この作品は、部分的にラヴァル大学の交わりによってサポートされていましたASJKに健康研究（CIHR）助成金のカナダの協会によってサポートされていました。 ASは出生後の神経発生におけるカナダリサーチチェアの受取人である。

<ol>
	<li>Kriegstein, A., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glial+nature+of+embryonic+and+adult+neural+stem+cells.">The glial nature of embryonic and adult neural stem cells.</a> Annu. Rev. Neurosci. 32, 149-184 (2009).</li><li>Kaneko, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+neurons+clear+the+path+of+astrocytic+processes+for+their+rapid+migration+in+the+adult+brain.">New neurons clear the path of astrocytic processes for their rapid migration in the adult brain.</a> Neuron. 67, 213-223 (2010).</li><li>Lois, C., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-distance+neuronal+migration+in+the+adult+mammalian+brain.">Long-distance neuronal migration in the adult mammalian brain.</a> Science. 264, 1145-1148 (1994).</li><li>Snapyan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vasculature+guides+migrating+neuronal+precursors+in+the+adult+mammalian+forebrain+via+brain-derived+neurotrophic+factor+signaling.">Vasculature guides migrating neuronal precursors in the adult mammalian forebrain via brain-derived neurotrophic factor signaling.</a> J. Neurosci. 29, 4172-4188 (2009).</li><li>Whitman, M. C., Fan, W., Rela, L., Rodriguez-Gil, D. J., Greer, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+vessels+form+a+migratory+scaffold+in+the+rostral+migratory+stream.">Blood vessels form a migratory scaffold in the rostral migratory stream.</a> J. Comp. Neurol. 516, 94-104 (2009).</li><li>Lledo, P. M., Saghatelyan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+new+neurons+into+the+adult+olfactory+bulb:+joining+the+network,+life-death+decisions,+and+the+effects+of+sensory+experience.">Integrating new neurons into the adult olfactory bulb: joining the network, life-death decisions, and the effects of sensory experience.</a> Trends Neurosci. 28, 248-254 (2005).</li><li>Bolteus, A. J., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+release+and+uptake+regulate+neuronal+precursor+migration+in+the+postnatal+subventricular+zone.">GABA release and uptake regulate neuronal precursor migration in the postnatal subventricular zone.</a> J. Neurosci. 24, 7623-7631 (2004).</li><li>Martini, F. J., Valdeolmillos, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actomyosin+contraction+at+the+cell+rear+drives+nuclear+translocation+in+migrating+cortical+interneurons.">Actomyosin contraction at the cell rear drives nuclear translocation in migrating cortical interneurons.</a> J. Neurosci. 30, 8660-8670 (2010).</li><li>Murase, S., Horwitz, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deleted+in+colorectal+carcinoma+and+differentially+expressed+integrins+mediate+the+directional+migration+of+neural+precursors+in+the+rostral+migratory+stream.">Deleted in colorectal carcinoma and differentially expressed integrins mediate the directional migration of neural precursors in the rostral migratory stream.</a> J. Neurosci. 22, 3568-3579 (2002).</li><li>Wilcock, A. C., Swedlow, J. R., Storey, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+spindle+orientation+distinguishes+stem+cell+and+terminal+modes+of+neuron+production+in+the+early+spinal+cord.">Mitotic spindle orientation distinguishes stem cell and terminal modes of neuron production in the early spinal cord.</a> Development. 134, 1943-1954 (2007).</li><li>Ono, M., Yanagawa, Y., Koyano, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+neurons+in+inferior+colliculus+of+the+GAD67-GFP+knock-in+mouse:+electrophysiological+and+morphological+properties.">GABAergic neurons in inferior colliculus of the GAD67-GFP knock-in mouse: electrophysiological and morphological properties.</a> Neurosci. Res. 51, 475-492 (2005).</li><li>Bozoyan, L., Khlghatyan, J., Saghatelyan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+Control+the+Development+of+the+Migration-Promoting+Vasculature+Scaffold+in+the+Postnatal+Brain+via+VEGF+Signaling.">Astrocytes Control the Development of the Migration-Promoting Vasculature Scaffold in the Postnatal Brain via VEGF Signaling.</a> J. Neurosci. 32, 1687-1704 (2012).</li><li>Bovetti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+vessels+form+a+scaffold+for+neuroblast+migration+in+the+adult+olfactory+bulb.">Blood vessels form a scaffold for neuroblast migration in the adult olfactory bulb.</a> J. Neurosci. 27, 5976-5980 (2007).</li><li>Platel, J. C., Heintz, T., Young, S., Gordon, V., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tonic+activation+of+GLUK5+kainate+receptors+decreases+neuroblast+migration+in+whole-mounts+of+the+subventricular+zone.">Tonic activation of GLUK5 kainate receptors decreases neuroblast migration in whole-mounts of the subventricular zone.</a> J. Physiol. 586, 3783-3793 (2008).</li><li>Nam, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+features+of+postnatal+subventricular+zone+cell+motility:+a+two-photon+time-lapse+study.">Dynamic features of postnatal subventricular zone cell motility: a two-photon time-lapse study.</a> J. Comp. Neurol. 505, 190-208 (2007).</li><li>Kim, Y., Comte, I., Szabo, G., Hockberger, P., Szele, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+mouse+subventricular+zone+stem+and+progenitor+cells+are+sessile+and+epidermal+growth+factor+receptor+negatively+regulates+neuroblast+migration.">Adult mouse subventricular zone stem and progenitor cells are sessile and epidermal growth factor receptor negatively regulates neuroblast migration.</a> PLoS One. 4, e8122 (2009).</li><li>Bortone, D., Polleux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KCC2+expression+promotes+the+termination+of+cortical+interneuron+migration+in+a+voltage-sensitive+calcium-dependent+manner.">KCC2 expression promotes the termination of cortical interneuron migration in a voltage-sensitive calcium-dependent manner.</a> Neuron. 62, 53-71 (2009).</li><li>Comte, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galectin-3+maintains+cell+motility+from+the+subventricular+zone+to+the+olfactory+bulb.">Galectin-3 maintains cell motility from the subventricular zone to the olfactory bulb.</a> J. Cell Sci. 124, 2438-2447 (2011).</li></ol>

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適切な脳領域に神経前駆細胞の正確なターゲティングは、神経回路の適切なアセンブリと関数の基礎となる基本的なプロセスです。細胞の大半は、胚発生中に移行し、唯一のそのようなOB、歯状回および小脳な​​どのいくつかの地域では、出生後の脳では、ニューロンの変位は、まだ行われます。出生後の脳内の細胞遊走を編成するメカニズムはよくわかっていない、しかし、残っている。成熟した神経組織において細胞遊走を誘導に関与する機序と分子経路の知識は、生後脳における神経ターゲティングの我々の理解を改善し、脳の病気の領域に神経細胞の動員を誘導するために新たな戦略を開発するための臨床的意義を持っているかもしれません。 本稿では、成体マウスの前脳の急性スライスにおける細胞移動を監視するためのプロトコルを記述します。我々は、成人SVZ-OBを使用しながらモデル系として経路は、同じ技術が胚および生後初期の脳内だけでなく、成人の脳損傷後における細胞遊走をフォローするために適用することができる。我々は、小脳や成人脳卒中後の線条体（Eiriz、グレード、マルバとSaghatelyan、未発表データ）、出生後早期のRMS 12における細胞遊走をフォローするために、このプロトコルを使用していました。 神経芽細胞遊走のイメージングは、細胞遊走が密接in vivo条件下で模倣微小環境で勉強できるようにするライブ急性スライスを用いて実施した。遊走細胞はウイルス粒子の定位注入により、または野生型マウスのSVZにレポーターマウスから神経芽細胞を移植のいずれかによって可視化した。マイグレーション分析が注射部位から遠く離れて行った。 RMSでの接線の移行を検討するために、我々は、神経芽細胞を標識した3-5日後に急性スライスを用意しました。 OBの放射状移動を研究するために、スライスは7-10日後に調製したステレオSVZにreotaxic注入。成体SVZにおける神経前駆細胞の標的化ウイルスはまた、勉強するために使用され、特定の分子手がかりをアップレギュレートまたはダウンレギュレートすることにより、移行プロセスに関与する因子を調節することができます。これは、RMSの異なる細胞型にラベルを付けたり、トレーサー充填血管4,13またはに沿ってウイルスまたは遺伝的に標識されたアストロ2、12に沿って神経芽細胞の移行パターンを解明するために、多波長イメージングを行うことも可能である。 イメージの神経芽細胞の移行に我々は、励起光の短パルス（30-100ミリ秒）後に別のz平面で比較的急速な買収を許可されたCCDカメラを搭載した電動蛍光正立顕微鏡を使用していました。これは、退色を防止し、神経芽細胞の移行の3次元可視化と干渉することなく、2以降の取得の時間間隔を減少させた。他のグループは、細胞MIGRを監視するために共焦点または二光子顕微鏡を使用していた生後RMS 14-16 ation。両方の技術は、主に時間的、空間的な解像度の問題に関連して、それぞれの長所と限界を持っています。私たちは、連続した買収の間で短い間隔（15〜30秒）を使用してお勧めします。新生ニューロンは、跳躍動作を持っており、移行相は4月10日分4と同じくらい簡単なことができ、静止期間によって中断されています。このタイプの移行を考えると、それは確実に移行し、固定相の先頭と末尾を識別するために、連続する時間ポイント間の比較的急速な取得間隔を（15-30秒）で使用することが重要です。これは2つの連続した​​買収の時間間隔は数分の範囲にあるときに達成することは困難である。定常と移行フェーズの正確な決定は明確に移行フェーズ4、17時のみに定量化されるべきである神経芽細胞の移行速度を定義する必要があります。ロング取得間隔アベレージを計算することができます距離などの電子速度も固定相を含み、一定の期間を旅行した。このメソッドによって定義された平均速度の変化は、しかし、移動速度の違い（移行フェーズでのみ定義されている）によって、または定常および移行フェーズ期間中のいずれかによって引き起こされる場合があります。取得パラメータでこれらの違いは、異なるグループによって報告された神経芽細胞の移行速度の不一致を根底にする可能性があります。 15〜30秒間隔で急速な買収を行うと移行フェーズで排他的に移動速度を決定することにより、我々はの取得間隔を使用して他のグループに対し、120から150ミクロン/ hの4,12の移行の速度を計算した3月7日分は、50〜100μmの/ hの14、15、18の速度を報告した。 CCDカメラ付き広視野蛍光イメージングの主な欠点は、それがスキャンシステムより低い空間分解能を提供することである。しかし、以来、神経芽細胞の移行は、コンパクトMORPを持ってソーマと短いリードするプロセスとhology、低い空間分解能は、細胞体および一流のプロセス（ 図3および動画1と2）のレベルで神経芽細胞の信頼性の高い識別と追跡することを妨げるものではない。 我々は本明細書に記載の技術は密接に分析される生体内の条件で模倣微小環境における神経芽細胞の移行は、RMSの異なるコンポーネント間の相互作用を研究することを可能にし、神経芽細胞の遊走におけるこれらの相互作用の役割が調査される。

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>スクロース</td><td>シグマ</td><td> S9378 </td></tr><tr><td>グルコース（ACSF） </td><td> EMD </td><td> DX0145-3 </td></tr><tr><td> NaClを</td><td>シグマ</td><td> S9625 </td></tr><tr><td> KCI </td><td>シグマ</td><td> P9541 </td></tr><tr><td> MgCI 2 x6H 2 O </td><td>シグマアルドリッチ</td><td> M2670 </td></tr><tr><td> NaHCO 3を </td><td>シグマ</td><td> S5761 </td></tr><tr><td>のNaH 2 PO 4·xH 2 Oを</td><td> EMD </td><td> SX0710-1 </td></tr><tr><td> CACI 2 x2H 2 O </td><td>シグマアルドリッチ</td><td> C3881 </td></tr><tr><td>デキストランTexasRed </td><td>インビトロジェン</td><td> D1864 </td></tr><tr><td>デキストランカスカdeBlue </td><td>インビトロジェン</td><td> D1976 </td></tr><tr><td>グルコース（40X溶液） </td><td>シグマ</td><td> G8769 </td></tr><tr><td>ナトリウムpyruvat </td><td>ギブコ</td><td> 11360-070 </td></tr><tr><td> HEPES </td><td>シグマ</td><td> H3375 </td></tr><tr><td> HBSS </td><td>ギブコ</td><td> 14170-112 </td></tr><tr><td> DNase Iを</td><td>シグマ</td><td> D-5025 </td></tr><tr><td>トリプシン-EDTA </td><td>ギブコ</td><td> 25300-054 </td></tr><tr><td> Neurobasal培地</td><td>ギブコ</td><td> 21103-049 </td></tr><tr><td> BSA </td><td> EMD </td><td> 2930 </td></tr><tr><td>ペニシリン/ストレプトマイシン</td><td>ライフテクノロジーズ</td><td> 15140-122 </td></tr><tr><td>ケタミン/キシラジン</td><td> CDMV </td><td> 5230 </td></tr><tr><td>パスツールピペット</td><td> VWR </td><td> 14672-380 </td></tr><tr><td> 15 mlコニカルチューブ</td><tD&gt;ザルスタット</td><td> 62.553.205 </td></tr><tr><td> 50 mlコニカルチューブ</td><td>ザルスタット</td><td> 62.547.205 </td></tr><tr><td>ガラスキャピラリー（定位注射） </td><td> WPI </td><td> 4878 </td></tr><tr><td>灯油</td><td> EMD </td><td> PX0045-3 </td></tr><tr><td> Proviodine </td><td>ルジェ</td><td> 65655-1370 </td></tr><tr><td>縫合</td><td> Stoelting </td><td> 50487 </td></tr><tr><td> Anafen </td><td> CDMV </td><td> 11508 </td></tr><tr><td> 20ccのシリンジ</td><td> VWR </td><td> SS-20L2 </td></tr><tr><td>ペトリ皿</td><td> VWR </td><td> 25384-094 </td></tr><tr><td>寒天</td><td>ラボラトリーマット</td><td> AP-0108 </td></tr><tr><td>グルー</td><td> Permabond </td><td> 910 </td></tr><tr><td> 95％O 2/5％CO 2で </td><td>リンデ</td><td> 24068835 </td></tr><tr><td>ブレード&lt;/ TD&gt; <td> WPI </td><td> 501901 </td></tr><tr><td>ナイロンメッシュ</td><td>ワーナー·インスツルメンツ</td><td> 64から0198 </td></tr><tr><td>遠心</td><td>エッペンドルフ</td><td> 5702 000.019 </td></tr><tr><td>ピペットプラー</td><td>サター·インストゥルメント</td><td> P-97 </td></tr><tr><td>ナノリットルインジェクタ</td><td> WPI </td><td> B203MC4 </td></tr><tr><td>定位注入装置</td><td> WPI </td><td> 502900 </td></tr><tr><td>マイクロドリルシステム</td><td> WPI </td><td> 501819 </td></tr><tr><td>ビブラトーム</td><td>サーモサイエンティフィック</td><td> 920110 </td></tr><tr><td>広視野の蛍光顕微鏡</td><td>オリンポス</td><td> BX61WIF </td></tr><tr><td> CCDカメラ</td><td> Photometrics </td><td> CS-HQ2-D </td></tr><tr><td>超静音イメージング室</td><td>ハーバード装置</td><td> 64から1487 </td></tr><tr><td> PH-1シリーズ20ヒータープラットフォーム</td><td>ハーバード装置</td><td> 64から0284 </td></tr><tr><td>暖房装置</td><td>ワーナー·インスツルメンツ</td><td> TC-344B </td></tr><tr><td> 40X水浸対物レンズ</td><td>オリンポス</td><td> 1-UM587 </td></tr><tr><td> 10Xの水浸対物レンズ</td><td>オリンポス</td><td> 1-UM583 </td></tr><tr><td>ラムダDG-4 </td><td>サターインスツルメンツ</td><td> DG-4/OF </td></tr><tr><td> MetaMorphソフトウェア</td><td>分子デバイス</td><td> 4万</td></tr><tr><td> Imarisソフトウェア</td><td>ビットプレン</td><td> BPI-IM70-F1 </td></tr></tbody></table>

time-lapse imaging of neuroblast migration in acute slices of the adult mouse forebrain

新しい機能性ニューロンが恒常的に成体哺乳類の脳の制限区域内の神経幹細胞の内因性のプールから生成されていることを示す証拠の実質的なボディがあります。脳室下帯（SVZ）から新生児神経芽細胞は、嗅球（OB）が1で、最終的な宛先に吻側渡り鳥ストリーム（RMS）に沿って移行します。 RMSで、神経芽細胞は、構造的な支持と移行4,5に必要な分子因子の供給源としての血管を使用して星状膠細胞の突起2,3によってensheathedチェーンで接線方向に移行します。 OBで、神経芽細胞はチェーンから切り離し、それらは介在ニューロンに分化し、既存のネットワーク1、6に統合異なる球層に放射状に移行します。 本稿では、我々は齧歯類の脳の急性スライスにおける細胞移動を監視するための手順を説明します。急性スライスの使用はassessmを許可密接にインビボ条件および in vivoイメージングにアクセスが困難な脳領域でに似ているという微小環境における細胞移動のENT。さらに、それは最終的に細胞の遊走特性を変化させることができる器官および細胞培養の場合のように、長い培養状態を回避できます。急性スライスにおける神経前駆細胞は、DIC光学または蛍光タンパク質を用いて可視化することができます。野生型マウスのSVZにレポーターマウスから神経芽細胞を移植し、神経芽細胞に蛍光タンパク質を発現するトランスジェニックマウスを用いてSVZにおける神経前駆細胞のウイルスラベリングは、神経芽細胞を可視化し、それらの移動を追跡するためのすべての適当な方法である。後者の方法は、しかし、個々の細胞が原因で標識された細胞の高密度の長時間追跡することはできません。我々は、比較的急速な取得間隔（1つのIMAを達成するためにCCDカメラを搭載した広視野の蛍光正立顕微鏡を使用確実に静止して渡り鳥のフェーズを識別するために、GEごとに15または30秒）。静止および移動相の持続時間の正確な同定結果の明確な解釈のために重要である。我々はまた、3Dでの神経芽細胞の移行を監視するために複数のzステップの買収を行いました。広視野の蛍光イメージングは、ニューロンの移動7-10を視覚化するために広く使用されています。ここで、我々は、神経芽細胞を標識成体マウスの前脳の急性スライスにおける神経芽細胞の遊走をリアルタイムにビデオ撮影を行うと、細胞遊走を分析するための詳細なプロトコルを記述します。説明されたプロトコルは成人RMSでの神経芽細胞の遊走を例示したが、それは、胚や初期生後脳における細胞移動を追跡するために使用することができます。

Watch this Scientific Journal Video about Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain at JoVE.com

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

我々は、マウスの前脳におけるニューロン移動のリアルタイムvideoimagingためのプロトコルを記述します。ウイルス標識又はグラフト神経前駆細胞の遊走は、固定と移動相の持続時間と速度を含む細胞遊走のさまざまなフェーズを、勉強するのは比較的急速な取得間隔と広視野蛍光イメージングを用いた急性スライスのライブで録音された移行。

成体マウスの前脳の急性スライスにおける神経芽細胞の移行のタイムラプスイメージング

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

time-lapse-imaging-neuroblast-migration-acute-slices-adult-mouse

Nanyang Technological University

Research

JoVE Journal

Neuroscience

14.8K ビュー.  Centre de Recherche Université Laval Robert-Giffard. 我々は、マウスの前脳におけるニューロン移動のリアルタイムvideoimagingためのプロトコルを記述します。ウイルス標識又はグラフト神経前駆細胞の遊走は、固定と移動相の持続時間と速度を含む細胞遊走のさまざまなフェーズを、勉強するのは比較的急速な取得間隔と広視野蛍光イメージングを用いた急性スライスのライブで録音された移行。

ビデオ: Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

出生後の脳における神経細胞の移動の高分解能タイムラプスイメージングのための器官型スライスアッセイ

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

神経科学

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

開発ゼブラフィッシュ前脳におけるクローン関連の神経前駆細胞のタイムラプスライブイメージング

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

カルシウムイメージングの急性脳室下帯スライスの調製

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

急性海馬スライスにおけるシナプス誘発アストログリアとニューロン活動の二重の電気生理学的記録

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish

The nervous system is often described as a hard-wired component of the body even though it is a considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity. Unlike the central nervous system (CNS), the peripheral nervous system (PNS) is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that govern this phenomenon. Using zebrafish as a model system, we have the unprecedented opportunity to couple regenerative studies with in vivo imaging and genetic manipulation. Peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. Axons are ensheathed by myelinating or non-myelinating Schwann cells, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. Following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris and re-innervate targets. To investigate the roles of all peripheral glia in PNS regeneration, we describe here an axon transection assay that uses a commercially available nitrogen-pumped dye laser to axotomize motor nerves in live transgenic zebrafish. We further describe the methods to couple these experiments to time-lapse imaging of injured and control nerves. This experimental paradigm can be used to not only assess the role that glia play in nerve regeneration, but can also be the platform for elucidating the molecular mechanisms that govern nervous system repair.

Although the peripheral nervous system (PNS) is capable of significant repair after injury, little is known about the cellular and molecular mechanisms that govern this phenomenon. Using live, transgenic zebrafish and a reproducible nerve transection assay, we can study dynamic glial cell behaviors during nerve degeneration and regeneration.

モーター神経切断とライブゼブラフィッシュにおけるグリア細胞の挙動のタイムラプスイメージング

The  nervous system is often described as a hard-wired component of the body even  though it is a considerably fluid organ system that reacts to external ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

生体産後エレクトロポレーションおよびマウスの急性脳スライスにおける神経芽細胞移動のタイムラプスイメージング中

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted area of the brain, neural stem cells proliferate and constantly generate neuroblasts that migrate tangentially in chains along the rostral migratory stream (RMS) to reach the olfactory bulb (OB). Once in the OB, neuroblasts switch to radial migration and then differentiate into mature neurons able to incorporate into the preexisting neuronal network. Proper neuroblast migration is a fundamental step in neurogenesis, ensuring the correct functional maturation of newborn neurons. Given the ability of SVZ-derived neuroblasts to target injured areas in the brain, investigating the intracellular mechanisms underlying their motility will not only enhance the understanding of neurogenesis but may also promote the development of neuroregenerative strategies.
This manuscript describes a detailed protocol for the transfection of primary rodent RMS postnatal neuroblasts and the analysis of their motility using a 3D in vitro migration assay recapitulating their mode of migration observed in vivo. Both rat and mouse neuroblasts can be quickly and efficiently transfected via nucleofection with either plasmid DNA, small hairpin (sh)RNA or short interfering (si)RNA oligos targeting genes of interest. To analyze migration, nucleofected cells are reaggregated in &#39;hanging drops&#39; and subsequently embedded in a three-dimensional matrix. Nucleofection per se does not significantly impair the migration of neuroblasts. Pharmacological treatment of nucleofected and reaggregated neuroblasts can also be performed to study the role of signaling pathways involved in neuroblast migration.

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

Neuroblast migration is a crucial step in postnatal neurogenesis. The protocol described here can be used to investigate the role of candidate regulators of neuroblast migration by employing DNA/small hairpin RNA (shRNA) nucleofection and a 3D migration assay with neuroblasts isolated from the rodent postnatal rostral migratory stream.

神経芽細胞の移行を検討するために、げっ歯類神経芽細胞のクレオ<em&gt;インビトロ</em

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

マウス蝸牛外植片の長期的タイムラプスイメージング

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

マウス鋤鼻器官の急性組織スライスで定義された細胞集団の中で、深さ生理学的解析

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

電気生理学的研究のための成体マウスの網膜の水平スライスの調製

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...