Name: in vitro time-lapse live-cell imaging to explore cell migration toward the organ of corti
Uploaded: 2020-12-04T14:45:00.000+00:00
Duration: 8 min 8 s
Description: Watch this Scientific Journal Video about In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti at JoVE.com

この研究は、韓国国立研究財団(NRF)とハリム大学研究基金の研究助成金(NRF-2018-R1D1A1B07050175、HURF-2017-66)によって支援されました。

ICRマウスを含むすべての研究プロトコルは、元州医科大学延世大学の施設動物管理・使用委員会(IACUC)によって承認されました。実験は世界医師会倫理規定に従って行われた。このプロトコルでは、妊娠中のICRマウスを12/12時間の明暗サイクルに保ち、食物および水に自由にアクセスした。
1. コクリー解剖
<ol><li>紫外線を30分間オンにして層流組織培養フードを殺菌し、使用前に70%エタノールで全ての表面をスプレーします。表面を乾かします。</li>
	<li>解剖器を70%エタノールに10分間入れ、使用する前に乾燥させます。</li>
	<li>手術用ブレードを使用して、出生後3-4日齢のマウスの首を切り落とします(図2A)。</li>
	<li>頭蓋骨を層流フードの実体顕微鏡の下に置き、組織を70%エタノールに浸します。</li>
	<li>組織を組織解剖液(1xハンクスバランス塩溶液、1 mM HEPES)に素早く浸し、エタノールを除去する。</li>
	<li>外科用ブレードで頭蓋骨の中心線を切る(図2B、C)。</li>
	<li>皮膚を前から引き下げ、耳の外耳道を切断して頭蓋骨を露出させる(図2D)。</li>
	<li>眼線を横切って頭蓋骨の前部から後部に切り取る(図2E)。</li>
	<li>頭蓋骨を開き、前脳、小脳、脳幹を鈍い鉗子で取り除く(図2F、G)。</li>
	<li>マイクロ鉗子を用いて、側頭骨から内耳を分離する(図2H)。</li>
	<li>組織解離液を含むペトリ皿にコクリーを移します。</li>
	<li>内の人工内軟組織のみを残して、すべての人工内臓のオティックカプセルを慎重に解剖する(図2I、J)。</li>
	<li>鉗子と別の鉗子のペアで人工内耳管の変化を保持し、ゆっくりと2つの組織を分離する(図2K)。</li>
	<li>ストリア血管症とテクトリアル膜を静かに剥がして取り除く(図2L、M)。</li>
	<li>新しい組織解剖液に殺菌プラスチックカバースリップを入れ、コルティの器官を直径9mmのカバースリップに置き、バジラー膜が下向きであることを確認する(図2N-P)。</li>
	<li>Reissnerの膜と残りの変形組織を鉗子でカバースリップに押し込んで組織を固定化する(図2N-P)。</li>
	<li>埋め込みティッシュのカバースリップを直径35mmの共焦点皿の中心に移す。</li>
	<li>皿の中央に位置する人工内草を持つガラスクローニングシリンダーを皿の上に置き、100 μLの外植培養培地(DMEM/F12、10%胎児血清(FBS)、1%N2サプリメント、アンピシリン(10μg/mL))をシリンダー内に10を加える(図2Q)。</li>
	<li>プレート5×マウス骨髄由来緑色蛍光タンパク質(GFP)タグ付きMSPの10細胞を2mLの培養培地(45%DMEM+ 45%DMEM/F12、10%FBS、1%N2サプリメント、アンピシリン10μg/mL)の外ガラスシリンダー外(図2R)<ol>
<li>MSCが80〜90%コンフルエントである場合、トリプシンエチレンジアミンテトラ酢酸でそれらを取り外すことによってそれらを通過させる。</li>
	</ol>
</li>
	<li>コンフォーカルディッシュを加湿インキュベーターに慎重に移し、5%CO2雰囲気で37°Cで一晩インキュベートします。</li>
	<li>すべての媒体をシリンダーの内側と外側に吸い込み、ガラスシリンダーをコンフォーカル皿から取り外します。</li>
	<li>共焦点皿に2mLの新鮮な培養培地を加え、分析の準備ができるまで加湿インキュベーターで組織培養皿をインキュベートする。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61947/61947fig2v2.jpg"> 図 2.マウスの内臓の解剖とコルチとMSCの器官の共培養.(A)マウスの切断、 (B) および (C) 頭部の中線矢頭解剖、 (D) および (E) 脳の角膜解剖、 (F) および (G) 脳と側頭骨の除去、 (H) 人工内壁の除去、 ( H)骨内壁の除去、 (J)人工内骨の分離、 ( (L) コルチの器官からのストリア血管症 (SV) とスパイラル靭帯 (SL) の分離、 (M) プレート膜の除去、 (N-P) プラスチックカバースリップ上の内骨膜の固定、(Q) コンフォーカルディッシュにおけるカバースリップとガラスシリンダーの位置、 (R) 白スケールバー (A-Ecm = 1)オレンジ(F、G、P)と黄色のスケールバー(H、I)=1mm;緑色のスケールバー(J-O)=0.5 mm.<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61947/61947fig2v2large.jpg" target="_blank">この図の大きなバージョンを表示するには、ここをクリックしてください。</a> 
2. タイムラプスイメージング
<ol><li>ここで示す実験では、ステージトップインキュベーターシステムを備えた共焦点顕微鏡システムを使用してください。</li>
	<li>共焦点顕微鏡、蛍光灯、コンピュータの電源を入れます。</li>
	<li>共焦点顕微鏡のステージ上に配置したステージトップインキュベーターの条件を37°C、5%CO2雰囲気に設定します。</li>
	<li>皿の固定容器の上にサンプル皿を置き、皿の固定蓋で覆い、上ヒーターの蓋でチャンバーを閉じます。</li>
	<li>視野のコルティとMSCの器官を局地化するためにズームとフォーカスを調整します。</li>
	<li>画像処理ソフトウェアを開きます。 [位置指定 ]オプションで 、20x Plan-Apochromat の目的(0.8 の数値) と 0.5x トリミング領域を選択します。</li>
	<li>[ 取得] で スマート セットアップ をクリックし 、[EGFP]を選択します。</li>
	<li>[取得] の下のチャネルタブを開き、レーザーパワーを0.2% に、ピンホールを44 μmに、マスター ゲインを750 Vに、デジタルゲインを1.0に設定します。</li>
	<li>イメージング設定の下の[ESID]をクリックし、ESIDゲインを4に、デジタルゲインを7.5に設定します。</li>
	<li>210タイルを生成するためにタイルとステークをクリックしてください。</li>
	<li>フォーカス戦略を開き、フォーカスモードを選択します。</li>
	<li>[ 時系列] で、 期間 を 24 時間 、 間隔 を 10 分に設定します。</li>
	<li>[取得] で、フレーム サイズを512 x 512 ピクセル、スキャン速度を8ピクセル、方向を双方向に設定し、平均を4倍に設定し、ピクセルあたりのビット数を16に設定します。</li>
	<li>実験を 開始 するをクリックして実験を開始します。</li>
</ol>3. 画像ファイルの変更
<ol><li>[ 処理] で [ ステッチ] をクリックし、 最小オーバーレイ を 5% に設定し、 最大シフト を 10%に設定します。</li>
	<li>[ ムービーのエクスポート] をクリックし、 非圧縮に設定し、 速度 を 7.5に設定します。</li>
</ol>4. 免疫染色
<ol><li>培地を慎重に吸引し、リン酸緩衝生理食塩分(PBS)でサンプルを5分間洗浄します。</li>
	<li>サンプルをPBSで4%ホルマリンで15分間固定し、サンプルをPBSで3回5分間洗浄します。</li>
	<li>PBSで0.1%トリトンX-100でサンプルを10分間透過し、PBSで3回5分間洗浄します。</li>
	<li>250 μLのファロイジン-iFluor 647試薬(PBSで1:1000希釈)を加え、シェーカーの室温でサンプルを1時間インキュベートします。</li>
	<li>サンプルをPBSで3回5分間洗います。</li>
	<li>カバースリップをガラススライドに移し、2滴の取り付け溶液を追加します。</li>
	<li>スライドにカバースリップをそっと置きます。</li>
	<li>透明なマニキュアでカバースリップを密封し、細胞が観察されるまで暗闇の中で4°Cで保管してください。</li>
	<li>励起/放出(Ex/Em)=650/665 nmのファロイジンおよびEGFPのEx/Em=488/507 nmで、適切なフィルターを備えた共焦点顕微鏡を使用してスライドをイメージします。</li>
</ol>

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著者らは開示するものは何もない。

損傷した細胞の再生を促進するために、損傷部位へのMSCの移植が広範囲に研究されており、治療効果が明らかである。移植とその後のMCの分化は、3-ニトロプロピオン酸13によって誘発される難聴を有するラットの聴力を回復させることが報告されている。Leeらはヒトに対してMSCをトランスベンで適用したが、彼らは聴覚14において有意な改善を達成しなか?...

難聴は先天的に起こり得るか、加齢、薬物、騒音を含むいくつかの要因によって徐々に引き起こされる可能性がある。難聴は、聴力を担う毛細胞が損傷した後に機能障害を回復することは非常に困難であるため、しばしば治療が困難である。世界保健機関(WHO)によると、世界の4億6,100万人が難聴と推定されており、これは世界人口の6.1%を占めています。難聴者のうち93%が成人で、7%が子供です。
多くのアプローチは、難聴を治療するために試みられてきた;特に、MSCを用いた再生アプローチが有望な治療法として登場している。組織が損傷すると、MSCは循環系に自然に放出され、損傷部位に移動し、そこで様々な分子を分泌して再生を促進する微小環境を形成する2。したがって、外部移植されたMSCを標的とする臓器への移行と、強力な免疫調節、血管新生、および抗アポトーシスを引き起こす分子のその後の分泌を通じて損傷した組織を治療する方法を開発することが重要であり、損傷した細胞機能3、4、5の回復を強化する。
MSCが損傷した組織に移行するホーミングプロセスは、克服すべき最も重要な障害である可能性があります。MSCには、テザリング/ローリング、活性化、逮捕、トランスマイグレーション/ダイアペデス、および移行6、7、8の順次ステップを備えた全身ホーミングメカニズムがあります。現在、これらのステップを改善する方法を特定するための取り組みが進行中です。遺伝子組み換え、細胞表面工学、インビトロプライミング、磁気誘導など、さまざまな戦略が6,7で試験されています。さらに、損傷した人工内毛部位にMSCをホーミングすることにより、聴覚毛細胞の保護と再生を促進するためにいくつかの試みがなされている。しかし、インビボでのMSCの追跡は時間と労力を要し、高度に専門的なスキルを必要とします 9.
この問題を解決するために、数時間にわたって細胞の移動を撮影するタイムラプス共焦点顕微鏡を介して、内腔内のMSCのホーミングを観察する方法が開発された(図1)。それは20世紀 初めに開発され、最近特定の細胞の移動を研究するための強力なツールとなっています。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61947/61947fig01.jpg" style="font-size: 14px;"> 図1:図1:(A)コルティの解剖された器官が鉗子を用いてプラスチックカバースリップに付着した後、カバースリップは35mmガラス底の共焦点顕微鏡皿に置かれ、(B)ガラスシリンダーが配置される。(C)ガラスシリンダーの内部を媒体で充填した後、(D)GFPラベル付きMSCを媒体で慎重にシリンダーの外に追加する。(E)一晩のインキュベーション後、ガラスシリンダーを取り外し、画像を共焦点顕微鏡で撮影する。略語: GFP = 緑色蛍光タンパク質;MSC = 間葉系幹細胞。<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61947/61947fig01large.jpg" style="font-size: 14px;" target="_blank">この図の大きなバージョンを表示するには、ここをクリックしてください。</a>

<table><tbody><tr><td>10X PBS Buffer</td><td>GenDEPOT</td><td>P2100-104</td><td></td></tr><tr><td>4% Formalin</td><td>T&amp;amp;I</td><td>BPP-9004</td><td></td></tr><tr><td>Ampicillin</td><td>sigma&amp;nbsp;</td><td>A5354-10ml</td><td></td></tr><tr><td>BSA</td><td>sigma&amp;nbsp;</td><td>A4503-100G</td><td></td></tr><tr><td>confocal dish</td><td>SPL</td><td>200350</td><td></td></tr><tr><td>confocal microscope&amp;nbsp;</td><td>ZEISS</td><td>LSM800</td><td></td></tr><tr><td>coverslip</td><td>SPL</td><td>20009</td><td></td></tr><tr><td>DMEM/F12</td><td>Gibco</td><td>10565-018</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Fisher scientific</td><td>16140071</td><td></td></tr><tr><td>Fluorsheild with DAPI</td><td>sigma&amp;nbsp;</td><td>F6057</td><td></td></tr><tr><td>Forcep</td><td>Dumont</td><td>0508-L5-P0</td><td></td></tr><tr><td>HBSS</td><td>Thermo Fisher scientific</td><td>14065056</td><td></td></tr><tr><td>HEPES</td><td>Thermo Fisher scientific</td><td>15630080</td><td></td></tr><tr><td>N2 supplement</td><td>Gibco</td><td>17502-048</td><td></td></tr><tr><td>Phalloidin-iFluor 647 Reagent</td><td>abcam</td><td>ab176759</td><td></td></tr><tr><td>Stage Top Incubator</td><td>TOKAI HIT</td><td>WELSX</td><td></td></tr><tr><td>Strain C57BL/6 mouse messenchymal stem cells with GFP</td><td>cyagen</td><td>MUBMX-01101</td><td></td></tr><tr><td>Triton X-100</td><td>sigma&amp;nbsp;</td><td>T8787</td><td></td></tr></tbody></table>

in vitro time-lapse live-cell imaging to explore cell migration toward the organ of corti

間葉系幹細胞(MCs)が細胞再生および治療に及ぼす影響を調べ、人工内皮との共培養後のMSC移行および形態学的変化を追跡する。コルティの器官は、解剖中に発生したライスナーの膜の一部を押すことによってプラスチックカバースリップに固定化された。ガラスシリンダーで閉じ込められたMSCは、シリンダーが取り外されたときに人工内皮上皮に移行した。彼らの主な局在は、コルチの器官の透析で観察され、神経線維と同様の方向に整列した。しかし、いくつかのMSCは、手足の領域に局在し、水平に細長い形状を示した。また、毛細胞領域への移動が増加し、カナマイシン処理後にMSCの形態が様々な形態に変化した。結論として、この研究の結果は、人工内皮とのMSCの共培養が細胞移植による治療薬の開発および様々な条件および要因を調べることができる細胞再生の研究に有用であることを示している。

3次元モードにおけるMSCのインビトロ移行は、トランスウェルシステムまたは従来の創傷治癒法によって評価され、2次元(2D)モード11での移動を観察する。コルティの器官は、ボエッチャー細胞、クラウディウス細胞、Deiters細胞、ピラー細胞、ヘンセン細胞、外毛細胞、内毛細胞、神経線維、バジル膜、および網状層層などの様々な細胞から構成され?...

Watch this Scientific Journal Video about In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti at JoVE.com

In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti

本研究では、共焦点顕微鏡を用いて、コルチの器官を含む人工内皮上皮を用いてex vivoインキュベーションによって損傷組織に向かって移動する細胞を観察するリアルタイムイメージング法を提示する。

コルチの器官に向かう細胞の移動を探るインビトロタイムラプスライブセルイメージング

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after ...

in-vitro-time-lapse-live-cell-imaging-to-explore-cell-migration

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

3.7K ビュー.  Yonsei University Wonju College of Medicine. 本研究では、共焦点顕微鏡を用いて、コルチの器官を含む人工内皮上皮を用いてex vivoインキュベーションによって損傷組織に向かって移動する細胞を観察するリアルタイムイメージング法を提示する。

ビデオ: In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

三次元マトリックスの蛍光タグ付きタンパク質を発現する遊走細胞の生細胞イメージング

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

生物工学

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

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

分析<em&gt;インビボ</emゼブラフィッシュ胚における細胞移植とタイムラプスイメージングを使用して&gt;細胞移動

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

発生生物学

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

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

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 ...

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture can be recorded under video microscopy. For analyzing cell migration in the developing brain, slice culture is commonly used to observe cell migration parallel to the slice section, such as radial cell migration. However, limited information can be obtained from the slice culture method to analyze cell migration perpendicular to the slice section, such as tangential cell migration. Here, we present the protocols for time-lapse imaging to visualize tangential cell migration in the developing chick optic tectum. A combination of cell labeling by electroporation in ovo and a subsequent flat-mount culture on the cell culture insert enables detection of migrating cell movement in the horizontal plane. Moreover, our method facilitates detection of both individual cell behavior and the collective action of a group of cells in the long term. This method can potentially be applied to detect the sequential change of the fluorescent-labeled micro-structure, including the axonal elongation in the neural tissue or cell displacement in the non-neural tissue.

We describe the methods for fluorescent labeling of tangentially migrating cells by electroporation, and for time-lapse imaging of the labeled cell movement in a flat-mount culture in order to visualize migrating cell behavior in the developing chick optic tectum.

発展途上ひよこ視蓋接線細胞移動の可視化

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture ...

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 ...

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 ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

共焦点肉眼検査を使用した生後小脳顆粒細胞の移動のex vivoイメージング

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

胚性マウス脳の器官型スライス培養における移行ニューロンの経時的共焦点イメージング<em&gt;ウテロの</em&gt;エレクトロポレーション

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

Ex 子宮エレクトロポレーションと gaba 作動性介在ニューロンの移行のライブ イメージング用マウス萌芽期の脳の脊髄スライス培養

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

コルチの器官に向かう細胞の移動を探るインビトロタイムラプスライブセルイメージング

要約

さらに動画を探す

この動画の章

三次元マトリックスの蛍光タグ付きタンパク質を発現する遊走細胞の生細胞イメージング

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

分析

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

発展途上ひよこ視蓋接線細胞移動の可視化

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

マウス蝸牛外植片の長期的タイムラプスイメージング

共焦点肉眼検査を使用した生後小脳顆粒細胞の移動のex vivoイメージング

胚性マウス脳の器官型スライス培養における移行ニューロンの経時的共焦点イメージング

Ex 子宮エレクトロポレーションと gaba 作動性介在ニューロンの移行のライブイメージング用マウス萌芽期の脳の脊髄スライス培養

コルチの器官に向かう細胞の移動を探るインビトロタイムラプスライブセルイメージング

要約

さらに動画を探す

この動画の章

三次元マトリックスの蛍光タグ付きタンパク質を発現する遊走細胞の生細胞イメージング

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

分析

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

発展途上ひよこ視蓋接線細胞移動の可視化

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

マウス蝸牛外植片の長期的タイムラプスイメージング

共焦点肉眼検査を使用した生後小脳顆粒細胞の移動のex vivoイメージング

胚性マウス脳の器官型スライス培養における移行ニューロンの経時的共焦点イメージング

Ex 子宮エレクトロポレーションと gaba 作動性介在ニューロンの移行のライブ イメージング用マウス萌芽期の脳の脊髄スライス培養

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