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
Summary
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.
Abstract
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).
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors would like to thank Simona Rodighiero for her priceless support with the FRET imaging analysis.
Materials
| Low Melting Point Agarose | Sigma-Aldrich | A9414 |
| DMSO | Sigma-Aldrich | W387520 |
| Riluzole | Sigma-Aldrich | R116 |
| Pfu Ultra HQ DNA polymerase | Agilent Technologies – Stratagene Products Division | 600389 |
| T3 Universal primer | Sigma-Aldrich | |
| Wizard SV Gel and PCR Clean-Up system | Promega | A9280 |
| Universal SmaI primer | Eurofins | |
| StrataClone Mammalian Expression Vector System / pCMV-SC blunt vector | Agilent Technologies – Stratagene Products Division | 240228 |
| SmaI | New England Biolabs | R0141S |
| T4 DNA ligase | Promega | M1801 |
| SalI | New England Biolabs | R0138S |
| EcoRV | New England Biolabs | R0195S |
| 35 mm, glass-bottomed imaging dish | Ibidi | 81151 |
| forceps | Sigma-Aldrich | F6521 |
| Stereomicroscope | Leica Microsystems | M10 F |
| Digital camera | Leica Microsystems | DFC 310 FX |
| Leica Application Suite 4.7.1 software | Leica Microsystems | |
| QuickTime Player, v10.4 | Apple | |
| Confocal microscope (inverted) | Leica Microsystems | TCS SP5 |
| Microinjector | Eppendorf | Femtojet |
| ImageJ macro Biosensor_FRET | ||
| GraphPad Prism 6.0c | GraphPad Software, Inc |
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