Name: biosensing motor neuron membrane potential in live zebrafish embryos
Uploaded: 2017-06-26T13:00:00
Description: Watch this Scientific Journal Video about Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos at JoVE.com

The authors would like to thank Simona Rodighiero for her priceless support with the FRET imaging analysis.

1. pHuC_Mermaidプラスミドの生成注：マーメイドは、 Ciona intestinalisの電圧感知ドメイン（VSD）をペアにして開発されたバイオセンサーです FRETパートナーフルオロフォアUmi-Kinoko Green（mUKG：ドナー）およびオレンジ発光蛍光タンパク質Kusabira Orange（mKOk：アクセプター）の単量体バージョンを用いて、ホスファターゼ（Ci-VSP）を含むCiona robusta ） <sup class="xre...

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	<li>Kn&#246;pfel, T., Gallero-Salas, Y., Song, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+voltage+indicators+for+large+scale+cortical+imaging+come+of+age.">Genetically encoded voltage indicators for large scale cortical imaging come of age.</a> Curr Opin Chem Biol. 27, 75-83 (2015).</li><li>Chemla, S., Chavane, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-sensitive+dye+imaging:+Technique+review+and+models.">Voltage-sensitive dye imaging: Technique review and models.</a> J Physiol Paris. 104 (1-2), 40-50 (2010).</li><li>Tsutsui, H., Karasawa, S., Okamura, Y., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+membrane+voltage+measurements+using+FRET+with+new+fluorescent+proteins.">Improving membrane voltage measurements using FRET with new fluorescent proteins.</a> Nat Methods. 5 (8), 683-685 (2008).</li><li>Benedetti, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=INaP+selective+inhibition+reverts+precocious+inter-+and+motorneurons+hyperexcitability+in+the+Sod1-G93R+zebrafish+ALS+model.">INaP selective inhibition reverts precocious inter- and motorneurons hyperexcitability in the Sod1-G93R zebrafish ALS model.</a> Sci Rep. 6, 24515 (2016).</li><li>Pennati, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+differences+between+larvae+of+the+Ciona+intestinalis+species+complex:+hints+for+a+valid+taxonomic+definition+of+distinct+species.">Morphological differences between larvae of the Ciona intestinalis species complex: hints for a valid taxonomic definition of distinct species.</a> PLoS ONE. 10 (5), 0122879 (2015).</li><li>Park, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+upstream+elements+in+the+HuC+promoter+leads+to+the+establishment+of+transgenic+zebrafish+with+fluorescent+neurons.">Analysis of upstream elements in the HuC promoter leads to the establishment of transgenic zebrafish with fluorescent neurons.</a> Dev Biol. 227 (2), 279-293 (2000).</li><li>Yuan, S., Sun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+mRNA+and+morpholino+antisense+oligonucleotides+in+zebrafish+embryos.">Microinjection of mRNA and morpholino antisense oligonucleotides in zebrafish embryos.</a> J Vis Exp. (27), (2009).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> J Vis Exp. (25), (2009).</li><li>Drapeau, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+locomotor+network+in+zebrafish.">Development of the locomotor network in zebrafish.</a> Prog Neurobiol. 68 (2), 85-111 (2002).</li><li>Brustein, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steps+during+the+development+of+the+zebrafish+locomotor+network.">Steps during the development of the zebrafish locomotor network.</a> J Physiol Paris. 97 (1), 77-86 (2003).</li><li>Drapeau, P., Ali, D. W., Buss, R. 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このプロトコールは、ゼブラフィッシュ胚脊髄運動ニューロンの電気的特性と、胚発生の約17hpfに現れ、24hpfまで持続する最も初期のステレオタイプ運動活性である自発的巻き取り行動との間の関連性を探索することを可能にした10 。 私たちのアプローチは、開発中の機能ネットワークにおける細胞間の相互作用の複雑さを完全に保ちながら、インタク?...

生体内の全身成分分析により、科学者は最も信頼性の高い方法で細胞の行動を調べることができます。膜電圧の変化が興奮性細胞間の伝達を促進する神経系のように、精査中の活性が細胞 - 細胞相互作用（接触および非接触依存）によって大きく影響を受ける場合、これは特に当てはまります。これらの電気信号によってコード化された情報の理解は、神経系が生理学的状態および疾患状態の両方で働く方法を理解するための鍵である。 最も非侵襲性の生理学的条件における細胞の電気的特性を研究するために、いくつかの遺伝的にコード化された電圧指示薬が最近開発されている1 。以前の世代の光電圧センサ（主に電圧感受性色素） 2とは対照的に、GEVIはインタクトな神経系の生体内解析を可能にし 、それらの発現は、特定の細胞型または集団に限定することができる。 ゼブラフィッシュの胚は、GEVIに起因する大きな潜在的可能性を利用するための生体内の 「基質」です。実際、ゼブラフィッシュモデルは、その光学的明瞭性と簡略化され、進化的に保存された神経系のおかげで、ネットワーク内のすべての細胞成分を簡単に同定し、操作することができます。実際、FRETに基づくGEVI Mermaid 3の採用により、筋萎縮性側索硬化症（ALS） 4のゼブラフィッシュモデルにおける脊髄運動ニューロン挙動の予兆的な変化が同定された4 。 以下のin vivoプロトコルは、ニューロン特異的様式でマーメイドを発現する完全なゼブラフィッシュ胚における脊髄運動ニューロンの電気的特性をどのように監視するかを記載する。また、どのように薬理学的に誘発されたchanこのような電気的性質の変化は、発達の非常に初期の段階でゼブラフィッシュの運動挙動を特徴付けるステレオタイプの運動活動である、胚性の自発的な巻き取りの頻度の変化に関連し得る。

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			<td>Leica Microsystems</td>
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			<td>Digital camera</td>
			<td>Leica Microsystems</td>
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			<td>Leica Application Suite 4.7.1 software&#160;</td>
			<td>Leica Microsystems</td>
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			<td>Microinjector&#160;</td>
			<td>Eppendorf&#160;</td>
			<td>Femtojet</td>
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biosensing motor neuron membrane potential in live zebrafish embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

FRETに基づくマーメイドバイオセンサーコード配列を有する発現ベクターは、pHuC汎ニューロンプロモーターの制御下で、神経系においてのみタンパク質の合成を駆動し、単一細胞の受精卵に、マイクロインジェクションによって送達された一過性のトランスジェニック胚を得るために（ 図1 、 左パネル）。マイクロインジェクション技術を?...

Watch this Scientific Journal Video about Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos at JoVE.com

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

生きたゼブラフィッシュ胚における運動ニューロン膜電位のバイオセンシング

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

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Unit 1

The Nervous System and Nervous Tissue

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Mechanical Vessel Injury in Zebrafish Embryos

ゼブラフィッシュ胚における機械的血管損傷

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

ゼブラフィッシュ胚からの単一細胞の単離とキャラクタリゼーション

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

ライブゼブラフィッシュ胚における有糸分裂とダイナミクスを観察します

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin ...

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

ゼブラフィッシュ胚からの内因性受容体の親和性ラベリング検出

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

熱ショック2処理とゼブラフィッシュ胚の倍数性マニピュレーション

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

生きた蛙およびゼブラフィッシュの胚における低酸素の誘導

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

ゼブラフィッシュ初期胚および腫瘍の細胞の電気的活動の可視化

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

大人のキューティクルをショウジョウバエ脚運動ニューロン軸索を可視化します。

The majority of work on the neuronal specification has been carried out in genetically and physiologically tractable models such as C. elegans, Drosophila ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

ゼブラフィッシュ胚と幼虫の全山免疫賦形化学

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography

ヘッドスペースガスクロマトグラフィーを用いたゼブラフィッシュ胚におけるエタノール濃度の定量

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies ...