私たちは、両教授に感謝したいと思います。広範なサポートのためのHuibert Mansvelderとバートザクマン、実りある議論と技術支援のために、ニューロンのトレース、およびブレンダンLodderを提供するための博士マルセルOberlaender。データは、寛大にR·ブルーノ（コロンビア大学、ニューヨーク州、米国）が提供する、LabVIEW用ntrode VIを使用して取得した。この研究は、マックスプランク協会と計算論的神経科学、チュービンゲンのためのバーンスタインセンターによってサポートされていました（教育研究のドイツ連邦環境省（BMBFの資金; FKZ：01GQ1002））（RTN）、Neurogenomicsと認知研究センター（CNCR） 、神経科学キャンパスアムステルダム（NCA）、CPJdK（NWO-ALW番号822.02.013とENC-ネットワーク＃P3-C3）とVU大学アムステルダムへの資金。

1。動物の作製全ての実験手順は、VU大学アムステルダム、オランダで現地の倫理委員会のオランダ法に基づいて、評価後に実施されています。 <ol><li>腹腔内注射（0.9％のNaCl、1.6〜1.7グラム/ kgの20％）ウレタンイソフルラン（酸素2-3％）で、続いてWistar系ラット（P25-P45、♂/♀）を麻酔。ピンチ撤退、まぶた反射神経、および鼻毛の動きを監視することによって、麻酔の深さを評価する。 </li><li>場合には、ウレタンの追加用量（初回用量の10％）を投与する動物は、十分な麻酔の深さに達していないか、鼻毛単収縮は、実験の過程の間に観察されるときはいつでも。 </li><li>加熱パッドを装備した定位フレームに麻酔をかけた動物を置き、直腸温度プローブを挿入し、加熱パッドにより37.5±0.5℃で、動物の体温を維持。 </li><lI&gt;定位フレームに動物の頭を置き、目に見える肌をレンダリングするためにハサミを使って頭の上に毛をトリミング。 </li><li>作業現場での局所麻酔用皮下（0.9％NaCl中）を100μlの1％リドカインを注入し、3〜5分待ちます。尾側から頭側方向に切断することにより、頭蓋骨の表面を覆う皮膚を除去し、残りの組織を清掃してください。 </li><li> 0.9％NaClおよび吸収性綿棒で広範囲頭蓋骨を清掃します。 </li><li> （：2.5ミリメートルの後部とブレグマ横5.5ミリメートル、一次体性感覚野（S1）の場合）左半球上のターゲット領域の座標を決定。外科皮膚マーカーペンで頭蓋骨上の位置をマークします。 </li><li>骨が透明になり、血管がはっきりと見えるようになるまで、関心領域から歯科用ドリルでそっと骨を削り取る。 </li><li>硬膜への損傷を回避し、外科用メス（＃11）を用いて薄くなった頭蓋骨切断窓、0.5ミリメートル×0.5ミリメートルの開頭術を行うや血管。オプション：可視性を向上させるために外科皮膚マーカーペンを使用して開頭のエッジをマークします。 </li><li>開頭術を囲む浴（ すなわち録音室）を構築するために、乾燥頭蓋骨、その後歯科用セメントに瞬間接着剤の薄層を適用します。 </li><li>ウィスカ包から正確に5ミリメートルに反対側のウィスカートリム。オプション：黒マスカラでトリミングウィスカーを強調表示します。 </li></ol> 2。 Juxtasomal記録とビオシチンラベリング<ol><li> 3-5MΩ抵抗を有する電極を得るために、〜1μmの内側先端径とホウケイ酸ガラスのパッチピペットを作る。注：juxtasomal記録のための理想的なピペット形態は徐々に細いテーパ、低円錐角、および約1μmの先端（ 図1）。 </li><li> （軟膜表面に対して）、記録深さに応じて、記録電極のテーパー寸法が調整されるべきである。非常に重要なことtがある彼は、直径が記録領域への機械的な損傷やストレスを避けるために、脳内のエントリーの時点で75μm未満である必要があり先細り。比較的深い皮質層からの記録は最大外で再び、1,500-1,800程度の長いテーパーを必要とするのに対し、これは表面的な皮質記録のために、最大限に75μmの外径が300〜500程度のテーパーが（ 図1A）が必要であることを示しています（電極先端から1800程度で）75μmの直径。 </li><li> 2％ビオシチンを補足し、正常なラットリンガー（NRR）でパッチピペットをロード（ソリューションおよび試薬については表1を参照）、マイクロマニピュレータに固定されたヘッドステージにパッチピペットをマウントします。 </li><li>特にラットS1のD2の列をターゲットに矢状面に対して34°の電極ホルダーの角度を設定します。 </li><li>ブリッジモード（電流クランプ）内のアンプにヘッドステージを接続します。 </li><li>パッチを配置します開頭に近接してピペット。 0.9％NaClで風呂に記入し、正の電流注入（1 nAの、オン/オフ200ミリ秒）で正方形のパルスを印加することによって評価MΩ、3〜5の間であるべきである電極抵抗を決定する。 </li><li>矩形パルス（1 nAの、オン/オフ200ミリ秒）などの正の電流を印加しながら、100〜150ミリバールの過圧を確立し、1ミクロンのステップでパッチピペットを進める。硬膜との接触を確立すると堅牢な抵抗変化を監視します。この時点で、正確な深さ測定を可能にするために &#39;ゼロ&#39;にマイクロマニピュレータの座標を設定する。 </li><li>パッチピペットは、電極抵抗の急激な低下によって示さ硬膜を貫通するまで、1ミクロン、ステップ·モードで進める。ピペットの保持圧力を取り除く。 </li><li> 1以下の手順で進めながら、単一のユニットを検索します。オン/オフのパルスに200ミリ秒を適用することにより、継続的に電極抵抗を監視します。電極抵抗のtypicの増加同盟国は、単一のニューロンの近接性を示している。正の活動電位（AP）〜2 mVの波形が（ 図2Aおよび図2B）が記録されるまで、電極を進める。 </li><li>オプション：ニューロンの自発的なスパイクパターンを記録し、ウィスカ誘発尾側の圧電素子18を使用して3.3°の角度で200ミリ、個々のウィスカーを偏向、たとえばによるスパイクを決める。 </li><li>抵抗値が25〜35MΩで、スパイクはjuxtasomal充填の最適な条件を得るために3-8 mVの振幅を持つまで、電極を進める。正の電流（1 nAの、オン/オフ200ミリ）の矩形パルスを印加することにより、juxtasomal充填を開始します。 AP波形と周波数（ 図2Aおよび2B）を監視しながら、ゆっくりと徐々に0.1 nAのステップで電流を増加させる。 </li><li>ブロックパルス（ 図2Cのオンフェーズ中にAPの頻度が明らかに増加のような膜の開口部を監視する</強い&gt;）。充填時のスパイク波形が増加幅と縮小後の過分極（ 図2Cおよび2D）を示している。追加のパラメータが増加し、ノイズや小さな（1-5 MV）負のDCシフトが含まれています。 </li><li>安定したビオシチン注入を維持するために充填しながら、現在の（1 NA）を増加または減少。例えば、ナトリウムイオンなどの細胞外イオンの過剰流入によって毒性を回避するために、APの周波数の急激な増加に応じた電流パルスを減らすか、または停止します。注：すべてのニューロンが標識化パラメータは、個々の記録条件に応じて調整する必要が電流注入とjuxtasomalに対して異なって応答する。 </li><li>密接に電流注入を停止した後の信号を監視します。充填セッションの後のスパイク波形は通常広がり、強く過分極後に減少を示している。スパイク波形が（元の特性のために、すなわち存在を返したときに明らかであるニューロンの回復を待つ通常のアフター過分極、 図2）。 </li><li>改善された染色の品質のためビオシチン負荷が増大するため、ニューロンの完全な回復の後にセッションを充填ビオサイチン繰り返します。 </li><li>スパイク振幅が細胞に機械的なストレスを減少させるために低下するまで1μmの段階でパッチピペットを後退させる。密接に動物の体温や呼吸を監視しながら、細胞内に拡散するビオサイチンために少なくとも1時間待ってください。 </li></ol> 3。動物を灌流し、脳を削除する<ol><li>灌流セットアップを準備し、すすぎ、0.9％NaClでチューブをプリロード。 </li><li>外科トレイに動物を置き、標準ラベルテープで固定します。麻酔の十分な深さを確保するため、足のピンチとまぶたの反射神経は存在しなければなりません。 </li><li>ただ胸郭の下の腹壁を通して切開（大人5〜6センチ）、横方向及び胸骨を露出させ、後方、前方方向に進行するように内側を作る。前方胸骨を引っ張り、慎重に横隔膜から肝臓を分離する。 </li><li>横隔膜の小さな切開を行い、下肋骨を切断し、心臓を露出さ腹腔の全長に沿って切開を続ける。 </li><li>心膜を取り除く。 </li><li>左心室に針を挿入し、右心房の切開部を作る。肝臓の脱色が完了するまでに、0.9％NaCl（約8 ml /分）で灌流する。 </li><li>足前部の剛性まで、4％パラホルムアルデヒド（PFA）に注入を切り替えて、下顎が明らかである。 </li><li>ハサミを用いてラットの首を切る</li><li>残りの首の筋肉を外し、完全に頭蓋骨を公開します。 </li><li>背側の脳幹内のはさみを置き、背位を維持し、矢状縫合糸に沿って丁寧に骨を切った。 </li><li>鉗子を用いて脳を露出するように矢状縫合の両側から骨を除去します。慎重にDを避けるために硬膜を削除amage。 </li><li>慎重に脳の腹側に鈍いへらを差し込んで、静かに脳を取り出します。 </li><li> 4℃のPFA中脳全体の一夜をポスト修正4℃での0.05 Mリン酸緩衝液（PB）およびストアに脳を切り替える</li></ol> 4。タンジェンシャルセクションで脳をスライスする<ol><li> 0.05 M PBから脳を取り、前方に直面したろ紙の上に置く。前頭面に沿って、小脳を遮断し、ミッド矢状平面で切断して半球を分離するために鋭利なカミソリの刃を使用してください。 </li><li>取り付けプラットフォームに瞬間接着剤を適用し、前方右に向けて、その矢状面上の左半球をマウントします。ビブラトーム上45°の角度で取り付け作業台を固定し、0.05 M PB中の脳を沈める。 </li><li>ビブラにカミソリの刃を固定し、脳表面との最初の接触は、半球の前後面の真ん中にあることを確認してください。 24は100μmを切るセクション0.05 M PBを含有する24ウェルプレート中でそれらを収集する。 </li></ol> 5。組織学的手順<ol><li>以前に記載された方法25に従って、チトクロームオキシダーゼ染色24とアビジン-ビオチン-ペルオキシダーゼ法についての組織学的なプロトコルを実行します。オプション：蛍光アビジン/ストレプトアビジン-Alexaの複合体を用いてビオサイチン視覚化。これは、さらに逆行または順行レーシング技術で二重染色することができます。 </li><li> S1の層4で樽を視覚化するチトクロームオキシダーゼ染色（L4）のための洗浄、0.05M PBでのセクション5×5分と3、3&#39;-ジアミノベンジジンテトラヒドロクロライド（DAB）を調製含有溶液（溶液については表2を参照してくださいと試薬）。 37℃で30〜45分間予熱した溶液中のPIAからのセクション6-12インキュベート</li><li> 6×5分間、0.05 M PBで切片をすすぎ、3％H 2内のすべてのセクションをインキュベートすることにより内因性ペルオキシダーゼ活性をクエンチ</室温（RT）で20分間、0.05 M PB中にサブ&gt; O 2。 </li><li> 5×10分間、0.05M PBでのセクションを洗浄します。 4℃で一晩（溶液および試薬については表3を参照）、ABC-溶液中でのセクションをインキュベート</li><li> （溶液および試薬については表4を参照）5×10分間、0.05M PBでのセクションをすすぎ、ビオシチンで満たされたニューロンを可視化するために、DAB-溶液を調製する。室温で45〜60分間ろ過した溶液中でのセクションをインキュベートする。 </li><li> 5×10分間、0.05M PBでのセクションを洗浄します。のMowiolと顕微鏡スライドとカバーガラスのマウント部分（ソリューションおよび試薬については、表5を参照）。 </li><li>光学顕微鏡を用いてラベルの品質を決定する。 </li></ol>

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	<li>Brown, S. P., Hestrin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-type+identity:+a+key+to+unlocking+the+function+of+neocortical+circuits.">Cell-type identity: a key to unlocking the function of neocortical circuits.</a> Curr. Opin. Neurobiol. 19 (4), 415-421 (2009).</li><li>DeFelipe, 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=New+insights+into+the+classification+and+nomenclature+of+cortical+GABAergic+interneurons.">New insights into the classification and nomenclature of cortical GABAergic interneurons.</a> Nat. Rev. Neurosci. 14 (3), 202-216 (2013).</li><li>Dean, P., Porrill, J., Ekerot, C. F., Jorntell, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cerebellar+microcircuit+as+an+adaptive+filter:+experimental+and+computational+evidence.">The cerebellar microcircuit as an adaptive filter: experimental and computational evidence.</a> Nat. Rev. Neurosci. 11 (1), 30-43 (2010).</li><li>Oberlaender, 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=Cell+type-specific+three-dimensional+structure+of+thalamocortical+circuits+in+a+column+of+rat+vibrissal+cortex.">Cell type-specific three-dimensional structure of thalamocortical circuits in a column of rat vibrissal cortex.</a> Cereb. Cortex. 22 (10), 2375-2391 (2012).</li><li>Dyer, M. A., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulating+proliferation+during+retinal+development.">Regulating proliferation during retinal development.</a> Nat. Rev. Neurosci. 2 (5), 333-342 (2001).</li><li>Klausberger, T., Somogyi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+diversity+and+temporal+dynamics:+the+unity+of+hippocampal+circuit+operations.">Neuronal diversity and temporal dynamics: the unity of hippocampal circuit operations.</a> Science. 321 (5885), 53-57 (2008).</li><li>Urban, N., Tripathy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroscience:+Circuits+drive+cell+diversity.">Neuroscience: Circuits drive cell diversity.</a> Nature. 488 (7411), 289-290 (2012).</li><li>Gentet, L. 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=Unique+functional+properties+of+somatostatin-expressing+GABAergic+neurons+in+mouse+barrel+cortex.">Unique functional properties of somatostatin-expressing GABAergic neurons in mouse barrel cortex.</a> Nat. Neurosci. 15 (4), 607-612 (2012).</li><li>Burgalossi, A., 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=Microcircuits+of+functionally+identified+neurons+in+the+rat+medial+entorhinal+cortex.">Microcircuits of functionally identified neurons in the rat medial entorhinal cortex.</a> Neuron. 70 (4), 773-786 (2011).</li><li>Bock, D. D., 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=Network+anatomy+and+in+vivo+physiology+of+visual+cortical+neurons.">Network anatomy and in vivo physiology of visual cortical neurons.</a> Nature. 471 (7337), 177-182 (2011).</li><li>Briggman, K. L., Helmstaedter, M., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wiring+specificity+in+the+direction-selectivity+circuit+of+the+retina.">Wiring specificity in the direction-selectivity circuit of the retina.</a> Nature. 471 (7337), 183-188 (2011).</li><li>Herfst, L., 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=Friction-based+stabilization+of+juxtacellular+recordings+in+freely+moving+rats.">Friction-based stabilization of juxtacellular recordings in freely moving rats.</a> J. Neurophysiol. 108 (2), 697-707 (2012).</li><li>Marx, M., Gunter, R. H., Hucko, W., Radnikow, G., Feldmeyer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+biocytin+labeling+and+neuronal+3D+reconstruction.">Improved biocytin labeling and neuronal 3D reconstruction.</a> Nat. Protoc. 7 (2), 394-407 (2012).</li><li>Bruno, R. M., Sakmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortex+is+driven+by+weak+but+synchronously+active+thalamocortical+synapses.">Cortex is driven by weak but synchronously active thalamocortical synapses.</a> Science. 312 (5780), 1622-1627 (2006).</li><li>Oberlaender, M., Ramirez, A., Bruno, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+experience+restructures+thalamocortical+axons+during+adulthood.">Sensory experience restructures thalamocortical axons during adulthood.</a> Neuron. 74 (4), 648-655 (2012).</li><li>Joshi, S., Hawken, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loose-patch-juxtacellular+recording+in+vivo-a+method+for+functional+characterization+and+labeling+of+neurons+in+macaque+V1.">Loose-patch-juxtacellular recording in vivo-a method for functional characterization and labeling of neurons in macaque V1.</a> J. Neurosci. Methods. 156 (1-2), 37-49 (2006).</li><li>Pinault, D. <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+single-cell+staining+procedure+performed+in+vivo+under+electrophysiological+control:+morpho-functional+features+of+juxtacellularly+labeled+thalamic+cells+and+other+central+neurons+with+biocytin+or+Neurobiotin.">A novel single-cell staining procedure performed in vivo under electrophysiological control: morpho-functional features of juxtacellularly labeled thalamic cells and other central neurons with biocytin or Neurobiotin.</a> J. Neurosci. Methods. 65 (2), 113-136 (1996).</li><li>de Kock, C. P., Bruno, R. M., Spors, H., Sakmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Layer+and+cell+type+specific+suprathreshold+stimulus+representation+in+primary+somatosensory+cortex.">Layer and cell type specific suprathreshold stimulus representation in primary somatosensory cortex.</a> J. Physiol. 581 (1), 139-154 (2007).</li><li>Egger, R., Narayanan, R. T., Helmstaedter, M., de Kock, C. P., Oberlaender, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+reconstruction+and+standardization+of+the+rat+vibrissal+cortex+for+precise+registration+of+single+neuron+morphology.">3D reconstruction and standardization of the rat vibrissal cortex for precise registration of single neuron morphology.</a> PLoS Comput. Biol. 8 (12), (2012).</li><li>Oberlaender, 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=Three-dimensional+axon+morphologies+of+individual+layer+5+neurons+indicate+cell+type-specific+intracortical+pathways+for+whisker+motion+and.">Three-dimensional axon morphologies of individual layer 5 neurons indicate cell type-specific intracortical pathways for whisker motion and.</a> Proc. Natl. Acad. Sci. U.S.A. 108 (10), 4188-4193 (2011).</li><li>Sakata, S., Harris, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminar+structure+of+spontaneous+and+sensory-evoked+population+activity+in+auditory+cortex.">Laminar structure of spontaneous and sensory-evoked population activity in auditory cortex.</a> Neuron. 64 (3), 404-418 (2009).</li><li>de Kock, C. P., Sakmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+frequency+action+potential+bursts+(&gt;or=+100+Hz)+in+L2/3+and+L5B+thick+tufted+neurons+in+anaesthetized+and+awake+rat+primary+somatosensory+cortex.">High frequency action potential bursts (&gt;or= 100 Hz) in L2/3 and L5B thick tufted neurons in anaesthetized and awake rat primary somatosensory cortex.</a> J. Physiol. 586 (14), 3353-3364 (2008).</li><li>de Kock, C. P., Sakmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spiking+in+primary+somatosensory+cortex+during+natural+whisking+in+awake+head-restrained+rats+is+cell-type+specific.">Spiking in primary somatosensory cortex during natural whisking in awake head-restrained rats is cell-type specific.</a> Proc. Natl. Acad. Sci. U.S.A. 106 (38), 16446-16450 (2009).</li><li>Wong-Riley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+visual+system+of+monocularly+sutured+or+enucleated+cats+demonstrable+with+cytochrome+oxidase+histochemistry.">Changes in the visual system of monocularly sutured or enucleated cats demonstrable with cytochrome oxidase histochemistry.</a> Brain Res. 171 (1), 11-28 (1979).</li><li>Horikawa, K., Armstrong, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+means+of+intracellular+labeling:+injection+of+biocytin+and+its+detection+with+avidin+conjugates.">A versatile means of intracellular labeling: injection of biocytin and its detection with avidin conjugates.</a> J. Neurosci. Methods. 25 (1), 1-11 (1988).</li><li>O'Connor, D. H., Peron, S. P., Huber, D., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+activity+in+barrel+cortex+underlying+vibrissa-based+object+localization+in+mice.">Neural activity in barrel cortex underlying vibrissa-based object localization in mice.</a> Neuron. 67 (6), 1048-1061 (2010).</li><li>Veinante, P., Deschenes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+study+of+motor+cortex+projections+to+the+barrel+field+in+rats.">Single-cell study of motor cortex projections to the barrel field in rats.</a> J. Comp. Neurol. 464 (1), 98-103 (2003).</li><li>Boudewijns, Z. S., 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=Layer-specific+high-frequency+action+potential+spiking+in+the+prefrontal+cortex+of+awake+rats.">Layer-specific high-frequency action potential spiking in the prefrontal cortex of awake rats.</a> Front. Cell. Neurosci. 7, 99 (2013).</li><li>Oberlaender, M., Bruno, R. M., Sakmann, B., Broser, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmitted+light+brightfield+mosaic+microscopy+for+three-dimensional+tracing+of+single+neuron+morphology.">Transmitted light brightfield mosaic microscopy for three-dimensional tracing of single neuron morphology.</a> J. Biomed. Opt. 12 (6), 064029 (2007).</li><li>Boudewijns, Z. S., 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=Semi-automated+three-dimensional+reconstructions+of+individual+neurons+reveal+cell+type-specific+circuits+in+cortex.">Semi-automated three-dimensional reconstructions of individual neurons reveal cell type-specific circuits in cortex.</a> Commun. Integr. Biol. 4 (4), 486-488 (2011).</li><li>Bruno, R. M., Hahn, T. T., Wallace, D. J., de Kock, C. P., Sakmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+experience+alters+specific+branches+of+individual+corticocortical+axons+during+development.">Sensory experience alters specific branches of individual corticocortical axons during development.</a> J. Neurosci. 29 (10), 3172-3181 (2009).</li><li>Schubert, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observing+without+disturbing:+how+different+cortical+neuron+classes+represent+tactile+stimuli.">Observing without disturbing: how different cortical neuron classes represent tactile stimuli.</a> J. Physiol. 581 (1), 5 (2007).</li><li>Neumann, E., Kakorin, S., Toensing, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+of+electroporative+delivery+of+drugs+and+genes).">Fundamentals of electroporative delivery of drugs and genes).</a> Bioelectrochem. Bioenerg. 48 (1), 3-16 (1999).</li><li>Haas, K., Sin, W. C., Javaherian, A., Li, Z., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+electroporation+for+gene+transfer+in+vivo.">Single-cell electroporation for gene transfer in vivo.</a> Neuron. 29 (3), 583-591 (2001).</li></ol>

authrosは、開示することは何もありません。

juxtasomal方法はビオシチン標識し事後細胞型の分類および/ ​​または3D再構成のための記録されたニューロンをするオプション（麻酔をかけ、目を覚ましヘッド固定または自由行動）行動条件にわたって、単一のユニットから生体内の活動電位スパイクの記録ができます。主な利点は、細胞外（従って、非侵襲的）構成における生理学的パラメータを得ることで、まだ、?...

神経回路網は、非常に特異的形態学的および生理学的特性1-7が特徴複数の細胞型で構成されます。その結果、個々の細胞型は（例えば参照Gentet ら 8とBurgalossi ら 9）は、ネットワーク内の特殊なタスクを実行します。私たちは、神経ネットワークを介して細胞種特異的な機能を理解し始めていると多くは発見されて残っている。このために、多くのラボでは、生理的パラメータが1,10-15を得られているのと同じニューロン集団の形態学的特性の分析を可能実験的なアプローチを実施しています。ここでは、ビオシチンで記録されたニューロンのエレクトロポレーションと組み合わせて、細胞外（したがって、非侵襲的）な構成で従来のパッチピペットを用いて電気生理学的記録を必要とするjuxtasomal標識法16,17を示しています。ザ·このアプローチの主な利点は、非侵襲的な性質は個々のニューロンの活動電位スパイク（ 例えば 、透析）細胞の細胞内含有量を変化させることなく記録されることを保証することである。エレクトロポレーションに続いて、juxtasomalアプローチは、構造（形態）にする機能（生理学）をリンクするための事後セル識別と復興のオプションを提供します。典型的には、形態学的再構成は、脊椎および/または神経繊維末端濃度の定量又は電子顕微鏡を用いてナノメートル分解能での神経形態であっても再構成に拡張することができ、樹状および軸索形態の再構成を伴う。ほとんどの研究は、マウスまたはラットのような小型げっ歯類での技術を適用しているがjuxtasomal記録技術は、皮質層を横切るまたは種の範囲内の皮質下領域における種々の細胞型のインビボでの記録のために使用することができる。我々の研究は、神経細胞を記録し、ラベルに焦点を当てていますラット一次体性感覚野（S1）から、記録したニューロン18の視覚的同定を含む、標準化された基準フレーム内の正確な位置合わせと組み合わせた樹状再建は、細胞種特異的ローカル特徴づけるために皮質ネットワーク4,19および軸索アーキテクチャの詳細な再構築をリバースエンジニアリングする長距離投影は20を対象としています。 覚醒ヘッド固定23、あるいは自由に動く動物9に 、別のインビボ記録技術（細胞内または全細胞）と比較して、juxtasomal録音は比較的安定であり、したがって、麻酔をかけ21,22を含む行動状況を横切って適用することができる、14鎮静。我々は選択の多くの製剤にこの技術の一般的な適用性を強調したがここでは、ウレタン麻酔したラットのS1でjuxtasomalラベルを示しています。

<table border="0" cellpadding="0" cellspacing="0" width="784">
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			<td>National Instruments, Austin, TX, USA</td>
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			<td>H3375</td>
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			<td style="width:119px;">60130</td>
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			<td style="width:275px;">Sigma Aldrich</td>
			<td style="width:119px;">22,350-6</td>
			<td></td>
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			<td style="width:119px;">63072</td>
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</table>

juxtasomal biocytin labeling to study the structure-function relationship of individual cortical neurons

大脳皮質は、一緒にネットワークとしての意思決定、感覚誘導動作やメモリなど、多くの高次認知機能に関与している複数の層と、多くの異なる細胞型を特徴としている。このような複雑な神経ネットワークは、このようなタスクを実行する方法を理解するために、重要なステップは、動物は、関連する認知タスクを実行しているときに優先的に、ネットワーク内の個々の細胞型の関数（または電気的活性）を決定することである。さらに、それは逆の皮質ネットワークを設計できるように、ネットワークの解剖学的構造と個々の神経細胞の形態学的なアーキテクチャを決定することも同様に重要である。今日利用可能な技術的なブレークスルーは、記録されたニューロンを特定事後の貴重なオプションを動物に行動する、覚醒中の細胞の活動を記録可能にする。ここでは、アクション·ポテンショを記録することを含むjuxtasomalビオシチン標識法を実証らは、従来のパッチピペットを用いて細胞外（またはルーズパッチ）の構成でスパイク。 juxtasomal記録構成は、麻酔をかけ鎮静、覚醒頭固定、さらには自由に動く動物を含め、行動条件にわたって比較的安定であり、適用されます。したがって、この方法は、個々のニューロン、最終的には、全体の皮質微小回路の復興に動物の行動の間に細胞型特異的活動電位スパイクを結ぶことができます。このビデオの原稿では、juxtasomal構成内の個々のニューロンが事後同定と形態学的再構成のためのウレタン麻酔ラットでビオサイチンで標識することができる方法を示しています。

個々のニューロンの三次元構造に関する詳細な知識は、神経ネットワークの組織的な原則を解明するために重要です。我々の方法は、それによって、 事後のニューロンの分類及び樹状と高解像度での単一ニューロンの軸索アーキテクチャの詳細な再構築を可能にする、in vivoでの準備から、高品質のビオシチンラベルを達成するためのパイプラインが含まれます。 juxtasomalラベル...

Watch this Scientific Journal Video about Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons at JoVE.com

Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons

神経回路網の構造を理解するために、個々のニューロンの機能的および形態学的特徴付けが必要である。ここで、我々はまだ、細胞内で樹状および軸索建築の事後再構築のために、ニューロンを標識する能力を維持し、細胞外の構成で電気生理学的記録を可能にするjuxtasomalビオシチンラベルを示しています。

Juxtasomalビオシチン標識は、個々の皮質ニューロンの構造機能相関を研究するために、

The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive ...

juxtasomal-biocytin-labeling-to-study-structure-function-relationship

Research

JoVE Journal

Neuroscience

13.5K ビュー.  VU University Amsterdam. 神経回路網の構造を理解するために、個々のニューロンの機能的および形態学的特徴付けが必要である。ここで、我々はまだ、細胞内で樹状および軸索建築の事後再構築のために、ニューロンを標識する能力を維持し、細胞外の構成で電気生理学的記録を可能にするjuxtasomalビオシチンラベルを示しています。 

ビデオ: Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons

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

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

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

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

初代ラット皮質ニューロンとグリアのBilaminarの共培養

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

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1).
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however, other options have been discussed as well5.
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.

マウス胚の脊髄の全載標本での分岐軸索勉強するDRGニューロンのDII -標識

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

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

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

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

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

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

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

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Optogenetic methods have emerged as a powerful tool for elucidating neural circuit activity underlying a diverse set of behaviors across a broad range of species. Optogenetic tools of microbial origin consist of light-sensitive membrane proteins that are able to activate (e.g., channelrhodopsin-2, ChR2) or silence (e.g., halorhodopsin, NpHR) neural activity ingenetically-defined cell types over behaviorally-relevant timescales. We first demonstrate a simple approach for adeno-associated virus-mediated delivery of ChR2 and NpHR transgenes to the dorsal subiculum and prelimbic region of the prefrontal cortex in rat. Because ChR2 and NpHR are genetically targetable, we describe the use of this technology to control the electrical activity of specific populations of neurons (i.e., pyramidal neurons) embedded in heterogeneous tissue with high temporal precision. We describe herein the hardware, custom software user interface, and procedures that allow for simultaneous light delivery and electrical recording from transduced pyramidal neurons in an anesthetized in vivo preparation. These light-responsive tools provide the opportunity for identifying the causal contributions of different cell types to information processing and behavior.

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

The recent development of neuroscience tools that combine genetics and optics, termed "optogenetics", enables control over neural circuit activity with an unprecedented level of spatial and temporal resolution. Here we provide a protocol for integrating in vivo recording with optogenetic manipulation of genetically-defined subsets of prefrontal cortical and subicular pyramidal neurons.

個々の錐体細胞のハイフィデリティOptogenetic制御法<em&gt;インビボ</em

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脳顆粒ニューロンの遺伝子操作<em&gt;体外</em&gt;と<em&gt;インビボ</em&gt;ニューロンの形態と移行を研究するために、

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

ライブ培養神経細胞の抗体供給した後細胞表面および内在化タンパク質の異なる標識化

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

レーザーキャプチャーマイクロダイセクション - 個々のドーパミンニューロンの単離と全体の腹側被蓋領域の実証

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly committed intermediate progenitor subtypes. Each of these subtypes expresses a set of specific molecular markers that, together with specific morphological criteria, can be used for their identification. Typically, immunofluorescent techniques are applied involving subtype-specific antibodies in combination with exo- or endogenous proliferation markers. We herein describe immunolabeling methods for the detection and quantification of all stages of adult hippocampal neurogenesis. These comprise the application of thymidine analogs, transcardial perfusion, tissue processing, heat-induced epitope retrieval, ABC immunohistochemistry, multiple indirect immunofluorescence, confocal microscopy and cell quantification. Furthermore we present&#160;a sequential multiple immunofluorescence protocol which circumvents problems usually arising from the need of using primary antibodies raised in the same host species. It allows an accurate identification of all hippocampal progenitor subtypes together with a proliferation marker within a single section. These techniques are a powerful tool to study the regulation of different progenitor subtypes in parallel, their involvement in brain pathologies and their role in specific brain functions.

This video article illustrates a comprehensive protocol to detect and quantify all stages of adult hippocampal neurogenesis within the same tissue section. We elaborated a method to overcome the limitations of indirect multiple immunofluorescence that arise when suitable antibodies from different host species are unavailable.

大人の海馬神経新生を研究するために、同じホスト種由来の抗体による免疫組織化学および複数のラベル

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly ...

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

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

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

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

ラット皮質ニューロンにおける繊維状アクチン（F-アクチン）涙点の定量化

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