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>
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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>

特別な利害関係は宣言されません。

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?...

<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

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 ...

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 ...

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 ...

Measurement of Oxygen Consumption Rate in Acute Striatal Slices from Adult Mice

Mitochondria play an important role in cellular ATP production, reactive oxygen species regulation, and Ca2+ concentration control. Mitochondrial dysfunction has been implicated in the pathogenesis of multiple neurodegenerative diseases, including Parkinson&#39;s disease (PD), Huntington&#39;s disease, and Alzheimer&#39;s disease. To study the role of mitochondria in models of these diseases, we can measure mitochondrial respiration via oxygen consumption rate (OCR) as a proxy for mitochondrial function. OCR has already been successfully measured in cell cultures, as well as isolated mitochondria. However, these techniques are less physiologically relevant than measuring OCR in acute brain slices. To overcome this limitation, the authors developed a new method using a Seahorse XF analyzer to directly measure the OCR in acute striatal slices from adult mice. The technique is optimized with a focus on the striatum, a brain area involved in PD and Huntington&#39;s disease. The analyzer performs a live cell assay using a 24-well plate, which allows the simultaneous kinetic measurement of 24 samples. The method uses circular-punched pieces of striatal brain slices as samples. We demonstrate the effectiveness of this technique by identifying a lower basal OCR in striatal slices of a mouse model of PD. This method will be of broad interest to researchers working in the field of PD and Huntington&#39;s disease.

Oxygen consumption rate (OCR) is a common proxy for mitochondrial function and can be used to study different disease models. We developed a new method using a Seahorse XF analyzer to directly measure the OCR in acute striatal slices from adult mice that is more physiologically relevant than other methods.