我々はpCAG-mCherryが構築提供するために博士隆彦松田に感謝します。 YCMはSchweppe財団からのキャリア開発賞とホワイトホール財団からの助成金によってサポートされています。

1。電源の準備（ないビデオ） <ol><li> 1X PBSでペニシリン/ストレプトマイシン（ペン/連鎖球菌）の新鮮な1X希釈を準備します。以上50 mLのが必要です。 0.22μmのシリンジフィルターでろ過する。 </li><li>所望のプラスミドコンストラクトを組み合わせることにより、注入構造を準備します。我々は〜10μLの最後のボリュームに適切な化学量論でpCAG-フラグベクトル内のすべての構成要素を入れた。また、pCAG-GFP 14またはpCAG-mCherryは、トラッキングエレクトロポレーションの効率化とエレクトロポレーションした細胞を追加する必要があります。我々はこの実験のためにpCAG-mCherryを使用しています。最後に、ニワトリ胚midbrainsに注入効率を視覚化するために0.05％の最終濃度でプラスミドDNAの混合物に0.22μmのろ過ファストグリーン染料を追加します。 </li></ol> 2。卵の準備（ビデオで） <ol><li> 11胚を取得HHステージにするために、卵は25から40パーセントの湿度と受精後約41時間°F（37.8℃）100℃でインキュベートする必要があります。場所彼らの側に卵と鉛筆ダッシュそれぞれのトップをマークします。予め温めておいた、加湿インキュベーター中トレイを置きます。ゆっくりにインキュベータープラットフォームの回転機能を設定することによって、卵を揺する &quot;自動&quot;湿度と水のレベルを24時間ごとにチェックしてください。 </li><li>右の実験を開始する前に、インキュベーターから卵を除去し、室温のまま。これは、開発を遅らせると開発上のを防ぐのに役立ちます。 </li><li> 70％エタノールでそれぞれの卵の表面をきれいにし、キムワイプで拭いてください。 </li><li>スコッチテープの2つの4センチの部分を取り、鉛筆のダッシュ上に大きな四角形を作るために卵の上に並べて貼り付けます。卵に対してテープを滑らかにします。ウィンドウが行われる場所です。テープは、ウィンドウが開いているカットされたときに落ちるから卵殻の小片を防ぐことができます。 </li><li> 10 mLの注射器と18½卵のアルブミンのうち5 mLを描画するためのゲージの針を使用しています。邪魔しないようにするようなシェルを貫通した後、卵黄から離れて針を角度アルブミン除去しながら、または胚を損傷するおそれがあります。卵の間に70％エタノールで針を清掃しますが、針が次の卵を貫通する前に乾燥していることを確認してください。 </li></ol> 3。ガラスの注射針の準備（ビデオで） <ol><li>ヒート670、プル120、ヴェル、40時間165：注射針は、フレーミング/ブラウンマイクロピペットプラー（サッターインストゥルメント·モデルP-97）に次の設定を使用して作られています。世界精密機器（API）4インチ1.0ミリメートルの薄壁ガラスキャピラリー（API TW100F-4）針を作るために使用されています。 </li><li> P10ピペットおよび長期の先端キャピラリーローディングピペットチップ（エッペンドルフ5242956.003）を使用して、プルダウンガラス針をロードします。注入する方法を多くの卵に応じて、5〜10μLをロードします。気泡のないプラスミド溶液を連続的にカラムを作成し、徐々にガラス針を記入してください。 </li><li>マイクロマニピュレーター（ナリシゲMM3）とPicospritzer IIIインジェクターに接続された針ホルダーにロードされた針を配置します。 dissectiの下に針を見る顕微鏡で、緑色の液体が停止した時点で針をトリミングします。それは良い針を可視化するために暗い背景を使用するのに役立ちます。トリミング適切な針は練習が付属しています。 </li></ol> 4。中脳/神経管注入（ビデオで） <ol><li>卵2〜2.5センチメートル径ウィンドウを削減する春のはさみを使用しています。アルブミンを描画するときに行われた前回の針穴からウィンドウを起動し、カットのように手で卵を回転させます。卵の間に70％エタノールで飽和したキムワイプではさみをきれいにしてください。 </li><li>解剖顕微鏡下でのニワトリ胚を表示します。それは目を訓練する必要があるかもしれません。それはニワトリ胚の下にPBSで0.22μmのろ過20パーセント、インドのインクを注入して胚を可視化するために役立つことがあります。しかし、我々は、可能であれば、それが生存率を低下する傾向にあるようにこのステップは、避けるべきであることを発見した。 </li><li>胚は針のパスに平行に配置されている場合は注射針の距離から頭で、最適に動作します。徐々に穴を開ける中脳後脳接合〜45度の角度で神経管。それだけで前脳と後脳の領域に重要な拡散せずに、プラスミドDNAとファストグリーン色素で脳を表す神経小胞を埋めるために非常に重要です。 25ミリ秒に設定されPicospritzerインジェクターの期間で注入を開始、これは針がトリミングに応じて変更する必要があります。一般的には、10から50 msの間使用しています。 </li></ol> 5。エレクトロポレーション（ビデオで） <ol><li>注射針を取得します。場所胚における卵のPBS溶液で1Xペン/連鎖球菌の3滴。 </li><li> BTX EMC830エレクトロの設定を確認します。 <table border="1"><tbody><tr><td> 電圧： </td><td> 20 V </td></tr><tr><td>パルスの長さ： </td><td> 25ミリ秒</td></tr><tr><td> ＃パルス： </td><td> 3 </td></tr><tr><td> INTERVAL： </td><td> 500ミリ秒</td></tr><tr><td>極性： </td><tD&gt;ユニポーラ</td></tr></tbody></table></li><li>別に2 3ミリメートルの電極間の中央に座っている胚の中脳と神経管の底と同じ水平レベルで2ミリメートル長いL字型の白金電極を配置します。神経管の下部に電極を整列する腹側中脳の効率的なエレクトロポレーションのために重要です。脳特有のエレクトロポレーションを達成するために、我々は短い2ミリメートル長いのヒントを持つ2つのカスタムメイドのプラチナL字型の電極と、元のBTX電極を置き換えられます。これらの白金電極は0.5mm径の白金線（アルファAesar社）で作られています。これらの白金電極だけで十分な長さくらい前脳や後脳領域のをターゲットにせずに長さ約0.9ミリメートルであり、ステージ11ニワトリ胚の脳をカバーしようとしています。 BTXフットスイッチを一度押します。気泡が成功するたびにエレクトロポレーションの電極の周りに生成する必要があります。慎重に卵から電極を取り外してあるelectrを拭く70％エタノールで覆われたキムワイプでオフオード。胚で卵の1Xペン/連鎖球菌のソリューションは、他の3滴を配置します。 </li><li>明確な包装テープのは5cm x 5cmの部分でウィンドウをカバーしています。よく、その卵を密封するために卵の内容がテープに接触しないようにしてください。 </li></ol> 6。インキュベートと収穫<ol><li>インキュベーターに戻した卵を置きます。インキュベータープラットフォームの転換がOFFになっていることを確認してください。希望するHHステージ23が達成されるまで卵をインキュベートします。 </li><li>収穫は胚を生きていると、各条件の一般的なPBSを充填したペトリ皿に入れます。画面には、mCherry式でのみ胚を維持し、蛍光解剖顕微鏡下で胚を回収した。 </li><li> 24ウェルディッシュの個々のウェルに胚を移します。たて2％パラホルムアルデヒド（PFA）を1 mLの各ウェルを記入し、4℃で2-3時間を修正することができ℃の</li><li> 10分PBSで各固定胚3回洗浄します。場所胚順次I平衡化するまで、nは15％および30％ショ糖。胚は、当初はスクロースに浮かぶだろうが、平衡時にシンクします。 </li><li>各胚についてmCherry発現パターンとレベルでメモを取り、蛍光解剖顕微鏡下で胚を確認してください。 OCTで各胚を埋め込むことができます。それは、その後の解析（ 図1）に最適な脳の断面を生成するために適切な方向の胚に非常に重要です。 </li><li>ライカCM3050Sのクライオスタットを用いて厚さ18μmの脳切片を作製します。適切な細胞型マーカーで免疫染色することによって脳の発達とドーパミン作動性ニューロンへの分化を分析します。 </li></ol> 7。代表的な結果ニワトリ胚中脳をelectroporatingと分析の概略を図1に示されています。が正常に注入し、エレクトロポレーションニワトリ胚では、mCherry式は、さらなる開発の後脳領域に表示する必要がある（ <stronG&gt;図2A、B、C）。 mCherryは、他の共同エレクトロ遺伝子の発現と相関すると仮定すると、適切なmCherryの発現を示す胚はその後修正され、さらなる研究（ 図2D）は中脳の凍結切片を調製するために埋め込 ​​むことができます。一般的には、腹側中脳神経前駆細胞の約50％が効率的に上記のプロトコルをトランスフェクトすることができます。エレクトロポレーション神経前駆細胞におけるドーパミン作動運命の仕様は、チロシンヒドロキシラーゼ（TH）（ 図3）ドーパミンニューロンマーカーとmCherry、Flag標識遺伝子の共局在して調べることができます。腹側中脳前駆細胞ドメインのパターニングは、異なる前駆細胞ドメインのマーカーとの共同immnostainingの凍結切片を調査することができます。 不十分なmCherry式は、異所性の遺伝子がおそらく効率的に発現されないことを示唆している。この条件の下で胚はさらなる研究のために使用すべきではありません。同様に、胚のその実験手順の最後に生存しなかったデータの収集と分析に使用することはできません。 <img alt="図1" src="/files/ftp_upload/4017/4017fig1.jpg" /> 図1。OVOエレクトロポレーションアッセイにおけるニワトリ胚中脳のイラスト。 HHステージ11ニワトリ胚の胚（）は、注入プロセスを可視化するために、高速グリーンと混合して目的の遺伝子と一緒にpCAG-mCherryを注入されています。 DNA構築物を充填した後、midbrainsは、方形波のエレクトロとエレクトロポレーションされています。希望するHHステージ23に到達するまでの胚はさらに培養されています。その後mCherry陽性胚は、収穫を固定し、組み込み（B）されています。中脳の凍結切片は、図1Bの中の白線で示された平面を切断して調製し、そのようなチロシンヒドロキシラーゼ（TH）（C）のような脳のマーカーで免疫染色によって分析されています。 <a href="http://www.jove.com/files/ftp_upload/4017/4017fig1large.jpg" target="_blank">ここをクリック</a>拡大図を表示します。 <img alt="図2" src="/files/ftp_upload/4017/4017fig2.jpg" /> 図2ニワトリ胚中脳から凍結切片の調製。 41時間のインキュベーションの後、mCherryと関心のある遺伝子を発現するHHステージ23ニワトリ胚は、（A、BおよびC）が収穫されるPFAで固定し、ショ糖で処理し、セクショニングのOCTに包埋した。ニワトリ胚中脳切片の準備を確実にするために慎重に配向している。中脳切片を得るための典型的な胚の形態と切断面は、（D）が表示されます。 <a href="http://www.jove.com/files/ftp_upload/4017/4017fig2large.jpg" target="_blank">拡大図を表示するには、ここをクリックしてください</a> 。 <img alt="図3" src="/files/ftp_upload/4017/4017fig3.jpg" /> 図3エレクトロ脳切片の分析。 mCherryの発現レベルの高い胚から凍結切片（A）は不格好なやつを準備し、染色される興味のあるFlag標識遺伝子（B）と中脳ドーパミン作動性ニューロンマーカーTH（C）を認識IES。胚midbrainsはpCAG-mCherryでエレクトロとpCAG -フラッグ空のベクトルは、コントロールとして機能します。 <a href="http://www.jove.com/files/ftp_upload/4017/4017fig3large.jpg" target="_blank">拡大図を表示するには、ここをクリックしてください</a> 。

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著者らは、開示する潜在的な利害を持っていません。

ニワトリ胚中脳の卵エレクトロポレーションで中脳ドーパミン作動性ニューロンの開発に遺伝子機能のin vivo試験で実行するトランスジェニックやノックアウト動物の世代に、低コストかつ迅速な代替手段を提供しています。胚の中脳特異的DNAインジェクションと一緒に短い2ミリメートル長いL字型の白金電極を使用すると、中脳ドーパミン作動性ニューロン前駆細?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>受精鶏卵</td><td>チャールス·リバー</td><td></td><td></td></tr><tr><td>卵インキュベーター</td><td> GQF製造</td><td> 1502スポーツマン</td><td></td></tr><tr><td> BTXエレクトロ</td><td> BTXハーバード装置</td><td> ECM830 </td><td></td></tr><tr><td>電極</td><td> BTXハーバード装置</td><td> 45から0162 L字型genetrodes </td><td> ECM830エレクトロで使用するために</td></tr><tr><td>プラチナイリジウム線</td><td>アルファAesar社</td><td> ＃10056 </td><td>直径0.5mm 2ミリメートル長さの電極を作るため</td></tr><tr><td>ガラス毛細管</td><td>世界の精密機器</td><td> TW100F-4 </td><td></td></tr><tr><td> Microloaderピペットチップ</td><td>エッペンドルフ</td><td> 5242 956.003 </td><td></td></tr><tr><td>墨</td><td>ステープルズ</td><td></td><td>使用前に20％をフィルタリングする</td></tr><tr><td>微量注入器</td><td>パーカーオートメーション、 パーカー·ハネフィンの協力</td><td> Picospritzer III </td><td></td></tr><tr><td>蛍光解剖顕微鏡</td><td>ライカ</td><td> MZ16F </td><td></td></tr><tr><td>マイクロピペットプラー</td><td>サッターインストゥルメント</td><td> D-97 </td><td></td></tr></tbody></table>

in ovo electroporation in chick midbrain for studying gene function in dopaminergic neuron development

腹側中脳のコントロールの動きにありますドーパミン作動性ニューロン、感情的な動作、および報酬のメカニズム1-3。腹側中脳ドーパミン作動性ニューロンの機能不全は、パーキンソン病、統合失調症、うつ病、認知症1-5に関与している。したがって、中脳ドーパミン作動性ニューロンの分化の調節を研究することだけ脳の開発と神経前駆細胞の運命仕様を制御するメカニズムに重要な洞察を提供することができませんでしたが、また人間の神経疾患の様々を治療するための新たな治療戦略の開発に役立つ。 ドーパミン作動性ニューロンは、胚腹側中脳の脳室帯の内側を覆う神経前駆細胞から分化する。神経前駆細胞の開発は、6,7の遺伝子発現プログラムによって制御されます。ここでは、（目のハンバーガーハミルトン（HH）ステージ11の脳に特異的に遺伝子を発現するエレクトロポレーションを利用する技術を報告するirteen体節、42時間）ニワトリ胚8,9。ニワトリ胚の外部の開発は、後の発達時点10月13日で決定の影響で、特定胚の段階で便利な実験操作が可能になります。以前のHHステージ13（19体節、48時間）以上のニワトリ胚神経管、神経系の異なる細胞型に分化することができる多能性神経前駆細胞で構成されています。 CMVプロモーターおよびニワトリβ-アクチンエンハンサーの両方が含まれていますpCAGベクトルは、旗やニワトリ胚神経管14内の他のエピトープタグ付き構造体の強い発現が可能になります。本稿では、DNAは胚の中脳領域に特異的に構築し、どのように小さなカスタムメイドの電極をエレクトロポレーションを特定するために注入する方法など、胚の中脳ドーパミン作動性ニューロン前駆細胞に地域制限の遺伝子発現を達成するための特別措置を強調しています。 Cの分析後の段階で田舎者の脳は、脳開発のプラスミドベクターによるゲインの関数と機能喪失研究のための生体システムに優れた提供しています。実験システムの変更が運命マッピング解析を実行するために、遺伝子発現の調節を調べるための神経系の他の部分に分析を拡張することができます。

Watch this Scientific Journal Video about In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development at JoVE.com

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

機能や中脳ドーパミン作動性ニューロンの開発中の遺伝子の調節を評価するために、我々は、関与の方法について説明します。<emOVOで&gt;</emニワトリ胚腹側中脳ドーパミン作動性ニューロン前駆細胞にプラスミドDNAコンストラクト&gt;エレクトロポレーション。この手法は、脳の発達とドーパミン作動性ニューロンへの分化のさまざまな側面を研究する目的遺伝子の効率的な発現を達成するために使用することができます。

ドーパミン作動性ニューロンの開発における遺伝子機能を研究するためのニワトリ中脳でのOVOエレクトロポレーションに

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

in-ovo-electroporation-chick-midbrain-for-studying-gene-function

Research

JoVE Journal

Neuroscience

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

11.3K ビュー.  Children's Hospital of Chicago Research Center. 機能や中脳ドーパミン作動性ニューロンの開発中の遺伝子の調節を評価するために、我々は、関与の方法について説明します。エレクトロポレーション。この手法は、脳の発達とドーパミン作動性ニューロンへの分化のさまざまな側面を研究する目?...

ビデオ: In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

子宮内エレクトロポレーションは、皮質ニューロンのサブセットで、遺伝子機能の研究のために初代神経培養した

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

神経科学

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.
Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.
Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.
The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.
We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.

In this video, we demonstrate how to apply a conductance into a dopaminergic neuron recorded in the whole cell configuration in rat brain slices. This technique is called the dynamic clamp.

ダイナミッククランプ法を用いたラット中脳ドーパミン作動性ニューロンにおけるNMDA受容体のコンダクタンスのアプリケーション

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

によるマウス網膜神経節細胞のトランスフェクション in vivoでエレクトロポレーション

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

マウス子宮内でエレクトロポレーション：制御時空間遺伝子導入

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

胚腹側中脳の器官型スライス培養：ドーパミン作動性ニューロンの発達を研究するための体制 in vitroで</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial and temporal control of ectopic microRNA expression. However, ventral brain structures like ventral midbrain are rather difficult to reach for any manipulations. Here, we demonstrate an efficient way to electroporate miRNA into ventral midbrain using thin platinum electrodes. This method offers a reliable way to transfect specific areas of the midbrain and a useful tool for in vivo studies.

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Ectopic expression is one technique to elucidate the microRNAs role in brain development. However, targeting specific areas using in ovo electroporation is challenging. Here, we show an efficient way to selectively electroporate ventral and dorsal midbrain regions.

腹チック中脳マイクロRNAの発現OVO中

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

ヒト多能性幹細胞からドーパミン作動性ニューロンの分化を監督

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

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 ...

Studying Brain Function in Children Using Magnetoencephalography

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the human brain. MEG is quiet and less likely to induce claustrophobia compared with magnetic resonance imaging (MRI). It is therefore a promising tool for investigating brain function in young children. However, analysis of MEG data from pediatric populations is often complicated by head movement artefacts which arise as a consequence of the requirement for a spatially-fixed sensor array that is not affixed to the child's head. Minimizing head movements during MEG sessions can be particularly challenging as young children are often unable to remain still during experimental tasks. The protocol presented here aims to reduce head movement artefacts during pediatric MEG scanning. Prior to visiting the MEG laboratory, families are provided with resources that explain the MEG system and the experimental procedures in simple, accessible language. An MEG familiarization session is conducted during which children are acquainted with both the researchers and the MEG procedures. They are then trained to keep their head still whilst lying inside an MEG simulator. To help children feel at ease in the novel MEG environment, all of the procedures are explained through the narrative of a space mission. To minimize head movement due to restlessness, children are trained and assessed using fun and engaging experimental paradigms. In addition, children's residual head movement artefacts are compensated for during the data acquisition session using a real-time head movement tracking system. Implementing these child-friendly procedures is important for improving data quality, minimizing participant attrition rates in longitudinal studies, and ensuring that families have a positive research experience.

This article introduces a child-friendly research protocol designed to improve data quality by reducing head movement during pediatric magnetoencephalography (MEG). We familiarize families with the MEG environment, train children to remain still using an MEG simulator, and correct for residual head movement artefacts using a real-time head movement detection system.

子供の脳磁図を用いた脳機能の研究

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

坐骨神経クラッシュ損傷のラットモデルにおける機能的評価のための3D運動学的解析

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...