより、この方法は、1つはリアルタイムでかつ空間的及び時間的解像度で、単一分子感度を有する一次ニューロンにお...

ニューロンは、高度が長く、多くの場合、非常に精緻化プロセスの確立及び神経回路の構造および機能を維持するための基本である細胞を分極されている。軸索はシナプスとの間で貨物を運ぶ重要な役割を果たしている。細胞体で合成されたタンパク質および細胞小器官は、神経機能をサポートするために、シナプス前末端に到達するために軸索を通って輸送される必要がある。これに対応して、遠位軸索で受信された信号は、形質導入し、細胞体に搬送する必要がある。これらのプロセスは、ニューロンの生存、分化および維持に必須である。いくつかのニューロンにおけるその軸索輸送が距離を通って1000倍以上細胞体の直径を行わなければならないでは、可能性は容易に小さくても赤字は著しく、神経回路機能に影響を与える可能性があることが想定される。 脳由来神経栄養因子（BDNF）、増殖因子のニューロトロフィンファミリーのメンバーは、ミリアンペアで存在する海馬、大脳皮質、および前脳基底核を含むニューヨークの脳領域、。 BDNFは、認知回路に関与するニューロンの生存、分化、および機能をサポートすることによって、認知および記憶形成において重要な役割を果たしている。 BDNFは、それがマイトジェン活性化プロテインキナーゼ/細胞外シグナル調節プロテインキナーゼ（MAPK / ERK）、ホスファチジルイノシトール3 - キナーゼを含むのTrkB媒介性シグナル伝達経路を活性化する軸索末端に、その受容体、チロシンキナーゼのTrkBに結合する（PI3K）およびホスホリパーゼC-ガンマ（PLCγ）。これらのシグナル伝達経路に関与するタンパク質を、逆行性神経細胞の細胞体に輸送され、エンドソーム1-6シグナリング BDNF / TrkBのを形成するために、エンドサイトーシス小胞構造にパッケージ化されています。 マイクロ流体培養チャンバーは、通常の条件下でだけでなく、怪我や病気7,8の設定で軸索生物学を研究するための非常に便利なプラットフォームです。軸索を分離することにより細胞体から、デバイスは、1つの軸索8-10で特異的に輸送を研究することができました。本研究で用いた450μmでマイクログルーブの障壁とPDMSベースのマイクロ流体プラットフォームは、商業的に（材料の表を参照）から購入した。 BDNF輸送を調べるために、モノビオチン化BDNF（mBtBDNF）を生成するための新規な技術を開発しました。私たちは（またAviTagをとして知られている）のビオチンアクセプターペプチドは、APを利用しました。これは、特に、大腸菌酵素ビオチンリガーゼ、BirAをすることによって、ビオチンに連結することができるリジン残基を含む15アミノ酸配列である。私たちは、PCR（ 図1A）、マウスプレproBDNF cDNAのC末端にAviTagを融合。構築物は、哺乳動物発現ベクター、をpcDNA3.1 mycを、彼のベクターにクローニングした。またをpcDNA3.1のmyc彼のベクターに細菌のBirA DNAをクローン化した。二つのプラスミドを一過両方のタンパク質を発現させるためにHEK293FT細胞に同時トランスフェクトした。 BirAをはビオのライゲーションを触媒するモノビオチン化BDNFモノマーを製造するために：1の比率、特にリジンの1でBDNFのC末端にAviTagを内に存在する。 〜18 kDaの分子量を有するビオチン化、成熟BDNFをNi-樹脂（ 図1C）を用いて培地から回収して精製した。イムノブロッティング（ 図1D）で修正されていないBDNFを検出することができないことによって判断されるようにBDNFのビオチン化は、完了した。ストレプトアビジン共役量子ドット、量子ドット655は、QD-BDNFを作るためにmBtBDNFを標識するために使用された。 mBtBDNFは、リン酸化のTrkB（ 図1E）を活性化し、組換えヒトBDNF（rhBDNF）程度に神経突起伸長（ 図1F）を刺激することができたとしてAviTagをの存在は、BDNFの活性を妨害しませんでした。免疫染色は、QD-BDNFはQD-BDNFは、生物活性（ 図1G）であることを示す、海馬軸索でのTrkBと共局在することが示さ。 BDNF輸送を研究するために、QD-BDNFは、遠位軸索区画に添加したラットE18海馬ニューロン（ 図2A）を含むマイクロ流体文化。軸索内のQD-BDNF逆行性輸送を赤色蛍光タグ（ 支援動画S1、S2）のリアルタイムライブイメージングによって捕獲された。生成されたカイモグラフを分析することによって、QD-BDNFは、約1.06ミクロン/秒（ 図3A）の移動速度で逆行性に輸送されることが観察された。 GFPまたはmCherryをタグ付きBDNFは、BDNFの軸索移動を追跡するために使用されている。主な欠点は、それらが単一分子研究のための十分に明るいではないということです。また、順行性および逆行性BDNFの動きの両方の存在は、それが困難な逆行性輸送BDNFは、ニューロトロフィン/受容体複合体にあったかどうかを評価することができる。 このビデオでは、一次ニューロンを用いたマイクロ流体チャンバ内にQD-BDNFのライブ人身売買を検査するために使用される技術を実証する。 ultrabrightnessと量子ドットのMAKの優れた光安定性エスことが可能BDNF輸送の長期的な追跡を実行する。これらの技術は、AD、HD、および他の神経変性疾患において、軸索機能の研究を強化するために利用することができる。

<table border="0" cellpadding="0" cellspacing="0" width="641">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:261px;">Name</td>
			<td style="width:205px;">Company</td>
			<td style="width:175px;">Catalog Number</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Platinum pfx DNA polymerase&#160;</td>
			<td>Invitrogen</td>
			<td>11708021</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EcoRI&#160;</td>
			<td>Fermentas</td>
			<td>FD0274</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BamHI&#160;</td>
			<td>Fermentas</td>
			<td>FD0054</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEK293FT cells</td>
			<td>Invitrogen</td>
			<td>R70007</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM-high glucose media</td>
			<td>Mediatech</td>
			<td>10-013-CV</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">d-biotin&#160;</td>
			<td>Sigma</td>
			<td>B4639</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TurboFect&#160;</td>
			<td>Fermentas</td>
			<td>R0531</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PMSF&#160;&#160;</td>
			<td>Sigma</td>
			<td>P7626</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">aprotinin</td>
			<td>Sigma</td>
			<td>A6279</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ni-NTA resins</td>
			<td>Qiagen</td>
			<td>30250</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">protease inhibitors cocktail</td>
			<td>Sigma</td>
			<td>&#160;S8820</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">silver staining kit&#160;</td>
			<td>G-Biosciences</td>
			<td>786-30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human recombinant BDNF</td>
			<td>Genentech</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microfluidic chambers</td>
			<td>Xona</td>
			<td>SND450</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">24x40 mm No. 1 glass coverslips&#160;</td>
			<td>VWR</td>
			<td>48393-060</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">poly-L-Lysine&#160;</td>
			<td>Cultrex</td>
			<td>3438-100-01</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS</td>
			<td>Gibco</td>
			<td>14185-052</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNase I</td>
			<td>Roche</td>
			<td>10104159001</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Gibco</td>
			<td>15090-046</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Neurobasal&#160;</td>
			<td>Gibco</td>
			<td>21103-049</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS&#160;</td>
			<td>Invitrogen</td>
			<td>16000-044</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GlutaMax&#160;</td>
			<td>Invitrogen</td>
			<td>35050-061</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27&#160;&#160;</td>
			<td>Gibco</td>
			<td>17504-044</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QD655-streptavidin conjugates</td>
			<td>Invitrogen</td>
			<td>&#160;Q10121MP</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-Avi tag antibody</td>
			<td>GenScript</td>
			<td>A00674</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">streptavidin-agarose beads&#160;</td>
			<td>Life Technology</td>
			<td>&#160;SA100-04</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">trichloroacetic acid</td>
			<td>Sigma</td>
			<td>T6399</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HRP-streptavidin&#160;</td>
			<td>Thermo Scientific</td>
			<td>N100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-pTrkB antibody</td>
			<td colspan="2">a generous gift from Dr M. Chao of NYU</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-TrkB antibody</td>
			<td>BD Science</td>
			<td>610101</td>
		</tr>
	</tbody>
</table>

real-time imaging of axonal transport of quantum dot-labeled bdnf in primary neurons

BDNFは、神経細胞の生存、分化、および機能のいくつかのファセットに重要な役割を果たしている。軸索における構造的および機能的障害は、ますますアルツハイマー病（AD）およびハンチントン病（HD）を含む神経変性疾患の早期の特徴とみなされる。未だ不明な軸索損傷が誘導される機構（単数または複数）である。私たちは、BDNFの軸索輸送を追跡するために使用することができ、BDNF（mBtBDNF）モノビオチン、生物学的に活性な生産する新規な技術の開発を報告した。量子ドットで標識されたBDNF（QD-BDNF）がmBtBDNFに量子ドット655を結合させることにより作製した。マイクロ流体デバイスは、ニューロン細胞体から軸索を単離するために使用した。軸索コンパートメントへのQD-BDNFを添加すると、軸索におけるBDNF輸送のライブイメージングを可能にした。私たちは、QD-BDNFは、約1.06ミクロン/秒の移動速度で、非常に少数のポーズで、逆行本質的に排他的に移動させることを実証した。このシステムは私を調査するために使用することができADまたはHDビデオで破砕軸索機能だけでなく、他の変性疾患のchanisms。

生産と生物学的に活性なモノビオチン化BDNFの精製 AviTagをシーケンス（GGGLNDIFEAQKIEWHE）と融合したBDNFの発現ベクターは、以前に公開されたプロトコル10に従って作成されました。完全長融合タンパク質の分子量は〜32kDaのが（であると予測された<a href="http://ca.expasy.org/tools/pi_tool.html" target="_blank">http://ca.expasy.org/tools/pi_tool.html</a> ）18 kDaの（ 図1A）の予測...

Watch this Scientific Journal Video about Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons at JoVE.com

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

BDNF、神経栄養因子の軸索輸送は、いくつかのニューロン集団の生存と機能に重要である。いくつかの変性疾患は、軸索の構造と機能の破壊でマークされています。私たちは、一次ニューロンを用いたマイクロ流体チャンバ内のQD-BDNFのライブ人身売買を検査するために使用される技術を実証した。

一次ニューロンにおける量子ドットで標識されたBDNFの軸索輸送のリアルタイムイメージング

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

real-time-imaging-axonal-transport-quantum-dot-labeled-bdnf-primary

Research

JoVE Journal

Neuroscience

15.0K ビュー.  University of California, San Diego.  BDNF、神経栄養因子の軸索輸送は、いくつかのニューロン集団の生存と機能に重要である。いくつかの変性疾患は、軸索の構造と機能の破壊でマークされています。私たちは、一次ニューロンを用いたマイクロ流体チャンバ内のQD-BDNFのライブ人身売買を検査するために使用される技術を実証した。 

ビデオ: Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

Mechanical Manipulation of Neurons to Control Axonal Development

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the 10-1000 &mu;dyne range1,2. Force measurements are obtained through observation of the Hookean bending of the glass needles, which are calibrated by a direct and empirical method3. Equipment requirements and procedures for fabricating, calibrating, treating, and using the needles on cells are fully described. The force regimes previously used and different cell types to which these techniques have been applied demonstrate the flexibility of the methodology and are given as examples for future investigation4-6. The technical advantages are the continuous 'visualization' of the forces produced by the manipulations and the ability to directly intervene in a variety of cellular events. These include direct stimulation and regulation of axonal growth and retraction7; as well as detachment and mechanical measurements on any type of cultured cell8.

Application and direct measurements of forces on neurons in the 2-1000 microdyne range are achieved with high precision using calibrated glass needles. This methodology can be used to control and measure several aspects of axonal development, including axonal initiation, axonal tension, velocity of axonal elongation, and force vectors.

軸索の開発を制御する神経細胞の機械的な操作

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the ...

神経科学

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 ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

アン<em&gt;エクスビボ</em&gt;レーザー誘起脊髄損傷モデルがリアルタイムで軸索変性のメカニズムを評価するために

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

管理のリアルタイム fMRI ニューロフィード バック トレーニングのプロトコル

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a connection between amyotrophic lateral sclerosis (ALS) and impairments in the nucleocytoplasmic pathway. ALS is a neurodegenerative disease affecting the motor neurons and resulting in paralysis and ultimately in death, within 2-5 years on average. Most cases of ALS are sporadic, lacking any apparent genetic linkage, but 10% are inherited in a dominant manner. Recently, hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene were identified as a genetic cause of ALS and frontotemporal dementia (FTD). Importantly, different groups have recently proposed that these mutants affect nucleocytoplasmic transport. These studies have mostly shown the final outcome and manifestations caused by HREs on nucleocytoplasmic transport, but they do not demonstrate nuclear transport dysfunction in real time. As a result, only severe nucleocytoplasmic transport deficiency can be determined, mostly due to high overexpression or exogenous protein insertion. 
This protocol describes a new and very sensitive assay to evaluate and quantify nucleocytoplasmic transport dysfunction in real time. The rate of import of a NLS-NES-GFP protein (shuttle-GFP) can be quantified in real time using fluorescent microscopy. This is performed by using an exportin inhibitor, thus allowing the shuttle GFP only to enter the nucleus. To validate the assay, the C9orf72 HRE translated dipeptide repeats, poly(GR) and poly(PR), which have been previously shown to disrupt nucleocytoplasmic transport, were used. Using the described assay, a 50% decrease in the nuclear import rate was observed compared to the control. Using this system, minute changes in nucleocytoplasmic transport can be examined and the ability of different factors to rescue (even partially) a nucleocytoplasmic transport defect can be determined.

This protocol describes a method to sensitively measure the nucleocytoplasmic transport rate within motor neuron-like NSC-34 cells by quantifying the real-time change in the nuclear import of a NLS-NES-GFP protein.

運動ニューロン様NSC-34細胞内のリアルタイムでの核細胞質輸送を測定するアッセイ

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

胎児運動ニューロンの軸索突起パターンの視覚化<em&gt;ショウジョウバエ</em

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal growth, development, and survival. We describe a detailed method for the use of microfluidic chambers (MFCs) for tracking axonal transport of fluorescently labeled organelles in MN axons. This method is rapid, relatively inexpensive, and allows for the monitoring of intracellular cues in space and time. We describe a step by step protocol for: 1) Fabrication of polydimethylsiloxane (PDMS) MFCs; 2) Plating of ventral spinal cord explants and MN dissociated culture in MFCs; 3) Labeling of mitochondria and acidic compartments followed by live confocal imagining; 4) Manual and semiautomated axonal transport analysis. Lastly, we demonstrate a difference in the transport of mitochondria and acidic compartments of HB9::GFP ventral spinal cord explant axons as a proof of the system validity. Altogether, this protocol provides an efficient tool for studying the axonal transport of various axonal components, as well as a simplified manual for MFC usage to help discover spatial experimental possibilities.

Axonal transport is a crucial mechanism for motor neuron health. In this protocol we provide a detailed method for tracking the axonal transport of acidic compartments and mitochondria in motor neuron axons using microfluidic chambers.

マイクロ流体室システムを用いた運動ニューロン培養におけるオルガネラの軸索輸送

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal ...

Real-Time fMRI Brain Mapping in Animals

Dynamic fMRI responses vary largely according to the physiological conditions of animals either under anesthesia or in awake states. We developed a real-time fMRI platform to guide experimenters to monitor fMRI responses instantaneously during acquisition, which can be used to modify the physiology of animals to achieve desired hemodynamic responses in animal brains. The real-time fMRI set-up is based on a 14.1T preclinical MRI system, enabling the real-time mapping of dynamic fMRI responses in the primary forepaw somatosensory cortex (FP-S1) of anesthetized rats. Instead of a retrospective analysis to investigate confounding sources leading to the variability of fMRI signals, the real-time fMRI platform provides a more effective scheme to identify dynamic fMRI responses using customized macro-functions and a common neuroimage analysis software in the MRI system. Also, it provides immediate troubleshooting feasibility and a real-time biofeedback stimulation paradigm for brain functional studies in animals.

Animal brain functional mapping can benefit from the real-time functional magnetic resonance imaging (fMRI) experimental set-up. Using the latest software implemented in the animal MRI system, we established a real-time monitoring platform for small animal fMRI.

動物におけるリアルタイムfMRI脳マッピング

Dynamic fMRI responses vary largely according to the physiological conditions of animals either under anesthesia or in awake states. We developed a ...

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal transport have devastating consequences for individual neurons and their networks, and contribute to a plethora of neurological disorders. As many of these conditions involve both cell autonomous and non-autonomous mechanisms, and often display a spectrum of pathology across neuronal subtypes, methods to accurately identify and analyze neuronal subsets are imperative.
This paper details protocols to assess in vivo axonal transport of signaling endosomes and mitochondria in sciatic nerves of anesthetized mice. Stepwise instructions are provided to 1) distinguish motor from sensory neurons in vivo, in situ, and ex vivo by using mice that selectively express fluorescent proteins within cholinergic motor neurons; and 2) separately or concurrently assess in vivo axonal transport of signaling endosomes and mitochondria. These complementary intravital approaches facilitate the simultaneous imaging of different cargoes in distinct peripheral nerve axons to quantitatively monitor axonal transport in health and disease.

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals.

軸索輸送の In Vivo イメージングのためのツールキットの拡張

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal ...

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

インビボ 無傷の背根神経節における一次感覚ニューロンのネットワークにおけるニューロンアンサンブルのカルシウムイメージング

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...