我々は、miR画像のためのこの映画とM. Nicolescuの初期段階に貢献したK. Mikicを認めている。 C.フーバーはUniverstitätsklinikumテュービンゲンのFORTUNEプログラムによるUniverstitätsklinikumテュービンゲンIZKF、A.アルウィンプレムアナンドの交わりによってサポートされていました。

1。 でのOVOエレクトロポレーションのための要件<ol><li> ベクター構築の準備：のmiRセンスおよびアンチセンスプライマーは、Ambion社の指示の後に設計をアニールおよびApaI / EcoRIでに連結したのpSilencer U6.1ベクトルを消化。成功した形質転換後、得られたクローンの一つは、成長したとのpSilencerプラスミドをキアゲンミディ調製キットを用いてアルカリ溶解法によって単離した。 </li><li> 電極：電極を購入することができたり、簡単13を構築すること。我々は電気穿孔したい地域によっては8より小さなタングステンワイヤ電極（Φ= 0.1 mm）を自作白金線電極（Φ= 0.2、0.25、または直径0.5mm）を使用するか、またはいくつかのケースでは。組織の灼熱のを回避するために、我々は組織電極シンナーに近いルールとして使用しています。中脳の広範なエレクトロポレーションのために、我々は真鍮/ステンレスチューブ状に半田付け0.5ミリメートルの白金線を使用シャフト、3〜5 mmでの距離（ 図1）と電極ホルダに固定されている。特定の領域をエレクトロポレーションするために、我々は、0.25ミリメートル白金線アノード0.2mmの白金線又は1mm以下のおおよその距離とカソード用の0.1mmのタングステンを使用する。 </li><li> 解決策：次の解決策を用意しておいてください。 <ol><li>オートクレーブしたチキンリンガー、pHは7.5（9.0グラムのNaCl、0.42グラムのKCl、0.24グラムの塩化カルシウムを、1リットルの蒸留水に溶解する）。 </li><li> PBS（8gのNaCl、0.2gのKCl、0.24グラムのKH 2 PO4、1.44グラムのNa 2 HPO 4、1リットルの蒸留H 2 Oに溶解する）。 </li><li>ファストグリーン：ストック溶液10 mg / mlの蒸留水</li><li>遅乾性インク（少ないシェラック、したがって胚にはあまり中毒）。注：カント·ソレルとアドラー14に記載のように、光ファイバ·ランプに取り付けられた青色ダイクロイックフィルタは、コントラストを高め、インクを注入する必要性をなくすことができる。 </li></ol></li></ol><img src="/files/ftp_upload/50024/50024fig1.jpg" fo:content-width="6in" fo:src="/files/ftp_upload/50024/50024fig1highres.jpg"/> 図1。電極と電極ホルダーの模式図。 （A、B、C）白金ワイヤはステンレススチール管状シャフトの一側に半田付けした。穴は、このようにそのピンプラグ（A）は赤と青のカラーワイヤがピンプラグに接続されていたインチ合うことができる管状シャフトの反対側に開けた。反対側の端にワイヤがエレクトロの電気ソケットを扱うバナナコネクタに装着した。（A、C）白金線は、90°の天使に折り曲げた。マニキュアの層は曲がり上記シャフトを単離した。 2。直接には、OVOエレクトロポレーション前の準備<ol><li>次の材料を準備します。 <ol><li>チキンリンガー1時06インクを希釈、Gibcから抗生物質 - 抗真菌溶液（100Xを追加O）1:100。 </li><li>ファストグリーン（1:20）を補充し、H 2 O中の特定のDNAあたり1〜2μgの/μL：ベクター溶液を準備します。 </li><li> 70％エタノールで、鉗子、はさみ、タングステン針と電極を滅菌する。 </li><li>ホウケイ酸ガラスキャピラリー（100ミリメートル×0.9ミリメートル、外径までの長さ）から核酸注入にマイクロピペットを引き出します。私たちは、 表1に示した設定で簡単なプラーPUL-1（WPI、ベルリン）を使用します。 </li><li> 表2に示すように、エレクトロのパラメータを設定します。 </li></ol></li></ol><table border="1" ><tbody><tr><td fo:width="1in"> パラメータ </td><td fo:width="1in"> 値 </td></tr><tr><td>熱</td><td> 350 </td></tr><tr><td>引っ張る</td><td> 100 </td></tr><tr><td>速度</tD&gt; <td> 50 </td></tr><tr><td>遅延</td><td> 200 </td></tr></tbody></table> 表1。 PUL-1の設定 <table border="1"><tbody><tr><td fo:width="1.5in"> パラメータ </td><td fo:width="1.5in"> 値 </td></tr><tr><td>周波数</td><td> 50 Hzの</td></tr><tr><td>遅延</td><td> 20から50ミリ秒* </td></tr><tr><td>幅</td><td> 20ミリ秒</td></tr><tr><td>電圧</td><td> 8-25のV * </td></tr><tr><td>パルス</td><td> 3から5倍</td></tr><tr><td colspan="2"> *電極の直径及び距離に応じ。どうぞいずれかのパラメータを変更する際にそれを考慮。 </td></tr></tbody></table> 表2。パルス刺激装置の設定 <ol start="2"><li>エレクトロポレーションのためのニワトリ胚を準備します。 <ol><li>ハンバーガー＆ハミルトン（HH）の間の胚を取得するために39時間、65％の湿度で37℃で、その長い側のニワトリ受精卵を培養するには、15 9月11日ステージ。胚は、卵子の最上部に落ち着くで登録して、鉛筆のラインでそれをマークする必要があります。 </li><li> 70％エタノールで卵を消毒。 </li><li>エアキャビティがどこにあるべきか、そのより広い側面に卵を貫通。 </li><li>卵白1〜2mlのを削除します。これは卵の殻から胚を切り離します。 </li><li>セロテープ胚に落下シェル破片を避けるために卵殻。鉛筆の線を中心に卵殻に小さなウィンドウをカットするはさみを使用してください。 </li><li>インクのビットを注入胚を可視化する暗域の下に。 </li><li>核酸注入およびエレクトロポレーションのための胚のより良いアクセシビリティを実現するために、鋭利なタングステン針と卵黄膜にカットをする。 </li></ol></li></ol> 3。エレクトロポレーションおよびインキュベーション<ol><li>胚上に暖かいリンゲル液を数滴を追加します。これは、胚を下げる組織への電極の過熱、粘着を防止し、両電極間の電流のための媒体を提供する。 </li><li>中脳レベルでの神経管の内腔にベクター溶液を注入する。あなたのDNA発現ベクターはなく、レポーター遺伝子（ 例えば、EGFP）が含まれていない場合は、DNA溶液にわずかに低い濃縮レポーター遺伝子ベクター（0.9μgの/μlのレポーター遺伝子ベクターにベクターを発現する1μgの/μlでのmiR）を加える。これは、後で8トランスフェクトした細胞を認識することを保証します。我々は、いわゆるpCAXベクトル、あちこちの親切な贈り物を使用するC.クルル13メートル。 </li><li>中脳の広範なトランスフェクションのために、中脳の高さで、胚の左側電極と右側に配置します。電極間の距離は3〜5 mmでなければなりません。他の設定は15V、20ミリ秒パルスとHH 10のため50ミリ秒の遅延である。少し中脳の背腹側（DV）軸に沿って2つの電極の位置を変更しても、細胞のより広範なトランスフェクションのDV軸クションを保証します。 </li><li>腹側トランスフェクションのために、卵黄膜から頭を上げ、腹側中脳の下にアノードを配置するためにいくつかのリンガーを追加します。頭が上がらない場合は、神経管のような暑さを避けるために、卵黄と胚を分離する膜の下に電極を押してください。陰極は陽極に反対背側方に、中脳の上に配置する必要があります。神経管に触れないでください。我々は、心臓の開発にあらゆる困難を避けるために、中脳の左腹側半分をトランスフェクト。電極間距離周りの1〜0.5ミリメートル、残りの設定された10V、20ミリ幅と50ミリ秒の遅延がある。 </li><li>それを冷却し、気泡を除去するために胚の頂部に生理食塩水の別の降下を適用する。 </li><li>テープで卵を密封し、少なくとも4時間インキュベートする。ベクトル表現のために必要な最小時間。 </li></ol> 4。ニワトリ胚の固定<ol><li>その卵から胚を取り出します。 </li><li>実体顕微鏡下で蛍光実体顕微鏡を用いたトランスフェクションの程度を評価きれいな胚。 </li><li> 4℃で4％PFA中で一晩胚を修正しました。トランスフェクトエリアの変化を分析するために（in situハイブリダイゼーション 、 免疫組織化学）切片および/ ​​または染色を進める。 </li></ol>

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我々は、開示することは何もありません。

このビデオでは、ニワトリ脳の特定の領域の神経上皮細胞にプラスミドをトランスフェクトする効果的な方法を示しています。低電圧の長方形の電気パルスは、OVO 6,16 内のひよこ神経管の細胞にDNAを導入することができます。しかし、DNAのターゲティングの精度は、多くの場合、比較的大きな電極（Φ= 0.5mm）を通って上昇幅の電界によって妨げられる。私たちは、百瀬<e...

遺伝子発現のための追加的な選手のような小さな非コードRNAの認識は、ゲノムのプログラミング/遺伝子調節に新しい複雑さを立ち上げました。非コードRNAの異なる種は、小さな非コードRNA 1-4を含む神経細胞における機能的重要性を有する。例えばマイクロRNA（MIRまたはmiRNA）は、脳5の開発にはっきりと変化する発現プロファイルを示している。ニワトリ胚のOVOエレクトロポレーションで対象とすることは、開発中の遺伝子発現と抑制の時間的·空間的制御のためのユニークな機会を提供します。 このビデオでは、エレクトロポレーション6月10日 OVOで使用したニワトリ脳の特定の領域にmiRの異所性発現を実施する様々なステップを示しています。細胞におけるこれらの小さな非コードRNAの長期持続効果を確実にするために、miRのDNA配列は、モノ - 又はバイシストロニックベクターにクローニングした。 OVOエレクトロポレーション中の場合は、のmiRは、Vを含む注入器は、卵の殻に小さな窓を作った後に胚を曝露することにより、中脳、神経管に注入される。中脳の小さなプラス（アノード）とマイナス（カソード）の特定の領域をトランスフェクトするために白金電極は、特定の位置に配置されている。腹側中脳トランスフェクションのために、アノードは、左腹側中脳と電流を適用する前に、中脳の右半分上記カソードの下に配置されている。卵殻の開口部をテープで閉じられ、胚であれば、どのような分析に必要とされるようにインキュベートする。この方法は、もともと村松ら 6で説明した特定領域のトランスフェクションのため百瀬ら 8向上した。 <img src="/files/ftp_upload/50024/50024schematic1.jpg" fo:content-width-="6in" fo:src="/files/ftp_upload/50024/50024schematic1highres.jpg"/> 回路図の概要。 <ol><li>卵内の胚は、目に小さなウィンドウを切断することによって公開されているE卵殻。 </li><li>溶解したベクター（単数または複数）は、マイクロキャピラリーを用いて中脳に注入される。 </li><li> 2つの電極は - 並列配置された、または胚の下で、上 - パルス電界を発生させる。 </li><li>電界は、時間的に陽極11,12に引き付け負に荷電したDNA（またはRNA）による細胞への侵入を促進する細胞膜に孔を作成する。 </li></ol>

<table border="1">
<tbody>
 <tr>
 <td>Name </td>
 <td>Company</td>
 <td></td>
 <td>Model</td>
 </tr>
 <tr>
 <td>Borosillicate glass capillaries </td>
 <td>Hartenstein</td>
 <td></td>
 <td>Model: 0.9 mm</td>
 </tr>
 <tr>
 <td>Microcapillary puller</td>
 <td>WPI, Berlin</td>
 <td></td>
 <td>Model: Pul1-E</td>
 </tr>
 <tr>
 <td>Electroporator</td>
 <td>Intracel</td>
 <td></td>
 <td>Model: TSSIC</td>
 </tr>
 <tr>
 <td>Stereomicroscope - fluorescence </td>
 <td>LEICA</td>
 <td></td>
 <td>Model: MZFLIII</td>
 </tr>
 <tr>
 <td>Stereomicroscope</td>
 <td>Zeiss</td>
 <td></td>
 <td>Model: Stemi</td>
 </tr>
 <tr>
 <td>Camera and software</td>
 <td>Zeiss</td>
 <td></td>
 <td>Model: Axiocam MRc/ Axiovision Re. 4.8</td>
 </tr>
 </tbody>
</table>

in ovo expression of microrna in ventral chick midbrain

非コードRNAは、遺伝子発現を調節する追加のプレーヤーである。特定分野のOVOエレクトロポレーションで対象となるには、異所性マイクロRNA発現の空間的および時間的制御のためのユニークなツールを提供しています。しかし、腹側中脳のような腹脳構造は、任意の操作のために到達することはかなり困難である。ここでは、薄い白金電極を用いて、腹側中脳へのmiRNAを電気穿孔するための効率的な方法を示しています。この方法では、中脳とのin vivo研究のための有用なツールの特定の領域をトランスフェクトするために信頼性の高い方法を提供しています。

のmiRをトランスフェクトした脳領域の範囲は、miR発現するベクター（ 図2）と一緒に注入されたレポーターベクターからのGFPの発現を介して見ることができる。直径0.5mmの白金電極を用い、後脳、中脳、間脳、時には（ 図2AおよびB）を含む、DVおよびAP-軸に沿った広い領域のトランスフェクションに通常中脳リードのAP軸に平行に配置された。広い電界における電極結...

Watch this Scientific Journal Video about In ovo Expression of MicroRNA in Ventral Chick Midbrain at JoVE.com

In ovo Expression of MicroRNA in Ventral Chick Midbrain

異所性発現は、脳の発達におけるマイクロRNAの役割を解明する一つの技術である。しかし、OVOエレクトロポレーションに使用して特定の領域を標的とすることは困難である。ここでは、選択的に腹側と背側中脳領域を電気穿孔するための効率的な方法を示しています。

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 ...

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Research

JoVE Journal

Neuroscience

In ovo Expression of MicroRNA in Ventral Chick Midbrain

10.4K ビュー.  University of Tübingen. 異所性発現は、脳の発達におけるマイクロRNAの役割を解明する一つの技術である。しかし、OVOエレクトロポレーションに使用して特定の領域を標的とすることは困難である。ここでは、選択的に腹側と背側中脳領域を電気穿孔するための効率的な方法を示しています。 

ビデオ: In ovo Expression of MicroRNA in Ventral Chick Midbrain

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 ...

神経科学

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

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

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

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

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 ...

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators' choice.
We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein 1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA 6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.
We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).

Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.

黒質や腹側被蓋野のドーパミン規制の包括的なプロファイリング

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

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 ...

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

エレクトロポひよこ胚脊髄における遺伝子発現差のRNA-配列解析

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, interactions with our environment can induce brain plasticity. Increasingly, research is trying to identify which environments stimulate brain plasticity beneficial for treating brain and behavioral disorders. Two environmental manipulations are described which increase or decrease the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in dopamine (DA) synthesis) neurons in the adult mouse midbrain. The first comprises pairing male and female mice together continuously for 1 week, which increases midbrain TH+ neurons by approximately 12% in males, but decreases midbrain TH+ neurons by approximately 12% in females. The second comprises housing mice continuously for 2 weeks in &#8216;enriched environments&#8217; (EE) containing running wheels, toys, ropes, nesting material, etc., which increases midbrain TH+ neurons by approximately 14% in males. Additionally, a protocol is described for concurrently infusing drugs directly into the midbrain during these environmental manipulations to help identify mechanisms underlying environmentally-induced brain plasticity. For example, EE-induction of more midbrain TH+ neurons is abolished by concurrent blockade of synaptic input onto midbrain neurons. Together, these data indicate that information about the environment is relayed via synaptic input to midbrain neurons to switch on or off expression of &#8216;DA&#8217; genes. Thus, appropriate environmental stimulation, or drug targeting of the underlying mechanisms, might be helpful for treating brain and behavioral disorders associated with imbalances in midbrain DA (e.g. Parkinson&#8217;s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction).

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

成体マウスにおける中脳ドーパミンニューロンの数の環境モジュレーション

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

マウスにおける低温感受性と適応を決定するためのシンプルで安価な方法

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated &#34;tagmentation&#34; to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

In Parkinson&#39;s Disease (PD), Substantia Nigra (SNc) dopaminergic neurons degenerate, leading to motor dysfunction. Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a Tyrosine Hydroxylase (TH) promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their transcriptome using RNA-seq.

RNA配列決定およびTH-プロモーター駆動EGFP発現を使用して中脳培養における生活ドーパミン作動性ニューロンの信頼性の高い同定

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...

An In Ovo Model for Testing Insulin-mimetic Compounds

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and &#946;-cell failure. Various strategies, including the injection of insulin or the usage of insulin-sensitizing drugs, were pursued to treat T2DM or at least reduce the symptoms. In addition, the application of herbal compounds has attracted increasing attention. Thus, it is necessary to find efficient test systems to identify and characterize insulin-mimetic compounds. Here we developed a modified chick embryo model, which enables testing of synthetic compounds and herbal extracts with insulin-mimetic properties. Using a fluorescence microscopy-based primary screen, which quantifies the translocation of Glucose transporter 4 (Glut4) to the plasma membrane, we were able to identify compounds, mainly herbal extracts, which lead to an increase of intracellular glucose concentrations in adipocytes. However, the efficacy of these substances requires further verification in a living organism. Thus, we used an in-ovo approach to identify their blood glucose-reducing properties. The approval by an ethics committee is not needed since the use of chicken embryos during the first two-thirds of embryonic development is not considered an animal experiment. Here, the application of this model is described in detail.

An In Ovo Model for Testing Insulin-mimetic Compounds

In this study, we describe an in-ovo system as a promising tool to test insulin-mimetic compounds in a living organism. The main advantages include adequate throughput rates and acceptable costs, which enable the identification of phytochemicals with insulin-mimetic properties.

インスリン模倣化合物のテスト用のOvo でモデル

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and ...

Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a premise for determining the role of neuron types within a neural circuit, and their significance in information encoding relative to learning. Likewise, the method can be used to test the physiological significance of two or more connected brain regions in awake and anesthetized animals. The current study demonstrates how VTA glutamate neurons modulate the firing rate of putative pyramidal neurons in the CA1 (hippocampus) of anesthetized mice. This protocol employs adeno-associated virus (AAV)-dependent labeling of VTA glutamate neurons for the tracing of VTA presynaptic glutamate terminals in the layers of the hippocampus. Expression of light-controlled opsin (channelrhodopsin; hChR2) and fluorescence protein (eYFP) harbored by the AAV vector permitted anterograde tracing of VTA glutamate terminals, and photostimulation of VTA glutamate neuron cell bodies (in the VTA). High-impedance acute silicon electrodes were positioned in the CA1 to detect multi-unit and single-unit responses to VTA photostimulation in vivo. The results of this study demonstrate the&#160;layer-dependent distribution of presynaptic VTA glutamate terminals in the hippocampus (CA1, CA3, and DG). Also, the photostimulation of VTA glutamate neurons increased the firing and burst rate of putative CA1 pyramidal units in vivo.

The current protocol demonstrates a simple method for tracing of ventral tegmental area (VTA) glutamate projections to the hippocampus. Photostimulation of VTA glutamate neurons was combined with CA1 recording to demonstrate how VTA glutamate terminals modulate CA1 putative pyramidal firing rate in vivo.

海馬の腹側テグメンタル領域グルタミン酸末端のインビボ解剖学的および機能的なトレース

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a ...