この作品は、R.ブリュースター（1R01GM085290 - 01A1）に授与されたNIHの助成金によって支えられている。

1.Microinjection <ol><li>希釈をコードするプラスミド膜をターゲットとした緑色蛍光タンパク質（MGFP、リチャードハーランドの礼儀）40 ng / mlの濃度に。 DNAはキアゲンのマキシプレップキットを使用して精製されたプラスミド（リチャードハーランドのmGFP/PCS2 +、礼儀）を線形化することによって調製される。ソリューションへのフェノールレッドの加算は、（総容積1 / 10に希釈）ソリューションを可視化することが好ましい。氷の上にDNA溶液を保管してください。 </li><li>累進的なスライドに針を合わせ、静かにきれいなかみそりを使用して直径が1μm以下である針の部分をタップすることで、実体顕微鏡下で、注射針を（カタログ番号NA - GD - 1 Narishinge科学から90ミリメートルのガラスキャピラリー）キャリブレーションブレード（適当な大きさの先端を得るために）。 </li><li>ピース上に配置フェノールレッドと水を含む溶液の0.5μlを使用してニードルを記入し、フロント、知られているボリューム（2nl）を注入する針を較正するために、マイクロインジェクション装​​置（ハーバード大学の装置PCI 100マイクロインジェクター）に針を接続パラフィンの。解決策は、フットペダルを使用して省略することができる。 7 kPaに圧力を設定し、それが解決策の0.5μlディスペンス、250ペダルを取るように（10ミリ秒30msec）に変わる。 </li><li>加湿100ミリメートルペトリ皿の中のワックスのストリップに校正針を慎重に置きます。料理を加湿するために、エッジの周りの湿ったキムワイプを（これは針で溶液の蒸発を防ぐ）に置きます。 </li><li>針の端（キャリブレーションされていないエンド）へのDNAの0.5〜1μlをロードするためにマイクロピペットを使用してください。 </li><li>ソリューションは、針の先端に達すると、マイクロインジェクション装​​置にロードするために使用された針の端を接続します。フットペダルを使用して約2 NL /注入を実現するためにインジェクターの設定を調整します。 </li><li>胚の培地（1.0ミリリットルハンクス証券第1位、0.1ミリリットルハンクの株式＃2、1.0ミリリットルハンクの証券第4位で満たされたペトリ皿の中央にある次の、位置のゼブラフィッシュの胚、95.9ミリリットルのddH 2 O、1.0ミリリットルハンクス証券第5位、1.0 mlの新鮮なハンクの株式＃6、pH7.2に約10滴1 M NaOHを使用してください）​​。 </li><li>胚が目的の段階に開発が完了したら、軽く細かい鉗子で絨毛膜をつまんで、注射の対象となる領域に針を位置づける。広い範囲のMGFP式の場合は、卵黄または1セルの段階で細胞質にDNAを注入する。小さい領域にMGFPの発現を制限するには、単一のセルの細胞質中に4（またはそれ以上）の細胞の段階でDNAを注入する。 </li><li>マイクロインジェクション装​​置のフットペダルを押すと、約2 NLを提供する、ソリューション（フェノールレッドの添加による目に見える）が適切に注入されることが保証されます。 </li><li>慎重に胚から針を取り除く。 </li><li>ペトリ皿内のすべての胚について繰り返します。 </li><li>胚は28.5で、目的の段階に° Cを開発することができます。 </li><li>優しく細かい鉗子を（胚は、胚の生存率を増加させるガラスシャーレにdechorionating、24hpf未満である場合）を使用して絨毛膜を剥離することによりDechorionate胚。 </li><li>ガラスピペットを用いてペトリ皿から空の絨毛膜を取り外します。 </li></ol> 2。イメージングMGFP標識ビブラトームセクション<ol><li> 1Xリン酸緩衝液（PBS）で固定液（4％パラホルムアルデヒドに胚を輸送するためにガラスのピペットを使用してください。4℃で一晩胚を修正しました。 </li><li>各5分間、1 × PBSで3回の胚を洗浄してください。 </li><li>シャーレに胚を置くと、微細な鉗子を用いて、胚を損なうことなく、卵黄を削除します。できる限り多くの卵黄を削除します。 </li><li> H 2 Oで4％低融点アガロースを準備する</li><li>プラスチック切片金型内に溶融アガロースを置きます。 </li><li>ペトリ皿から注入された胚を持ち上げ、アガロース内の場所と向きにするために微細な鉗子を使用してください。胚は、金型の一方の端に向かって配置する必要があります。 </li><li>アガロースが硬化されると、金型からアガロースブ​​ロックを削除するには、そのようなヘラのように、平面のツールを使用してください。 </li><li>それが偶数になるようにかみそりの刃を持つアガロースブ​​ロックの下端をカット。 </li><li>ビブラトーム舞台にスーパー接着剤のドロップを追加し、その上にアガロースブ​​ロック（剃刀で横ばいしたブロックの側面に接着剤を直視しなければならない）を置きます。複数の胚を切片の場合は、ステージ上にいくつかのアガロース包埋標本をマウント。 </li><li>ビブラトーム（ビブラトーム社）にビブラトームステージを取り付けます。 </li><li>ビブラトームのパラメータを設定します（一般的な設定：速度-3、厚さ50μmの）。複数の胚をセクショニングの場合、ステージ上でアガロースブ​​ロックを合わせ、ブレードの全胚経由ではなく、アガロースブ​​ロック（同じ胚に対応するセクションが一緒に残ることなど）の全体幅を通過することを確認してください。 </li><li>ビブラトーム段階での1X PBSを注ぐ。 </li><li>セクション。 </li><li>胚は、切開されるとNED、ビブラトーム段階からアガロースブ​​ロック（複数可）を削除します。 </li><li>慎重にアガロースブ​​ロック（複数可）の後ろを切断することにより、各セクションを区切ります。 </li><li> 1X PBSを充填したマルチウェルディッシュ（別の胚に対応するセクションに別の井戸を使用）に必要なセクションと場所を選択するために微細な鉗子を使用してください。 </li><li>各胚について、この手順を繰り返します。 </li><li>抗体標識を続行、MGFPイメージング（セクションは準備の一日か二日以内にイメージングされた場合は、この手順が不要な場合もあります）高めるために、23も19を繰り返します。 </li><li>非特異的結合をブロックするために1 × PBSでブロッキング溶液で、室温で30分間のセクションを扱います。 </li><li>一次抗体（ウサギA - GFP、Invitrogen社カタログ番号A11122）4、5、室温で数時間または一晩シェーカー上でブロッキング溶液で@ 1:200に希釈℃の切片をインキュベートします</li><li> 4回（10分、15分、30分および60分、各）1％BSA/1X PBS / 1％DMSO溶液で洗浄する。 </li><li> 4℃、室温または一晩で5時間ブロッキング溶液に適当な二次抗体を持つセクションを扱う℃に</li><li> 4回（10分、15分、30分および60分、各）1％BSA/1X PBS / 1％DMSO溶液で洗浄する。 </li><li>核を染色するために30分間、DAPI（Invitrogen社カタログ番号D1306 50ミリリットル1X PBSで0.1％DAPI）を使用してセクションを扱う。 </li><li> 5分間ずつ1X PBSで3回洗浄する。 </li><li>スライド上にマウントします。 </li><li>共焦点顕微鏡を使用してイメージ。 </li></ol> 3。ライブイメージング<ol><li>ライブDechorionate、それらが所望の発達段階に到達する30分前に胚を注入した。 15分間鎮静剤溶液中での胚（胚の培地中0.01％Tricaine）を置きます。 </li><li> （;品番P35G - O - 20 - Cマテック）35ミリメートルのガラス底の培養皿上に1％アガロースの低下を置きます。 </li><li>アガロースを固化することができます。 </li><li>アガロースの3つの穴を（そのようなマイクロピペットの先端のような他のオブジェクトは穴を作るために使用されることがあります）ように炎上熱毛細管。残存アガロースは、穴の底ではないことを確認してください。 </li><li>胚培地でシャーレを埋める。 </li><li>穴に胚を配置するガラスピペットを使用してください。 </li><li>次に、オリエント胚は（背側頭部）の領域がイメージを作成するように微細な鉗子を使用して、ガラスと接触している。 </li><li>注意深く顕微鏡ステージ（蒸発を防ぐために、ペトリ皿上に蓋を置く）にペトリ皿を運ぶ。 </li><li>イメージングしながら、胚は℃、28.5になっていることを確認してください</li></ol> 4。結果ここでは、モザイクの表現を用いてゼブラフィッシュ神経管の画像単一細胞への直接的なアプローチを説明します。ゼブラフィッシュ胚の光透過性にもかかわらず、我々は神経細胞の可視化が大幅にビブラトームを用いて得られた断面に増強されることを見出した。これらのセクションでは、共焦点顕微鏡を用いて異なる焦点面内の特定の細胞から細胞の拡張子のイメージングを可能にする（50 mm）の厚さです。この方法論を使用して結果を別の場所で2公開されているが、WT胚（図1）とN -カドヘリン（N - CAD）変異体（図2）の神経管の細胞の代表的な画像が提供されています。後者は、N - CADの変異体は正中線（図2）に向けて適切に方向付けるために失敗に神経板の外側の領域でその細胞を明らかにする。このモザイクのラベリングとは対照的に、このようなβ-カテニンなどの一般的な細胞表面マーカーと上皮を免疫染色すると、個々の細胞の形態は（図3）可視化することはできません。 ライブ胚のタイムラプスイメージングが神経胚形成の間に起こる細胞のダイナミクスの理解を可能にする、固定標本のモザイクのラベルを補完。ムービー1はWT細胞が積極的に内方に向いた膜の突起を拡張することで、正中線に向かって移行することを明らかにした。 <img src="/files/ftp_upload/1976/1976fig1.jpg" alt="図1" /> WTゼブラフィッシュ胚の神経管に図1。MGFP式。 <img src="/files/ftp_upload/1976/1976fig2.jpg" alt="図2" /> N -カドヘリンのゼブラフィッシュ胚の神経管2。MGFP式図 。 <img src="/files/ftp_upload/1976/1976fig3.jpg" alt="図3" /> 神経上皮の図3。B -カテニン免疫染色。

<ol>
	<li>N&#252;sslein-Volhard, C., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish Practical approach series. , (2002).</li><li>Hong, E., Brewster, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-cadherin+is+required+for+the+polarized+cell+behaviors+that+drive+neurulation+in+the+zebrafish.">N-cadherin is required for the polarized cell behaviors that drive neurulation in the zebrafish.</a> Development. 133, 3895-3905 (2006).</li><li>Andersen, E., Asuri, N., Clay, M., Halloran, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Imaging+of+Cell+Motility+and+Actin+Cytoskeleton+of+Individual+Neurons+and+Neural+Crest+Cells+in+Zebrafish+Embryos.">Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos.</a> J Vis Exp. , (2010).</li></ol>

利害の衝突は宣言されません。

結論では、標識技術は、ゼブラフィッシュ胚における形態形成のプロセスの単一細胞の分析を可能にするここで説明する。このプロトコルの主な重点は、ユビキタスプロモーターの制御下MGFPを使用して、神経管のイメージング標識細胞のための方法になります。この一過性発現アッセイのリーダーの追加のアプリケーション用にアンデルセンらによる最近の論文を参照する必要があり...

A correction was made to <a href="http://www.jove.com/index/Details.stp?ID=1976">Labeling and Imaging Cells in the Zebrafish Hindbrain</a>. There was an error in the authors affiliations. The authors have been corrected to:
Pradeepa Jayachandran1, Elim Hong2, Rachel Brewster1 1Department of Biological Sciences, University of Maryland, Baltimore County 2Center for Neuroscience, Children's National Medical Center
instead of:
Pradeepa Jayachandran1, Elim Hong2, Rachel Brewster2 1Center for Neuroscience, Children's National Medical Center 2Department of Biological Sciences, University of Maryland Baltimore County

A correction to the author affiliations has been made for the article Labeling and Imaging Cells in the Zebrafish Hindbrain.

Erratum: Labeling and Imaging Cells in the Zebrafish Hindbrain

ソリューション<ul><li>ハンクの株式第1位：8.0グラムのNaClを100ml DD H 2 Oで0.4グラムのKCl </li><li>ハンクの株式＃2：0.358グラムのNa 2 HPO 4、無水、100ミリリットルのddH 2 Oで0.60グラムのKH 2 PO 4 </li><li>ハンクの証券第4位：50ミリリットルのddH 2 Oで0.72グラムのCaCl 2 </li><li>ハンクの株式第5位：50ミリリットルDD H 2 Oで1.23グラムのMgSO 4 x7H 2 O </li><li> 1 Xリン酸緩衝液：0.8％のNaCl、0.02％のKCl、0.02 M PO4、pH7.3に</li><li>ブロッキング溶液：2％正常ヤギ血清、1％ウシ血清アルブミン（BSA）、1％dimethysulfoxide（DMSO） </li></ul>

labeling and imaging cells in the zebrafish hindbrain

初期の脊椎動物の胚を形成する形態形成のプロセスを理解するための鍵は、高解像度での画像セルにできることです。ゼブラフィッシュ胚、モザイク式の中のプラスミドDNAの結果の注入は、単一の細胞または細胞の小さなクラスターの可視化を可能に<sup&gt; 1</sup&gt;。我々は、ユビキタスプロモーターの制御下でプラスミドDNAのエンコーディング膜をターゲットとする緑色蛍光タンパク質（MGFP）の注入は神経胚形成を受けているイメージング細胞に使用できる方法について説明します。このプロトコルの中心は、セクションでもリアルタイムで高分解能でイメージング標識された細胞のための方法論です。このプロトコルは、若いゼブラフィッシュ胚にMGFPのDNAの注入を必要とする。胚はその後ビブラトーム切片、抗体標識と共焦点顕微鏡によるイメージングのために処理されます。また、MGFPを表現するライブ胚をタイムラプス共焦点顕微鏡を用いて画像化することができます。我々は以前、ゼブラフィッシュ胚の後脳領域で神経管の形成を駆動する細胞の挙動を分析するためにこの簡単なアプローチを使用している<sup&gt; 2</sup&gt;。ライブイメージングは​​、神経胚形成の間に起こる細胞のダイナミクスの理解を可能にするこのアプローチを補完しながら神経管の細胞の形状や組織の前例のない可視化のために許容される固定の準備。

Watch this Scientific Journal Video about Labeling and Imaging Cells in the Zebrafish Hindbrain at JoVE.com

Labeling and Imaging Cells in the Zebrafish Hindbrain

初期胚を形成する形態形成のプロセスを理解するための鍵は、高解像度での画像セルにできることです。ここでは、細胞膜をターゲットとする緑色蛍光タンパク質で全体のゼブラフィッシュの胚では細胞の単一細胞または小さなクラスターを標識するためのテクニックを説明します。

ゼブラフィッシュ後脳における標識およびイメージング細胞

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

labeling-and-imaging-cells-in-the-zebrafish-hindbrain

Research

JoVE Journal

Neuroscience

11.2K ビュー.  University of Maryland, Baltimore County. 初期胚を形成する形態形成のプロセスを理解するための鍵は、高解像度での画像セルにできることです。ここでは、細胞膜をターゲットとする緑色蛍光タンパク質で全体のゼブラフィッシュの胚では細胞の単一細胞または小さなクラスターを標識するためのテクニックを説明します。

ビデオ: Labeling and Imaging Cells in the Zebrafish Hindbrain

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 ...

神経科学

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

無傷の感覚器官の前駆細胞の生細胞イメージングショウジョウバエ蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

ニワトリ胚における軸索軌道とシナプスターゲットを追跡する後脳のエレクトロ

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

の中枢神経系における単一細胞のラベリング<em&gt;キイロショウジョウバエ</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

蛍光色素で大人のゼブラフィッシュの網膜神経節細胞の逆行性標識

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

The Mouse Hindbrain As a Model for Studying Embryonic Neurogenesis

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded forebrain neuroepithelium is not amenable to wholemount analysis to examine organ-wide neurogenesis patterns. Moreover, defining the mechanisms of forebrain neurogenesis is not necessarily predictive of neurogenesis in other parts of the brain; for example, due to the presence of forebrain-specific progenitor subtypes. The mouse hindbrain provides an alternative model for studying embryonic neurogenesis that is amenable to wholemount analysis, as well as tissue sections to observe the spatiotemporal distribution and behavior of neural progenitors. Moreover, it is easily dissected for other downstream applications, such as cell isolation or molecular biology analysis. As the mouse hindbrain can be readily analyzed in the vast number of cell lineage reporter and mutant mouse strains that have become available, it offers a powerful model for studying the cellular and molecular mechanisms of developmental neurogenesis in a mammalian organism. Here, we present a simple and quick method to use the mouse embryo hindbrain for analyzing mammalian neural progenitor cell (NPC) behavior in wholemount preparations and tissue sections.

This article demonstrates how the mouse embryonic hindbrain can be used as a model for studying developmental neurogenesis in both whole organ and tissue section preparations.

萌芽期の神経を研究するためのモデルとしてのマウス脳

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

ゼブラフィッシュ稚魚における側線有毛細胞の生体内カルシウム イメージング

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

シアログリカンの代謝標識による原発神経幹および前駆細胞の細胞表面マーカーの同定

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). Therefore, detection of the presence of A&#946; in AD brain tissue is a valuable tool for developing new treatments to prevent the progression of AD. Several classical amyloid binding dyes, fluorochrome, imaging probes, and A&#946;-specific antibodies have been used to detect A&#946; histochemically in AD brain tissue. Use of these compounds for A&#946; detection is costly and time consuming. However, because of its intense fluorescent activity, high-affinity, and specificity for A&#946;, as well as structural similarities with traditional amyloid binding dyes, curcumin (Cur) is a promising candidate for labeling and imaging of A&#946; plaques in postmortem brain tissue. It is a natural polyphenol from the herb Curcuma longa. In the present study, Cur was used to histochemically label A&#946; plaques from both a genetic mouse model of 5x familial Alzheimer&#39;s disease (5xFAD) and from human AD tissue within a minute. The labeling capability of Cur was compared to conventional amyloid binding dyes, such as thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC), as well as A&#946;-specific antibodies (6E10 and A11). We observed that Cur is the most inexpensive and quickest way to label and image A&#946; plaques when compared to these conventional dyes and is comparable to A&#946;-specific antibodies. In addition, Cur binds with most A&#946; species, such as oligomers and fibrils. Therefore, Cur could be used as the most cost-effective, simple, and quick fluorochrome detection agent for A&#946; plaques.

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

天然ポリフェノールクルクミンを用いた脳組織におけるアミロイドプラークの標識とイメージング

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...

In vivo Imaging of Fully Active Brain Tissue in Awake Zebrafish Larvae and Juveniles by Skull and Skin Removal

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at cellular and subcellular resolution. Advances in molecular and imaging technologies have allowed us to gain numerous detailed insights into cellular and molecular mechanisms of brain development in the transparent zebrafish embryo. Recently, processes of refinement of neuronal connectivity that occur at later larval stages several weeks after fertilization, which are for example control of social behavior, decision making or motivation-driven behavior, have moved into focus of research. At these stages, pigmentation of the zebrafish skin interferes with light penetration into brain tissue, and solutions for embryonic stages, e.g., pharmacological inhibition of pigmentation, are not feasible anymore.
Therefore, a minimally invasive surgical solution for microscopy access to the brain of awake zebrafish is provided that is derived from electrophysiological&#160;approaches. In teleosts, skin and soft skull cartilage can be carefully removed by micro-peeling these layers, exposing underlying neurons and axonal tracts without damage. This allows for recording neuronal morphology, including synaptic structures and their molecular contents, and the observation of physiological changes such as Ca2+ transients or intracellular transport events. In addition, interrogation of these processes by means of pharmacological inhibition or optogenetic manipulation is feasible. This brain exposure approach provides information about structural and physiological changes in neurons as well as the correlation and interdependence of these events in live brain tissue in the range of minutes or hours. The technique is suitable for in vivo brain imaging of zebrafish larvae up to 30 days post fertilization, the latest developmental stage tested so far. It, thus, provides access to such important questions as synaptic refinement and scaling, axonal and dendritic transport, synaptic targeting of cytoskeletal cargo or local activity-dependent expression. Therefore, a broad use for this mounting and imaging approach can be anticipated.

Here we present a method to image the zebrafish embryonic brain in vivo upto larval and juvenile stages. This microinvasive procedure, adapted from electrophysiological approaches, provides access to cellular and subcellular details of mature neuron and can be combined with optogenetics and neuropharmacological studies for characterizing brain function and drug intervention.

目覚めしたゼブラフィッシュ幼虫と頭蓋骨と皮膚除去による若年組織の完全に活性な脳組織のインビボイメージング

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at ...