This work is supported by startup funds from The Jackson Laboratory.

1。神経培養エアコン用マウスグリア文化の準備<ol><li> T75フラスコは、コラーゲン溶液（蒸留H 2 O中Purecolの1:3希釈）でコーティングされています。フラスコを立設し、組織培養フード中で一晩乾燥させています。 </li><li>解離バッファ（ACSF：119 mMのNaCl、カルシウムなし5mMのKCl、1mMのMgCl 2、30mMのデキストロース、25mMのHEPES、pH7.4で）やグリア培地（DMEM、10％FBS、10単位/ mlペニシリン、10 μg/ mlストレプトマイシン、およびグルタマックスは）℃使用直前まで準備し、4℃で保存されています。 </li><li>グリア文化の日に、グリアメディアがマウスの脳の解剖の前に37℃の水浴中で少なくとも30分間加温する。胚または新生仔マウスは、急速な断頭により安楽死させる。脳皮質を解剖顕微鏡下で解剖されています。 </li><li>解剖した脳組織を37℃パパイン溶液（100mlパパインおよび2 ml郭清を緩衝液中の20μlの1パーセントのDNase I）°CのFOで消化されるR 20分。 </li><li>パパイン消化後、脳組織を10mlの予め温めグリアメディアでリンスしています。新鮮グリアメディア（2 ml）を消化した脳組織に追加され、組織はファイアーポリッシュガラスパスツールピペットを用いて摩砕しています。 </li><li>新鮮グリアメディア（8ml）を解離組織に追加されます。 70μmのセルストレーナーは、解離組織懸濁液をフィルタリングするために使用されます。次いで、細胞を、T75フラスコ（通常2-3胚ができるシード1 T75フラスコ）に接種し、37℃のインキュベーターに入れられる。 </li><li>翌日、T75フラスコから培地を吸引しています。フラスコを10mlのPBSで洗浄する。新鮮グリアメディア（10-15 ml）を各フラスコに添加されています。 </li><li> 10から12日以内に、T75フラスコのグリアがコンフルエントになる必要があります。グリア細胞は、PBSで洗浄し、0.05％トリプシン5mlをそれぞれT75フラスコに追加されます。 </li><li> 5分間37℃トリプシンのインキュベーション後、グリア細胞はそれぞれT75フラスコから解離およびグリア細胞を2つに分割され異なるコラーゲンコートT75フラスコ。 </li><li>培養インサート（ミリポアPICM03050）を6 - ウェルプレート、コラーゲンでコーティングして、一晩空気乾燥にそれらを置くことによって調製される。グリアメディア（各2ml）を各培養インサートの下に配置されています。 </li><li>コンフルエントT75フラスコからグリアをトリプシン処理し、（6ウェルプレートあたり1 T75フラスコ、各2ml挿入）培養インサート上に播種されています。これらのグリア細胞培養インサートは、条件ニューロン培養するのに使用されます。 </li></ol> 2。ニューロンの文化<ol><li>神経細胞培養用のカバーガラスは、70％で少なくとも一晩HNO 3を浸漬することによって清掃されています。カバーガラスは、その後ddH 2 Oを少なくとも3回洗浄し、（&gt; 200℃）で一晩Isotempオーブンで乾燥させます。 </li><li>洗浄カバーガラスを6ウェルプレート1ウェルあたり1カバースリップに配置されます。カバースリップを37℃のインキュベーターで一晩、ポリ-L-リジン溶液（2ml、100mMホウ酸、pH = 8.5で0.1％）でコーティングされています。 </li><li>ザポリ-L-リジン溶液は、カバーガラスとカバーガラスから取り外されオートクレーブ滅菌蒸留H 2 Oで3回洗浄するカバーガラスは、その後37でインキュベートされる（Neurobasalが5％熱不活化ウマ血清、10単位/ mlペニシリン、10μg/ mlのストレプトマイシン、グルタマックスを補ったプレート培地で一晩℃のインキュベーター、メッキ用培地に添加されている2パーセントB27サプリメント当日）が使用されます。 </li><li>翌日、プレート培地を除去し、B-27に、新鮮なプレート培地2mlのガラスカバースリップを持って各ウェルに添加する。 </li><li>妊娠マウス（E15-18）は、CO 2で安楽死され、マウス胎児は断頭により安楽死させる。海馬や線条体は、それぞれ脳組織​​から解剖し、海馬ニューロンまたは線条体中型有棘神経細胞の培養のために、氷冷解剖緩衝液を用いて15 mlコニカルチューブに入れられます。 </li><li>ステップ1.4）、1.5）で説明したように解剖した脳組織は、パパインによる解離する。 </li><li> F解離ollowing、各ニューロンの懸濁液中の細胞数は血球計算板でカウントされます。神経細胞は、その後、6ウェルプレートのウェル当たり0.4×10 6細胞の密度で各カバースリップ上に播種し、37℃のインキュベーターで一晩培養する（ニューロンがDIV 0アール）。 </li><li>コンディショニングのグリア培養インサートニューロン培養を準備するには、グリアメディアは、グリア培養インサートから削除され、新鮮なメッキ媒体（インサート内の2ml及びインサートの下に2ミリリットル）を添加する。 </li><li>翌日（DIV 1）は、ニューロンとグリアカバースリップを培養インサート、各ウェル中の1カバースリップの下に配置されます。 </li><li>彼らは、トランスフェクションおよびイメージング実験の準備が整うまで、ニューロンは新鮮予め温めておいた餌メディア（B-27）3〜4日ごとに半分の容積が供給される。 </li></ol> 3。トランスフェクション<ol><li>神経細胞のトランスフェクションは、リポフェクタミン2000を使用して実行されます。各ニューロンの培養用（カバースリップあたり一つの文化）、2μlのリポフェクタミン2000と1μgのDNAを使用しています。ノーサプリメントで新鮮Neurobasal培地はリポフェクタミンとDNAを希釈するために使用されます。 </li><li> DNAとリポフェクタミンを混合した後、室温で20分間インキュベートする。 </li><li>各グリア培養インサート下からのメディアの1ミリリットルを保存し、各インサートの内側から1ミリリットル、円錐管にメディアを転送する。 37℃で保存した培地でインキュベート℃にメディア送りの同じボリューム+ B27を追加この培地は、トランスフェクション後の神経細胞培養のために使用されます。 </li><li> 20分リポフェクタミン/ DNAのインキュベーションの後、グリア培養インサートを6ウェルプレートから瞬間的に削除されます。 200μlのリポフェクタミン/ DNA混合物をニューロンと各カバースリップに追加されます。グリア細胞培養インサートはその後ニューロン上に、各ウェルに返されます。培養インサートの膜の下の気泡の発生は避けるべきである。ニューロンは2〜4時間、37℃でリポフェクタミン/ DNA混合物と共にインキュベートする。 </li><li>目の後E 2〜4時間のインキュベーション期間、グリア挿入が再び削除されます。リポフェクタミン/ DNA混合物と培養培地を各ウェルから除去されます。ステップ3.3）に保存されたメディアは、ニューロン（ウェル当たり2ミリリットル）に追加されます。グリア細胞培養インサート）を各ウェルに返され、各インサートからグリアメディアが削除され、ステップ3.3からのメディアの2ミリリットル各インサートに追加されます。 </li><li>ニューロンは、全反射イメージングは​​、これらのニューロン上で実行される前に、トランスフェクトされたcDNAの発現を可能にするために、追加の24から72時間培養する。 </li></ol> 4。 TIRFイメージング<ol><li>人工脳脊髄液（ACSF：119 mMのNaCl、2.5mMのKCl、30mMのグルコース、2mMのCaCl 2、1mMのMgCl 2、25mMのHEPES緩衝液、pH = 7.4）をライブイメージング実験のために使用されます。当社の全反射イメージングシステムは、検出器のための100 mWのシアン励起用レーザー、電子増倍電荷結合素子（EMCCD）カメラでセットアップカスタムです。 </li><li>イメージング実験に先立ち、ACSFは37＆dで警告される例えば、少なくとも30分間℃の水浴。ライブイメージング用加熱インサートは、顕微鏡ステージ上に配置され、加熱挿入、レーザー、カメラはイメージング実験の開始前に少なくとも30分間温めています。 </li><li>トランスフェクトされたニューロンとカバースリップをライブイメージング室に組み立てられる。チャンバーを組み立てた後あらかじめ温めておいたACSFはチャンバ内に配置され、このステップはできるだけ迅速に完了する必要があります。 </li><li>チャンバーは顕微鏡ステージ上で加熱インサート上に置かれると、トランスフェクトされたニューロンは、エピ蛍光イメージングモードで視覚的に識別されます。 </li><li>健康なトランスフェクトされたニューロンは（ 図1及び図2参照）を同定した後、原形質膜上のpHluorin-受容体は非常に高い蛍光レベル時を持っているように、我々は一般的に、細胞膜上の既存pHluorin蛍光を排除するために、光漂白剤の1分を実行TIRF画像が最初に開始され、これは、個々のプラグインの可視化を妨げるertionイベント。 </li><li>光退色後、EMCCDカメラで電子増倍管のゲイン設定が最大に設定されており、記録は、10 Hzで1分間実行されます。 </li><li>各記録は600の画像が含まれています。個々の挿入イベントがImageJを用いた記録バックを再生することにより識別されます。個々の挿入イベントは手動でImageJを用いて分析する。 </li></ol> 5。代表的な結果一貫性のあるニューロンの培養は成功したライブイメージング実験の鍵となります。 図1に 18をdivに、DIV 11から我々のプロトコルを使用して培養したマウス海馬神経細胞を示しています。これは、神経細胞が広範囲プロセスを開発していることと、樹状突起は樹状突起棘、これらのニューロンは、文化の中で健康であることの表示で密に覆われている画像から明らかである。私たちの文化プロトコルはまた、脳内の神経細胞の他のタイプを使用することができる。例えば、我々は最近、文化に、このプロトコルを使用培養マウス線条体中型有棘ニューロン9にドーパミンD2受容体（DRD2）の挿入を検討する研究では線条体中型有棘ニューロン図2は、DIV 8時に我々のプロトコルを使用して培養したマウス線条体中型有棘ニューロンを示しています。我々はまた、正常ニューロンのこのタイプのsuperecliptic pHluorinタグ付きDRD2sの全反射イメージングを行った。 図3はpH DRD2このニューロンにおける挿入の中型有棘ニューロンと可視化でpHluorinタグ付きDRD2（PH-DRD2）の発現を示す。 <img alt="図1" src="/files/ftp_upload/4450/4450fig1.jpg" /> インタフェース培養法を用いて図1。マウス海馬培養。 DIV：in vitroでの日、文化の日は、DIV 0とみなされます。画像から、それは明らかであるDIVとDIV 11〜15の間の文化のこのタイプの成熟した海馬ニューロン。 DIVの11と13で、ニューロンの樹状突起はfilipodiaと未熟な棘で覆われている。 DIVの15時と18は、ニューロンの樹状突起は、大きな棘は、より成熟した神経細胞の指標で覆われている。左パネル：異なる年齢のマウス海馬ニューロンの低倍率像。右のパネル：高倍率画像（左パネルのニューロンの樹状突起上でズーム）は、異なる年齢での樹状突起と神経細胞樹状突起の。 <img alt="図2" src="/files/ftp_upload/4450/4450fig2.jpg" /> インターフェイス培養法を用いて図2マウス線条体中型有棘ニューロン文化。画像内のニューロンは、DIV 8時です。 <img alt="図3" src="/files/ftp_upload/4450/4450fig3.jpg" /> 図3：スーパー黄道pHluorinタグ付きドーパミンD2受容体（PH-DRD2）を用いてトランスフェクトマウス線条体中型有棘ニューロン。左の画像は、タイムラプス記録の最大値投影（600画像、画像あたり100ミリ秒）です。白い矢印は、pH-Dの個々の小胞の挿入を示すRD2。この画像は、xyディメンションの挿入情報を保持しますが、時間情報が失われます。右の画像はXYTスタックがy軸の周りに90度回転させ、各x軸線上の最大ピクセルは、単一のy軸の画素に投影された、YT最大値投影を示しています。この画像はYT軸に沿って挿入情報を保持するが、x軸の情報が失われます。

<ol>
	<li>Thomas, G. M., Hayashi, T., Chiu, S. L., Chen, C. M., Huganir, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitoylation+by+DHHC5/8+targets+GRIP1+to+dendritic+endosomes+to+regulate+AMPA-R+trafficking.">Palmitoylation by DHHC5/8 targets GRIP1 to dendritic endosomes to regulate AMPA-R trafficking.</a> Neuron. 73, 482-496 (2012).</li><li>Anggono, V., Huganir, R. L. <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+AMPA+receptor+trafficking+and+synaptic.">Regulation of AMPA receptor trafficking and synaptic.</a> Curr. Opin. Neurobiol. , (2012).</li><li>Makuch, L., Volk, L., Anggono, V., Johnson, R. C., Yu, Y., Duning, K., Kremerskothen, J., Xia, J., Takamiya, K., Huganir, R. L. <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+AMPA+receptor+function+by+the+human+memory-associated+gene+KIBRA.">Regulation of AMPA receptor function by the human memory-associated gene KIBRA.</a> Neuron. 71, 1022-1029 (2011).</li><li>Thomas, G. M., Lin, D. T., Nuriya, M., Huganir, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+bi-directional+regulation+of+AMPA+receptor+phosphorylation+and+trafficking+by+JNK.">Rapid and bi-directional regulation of AMPA receptor phosphorylation and trafficking by JNK.</a> EMBO J. 27, 361-372 (2008).</li><li>Lin, D. T., Huganir, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PICK1+and+phosphorylation+of+the+glutamate+receptor+2+(GluR2)+AMPA+receptor+subunit+regulates+GluR2+recycling+after+NMDA+receptor-induced+internalization.">PICK1 and phosphorylation of the glutamate receptor 2 (GluR2) AMPA receptor subunit regulates GluR2 recycling after NMDA receptor-induced internalization.</a> J. Neurosci. 27, 13903-13908 (2007).</li><li>Hayashi, T., Rumbaugh, G., Huganir, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+AMPA+receptor+subunit+trafficking+by+palmitoylation+of+two+distinct+sites.">Differential regulation of AMPA receptor subunit trafficking by palmitoylation of two distinct sites.</a> Neuron. 47, 709-723 (2005).</li><li>Lin, D. T., Makino, Y., Sharma, K., Hayashi, T., Neve, R., Takamiya, K., Huganir, R. L. <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+AMPA+receptor+extrasynaptic+insertion+by+4.1N,+phosphorylation.">Regulation of AMPA receptor extrasynaptic insertion by 4.1N, phosphorylation.</a> 12, 879-887 (2009).</li><li>Araki, Y., Lin, D. T., Huganir, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+membrane+insertion+of+the+AMPA+receptor+GluA2+subunit+is+regulated+by+NSF+binding+and+Q/R+editing+of+the+ion+pore.">Plasma membrane insertion of the AMPA receptor GluA2 subunit is regulated by NSF binding and Q/R editing of the ion pore.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 11080-11085 (2010).</li><li>Li, Y., Roy, B. D., Wang, W., Zhang, L., Zhang, L. F., Sampson, S. B., Yang, Y. P., Lin, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Two+Functionally+Distinct+Endosomal+Recycling+Pathways+for+Dopamine+D2+Receptor.">Identification of Two Functionally Distinct Endosomal Recycling Pathways for Dopamine D2 Receptor.</a> Journal of Neuroscience. , (2012).</li><li>Yu, Y. J., Dhavan, R., Chevalier, M. W., Yudowski, G. A., von Zastrow, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+delivery+of+internalized+signaling+receptors+to+the+somatodendritic+surface+by+sequence-specific+local+insertion.">Rapid delivery of internalized signaling receptors to the somatodendritic surface by sequence-specific local insertion.</a> J. Neurosci. 30, 11703-11714 (2010).</li><li>Yudowski, G. A., Puthenveedu, M. A., von Zastrow, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+modes+of+regulated+receptor+insertion+to+the+somatodendritic+plasma+membrane.">Distinct modes of regulated receptor insertion to the somatodendritic plasma membrane.</a> Nat. Neurosci. 9, 622-627 (2006).</li><li>Yudowski, G. A., Puthenveedu, M. A., Leonoudakis, D., Panicker, S., Thorn, K. S., Beattie, E. C., von Zastrow, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+of+discrete+exocytic+events+mediating+surface+delivery+of+AMPA+receptors.">Real-time imaging of discrete exocytic events mediating surface delivery of AMPA receptors.</a> J. Neurosci. 27, 11112-11121 (2007).</li><li>Ashby, M. C., De La Rue, S. A., Ralph, G. S., Uney, J., Collingridge, G. L., Henley, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+AMPA+receptors+(AMPARs)+from+synapses+is+preceded+by+transient+endocytosis+of+extrasynaptic+AMPARs.">Removal of AMPA receptors (AMPARs) from synapses is preceded by transient endocytosis of extrasynaptic AMPARs.</a> J. Neurosci. 24 (AMPARs), 5172-5176 (2004).</li><li>Kopec, C. D., Real, E., Kessels, H. W., Malinow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GluR1+links+structural+and+functional+plasticity+at+excitatory+synapses.">GluR1 links structural and functional plasticity at excitatory synapses.</a> J. Neurosci. 27, 13706-13718 (2007).</li><li>Makino, H., Malinow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPA+receptor+incorporation+into+synapses+during+LTP:+the+role+of+lateral+movement+and+exocytosis.">AMPA receptor incorporation into synapses during LTP: the role of lateral movement and exocytosis.</a> Neuron. 64, 381-390 (2009).</li><li>Makino, H., Malinow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentalized+versus+global+synaptic+plasticity+on+dendrites+controlled+by+experience.">Compartmentalized versus global synaptic plasticity on dendrites controlled by experience.</a> Neuron. 72, 1001-1011 (2011).</li><li>Wang, Z., Edwards, J. G., Riley, N., Provance, D. W., Karcher, R., Li, X. D., Davison, I. G., Ikebe, M., Mercer, J. A., Kauer, J. A., Ehlers, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+Vb+mobilizes+recycling+endosomes+and+AMPA+receptors+for+postsynaptic+plasticity.">Myosin Vb mobilizes recycling endosomes and AMPA receptors for postsynaptic plasticity.</a> Cell. 135, 535-548 (2008).</li><li>Miesenbock, G., De Angelis, D. A., Rothman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+secretion+and+synaptic+transmission+with+pH-sensitive+green+fluorescent+proteins.">Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.</a> Nature. 394, 192-195 (1998).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nat. Protoc. 1, 2406-2415 (2006).</li><li>Sekine-Aizawa, Y., Huganir, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+receptor+trafficking+by+using+alpha-bungarotoxin-binding-site-tagged+receptors.">Imaging of receptor trafficking by using alpha-bungarotoxin-binding-site-tagged receptors.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 17114-17119 (2004).</li></ol>

特別な利害関係は宣言されません。

For unknown reasons, mouse neurons are always more difficult to culture than rat neurons. In our experience, a mixed culture of neurons and glia works well for primary cultured mouse neurons. However, such a mixed culture is not suitable for TIRF imaging experiments, as in this type of culture neurons and their processes tend to grow on top of glia cells, situating the neuronal somata and dendritic processes beyond the reach of TIRF microscopy. Therefore, a lower-density neuronal culture with few glia on the covers...

<table border="1">
	<tbody>
 <tr>
 <td>Name of Reagent</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>purified bovine collagen solution (Purecol)</td>
 <td>Advanced Biomatrix</td>
 <td>5005-B</td>
 <td></td>
 </tr>
 <tr>
 <td>Hank's Balanced Salt Solution (HBSS)</td>
 <td>GIBCO</td>
 <td>14185-045</td>
 <td></td>
 </tr>
 <tr>
 <td>penicillin-streptomycin (Pen Strep)</td>
 <td>GIBCO</td>
 <td>15140-122</td>
 <td></td>
 </tr>
 <tr>
 <td>sodium pyruvate</td>
 <td>GIBCO</td>
 <td>11360-070</td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM High Glucose</td>
 <td>GIBCO</td>
 <td>10313-021</td>
 <td></td>
 </tr>
 <tr>
 <td>fetal bovine serum (FBS)</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>GlutaMAX</td>
 <td>GIBCO</td>
 <td>35050-061</td>
 <td></td>
 </tr>
 <tr>
 <td>papain</td>
 <td>Worthington Biochemical Corp.</td>
 <td>LS003126</td>
 <td></td>
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 <tr>
 <td>Deoxyribonuclease I from bovine pancreas (DNase I)</td>
 <td>SIGMA</td>
 <td>DN25-10MG</td>
 <td></td>
 </tr>
 <tr>
 <td>Dulbecco's Phosphate Buffered Saline (DPBS)</td>
 <td>GIBCO</td>
 <td>14190-144</td>
 <td></td>
 </tr>
 <tr>
 <td>0.05% trypsin</td>
 <td>GIBCO</td>
 <td>25300-054</td>
 <td></td>
 </tr>
 <tr>
 <td>poly-l-lysine hydrobromide</td>
 <td>SIGMA</td>
 <td>P2636-1G</td>
 <td></td>
 </tr>
 <tr>
 <td>boric acid</td>
 <td>Fisher-Scientific</td>
 <td>BP 168-500</td>
 <td></td>
 </tr>
 <tr>
 <td>Neurobasal Medium</td>
 <td>GIBCO</td>
 <td>21103-049</td>
 <td></td>
 </tr>
 <tr>
 <td>B-27 Serum-Free Supplement</td>
 <td>GIBCO</td>
 <td>17504-044</td>
 <td></td>
 </tr>
 <tr>
 <td>heat inactive horse serum</td>
 <td>GIBCO</td>
 <td>26050-070</td>
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 <td>Lipofectamine 2000</td>
 <td>Invitrogen</td>
 <td>11668 019</td>
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 <tr>
 <td>HEPES</td>
 <td>Fisher-Scientific</td>
 <td>BP310-500</td>
 <td></td>
 </tr>
 <tr>
 <td>Culture Insert</td>
 <td>Millipore</td>
 <td>PICM03050</td>
 <td></td>
 </tr>
	 </tbody>
 </table>

imaging phluorin-tagged receptor insertion to the plasma membrane in primary cultured mouse neurons

原形質膜（PM）と細胞内コンパートメント間の受容体の輸送を支配するメカニズムの理解は、優れた空間的および時間的な解像度で実験的なアプローチを必要とします。さらに、そのようなアプローチは、細胞内区画のものからPMに局在受容体を区別する能力を持っている必要があります。各小胞が受容体の数が少ないを運ぶので、最も重要なのは、単胞の受容体を検出することは、卓越した検出感度を必要とします。と細胞の化学固定を必要とするため、十分な時間分解能1-6を欠いている免疫標識表面受容体の蛍光顕微鏡検査、、受容体の輸送を調べるための標準的なアプローチは両方必要な空間および時間解像度を欠いている生化学的検出、続いて表面ビオチン化が含まれています。これらの制限を克服するために、私たちと他の人が開発され、新しいストラを採用してきた7月17日優れた空間的および時間的な解像度でPMに受容体の動的挿入の可視化を可能にtegy。アプローチでは、受容体のN末端 ​​細胞外ドメインにpH感受性GFP、superecliptic pHluorin 18のタグが含まれています。 Superecliptic pHluorinは、酸性pHで中性pH及び非蛍光液（pH &lt;6.0）での蛍光性のユニークな特性を持っています。したがって、細胞内輸送小胞またはエンドソーム区画の酸性ルーメン内タグ付けされた受容体は、非蛍光性であり、PMの外表面に、細胞外pH中性の環境にさらされた場合にのみ、それらは容易に可視になります。当社の戦略は、結果的に私たちは細胞内輸送小胞の中でそれらからPM表面受容体を区別することができます。十分な空間的および時間的な解像度だけでなく、受容体の動的輸送を研究するために必要な感度を達成するために、我々は採用反映内部合計私たちは光イメージング（〜170 nm）の最適な空間分解能を達成することができイオン蛍光顕微鏡（TIRFM）は、ビデオレート顕微鏡（30フレーム/秒）、感度の時間分解能は、単一GFPの蛍光を検出する分子。 TIRFM下イメージングpHluorinタグ付きの受容体によって、我々は直接培養神経細胞におけるPMに個々の受容体挿入イベントを可視化することができました。このイメージングアプローチは、潜在的にsuperecliptic pHluorinで標識することができる細胞外ドメインを持つ任意の膜タンパク質に適用することができ、異なる膜タンパク質（受容体、イオンチャネル、トランスポーター、など）の挿入をするために、管理キー詳細なメカニズムの解剖を許可します午後。

Watch this Scientific Journal Video about Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons at JoVE.com

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

イメージング初代培養マウス神経細胞における形質膜に受容体の挿入をpHluorinタグ付き

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

imaging-phluorin-tagged-receptor-insertion-to-plasma-membrane-primary

Research

JoVE Journal

Neuroscience

13.1K ビュー.  The Jackson Laboratory. By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

ビデオ: Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

複数のマウスの神経解剖学的磁気共鳴イメージング

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

神経科学

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

FM染料を用いて培養小脳顆粒神経細胞のシナプス小胞プールの補充の定量分析

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Inducing Dendritic Growth in Cultured Sympathetic Neurons

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs 4-6. Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models 7, and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders 8-10 and neurodegenerative diseases 11-13. Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases.
Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture 14,15. However, the addition of either bone morphogenetic protein-7 (BMP-7) 16,17 or Matrigel 18 to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons 19 that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies 17,18,20,21. Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth 17,18.
Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

培養交感神経ニューロンの樹状成長を誘導する

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

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 ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

脳スライスビオチン：<em&gt;生体外</emアダルトニューロンにおいて地域固有の細胞膜のタンパク質輸送を測定するために&gt;の取り組み

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

マウスドーパミン作動性ニューロンの初代培養

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

軸索分岐と神経形態形成中に形質膜配信の役割を定義するために組み合わせる方法論を活用

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

神経細胞死とマウス プライマリ小脳顆粒ニューロンの変性をモデリング

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

GLUT4 タンパク質デコンボリューション顕微鏡を用いたマウス一次視床下部ニューロンにおける人身売買のライブ映像

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

胚性マウス小脳から単離されたニューロンの一次培養

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...