生物传感运动神经元膜电位在活斑马鱼胚胎
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
体内全身成分分析允许科学家以最可靠的方式研究细胞行为。当细胞细胞相互作用（接触和非接触依赖性）受到严重影响时，尤其是在神经系统中，膜电压变化驱动可激活细胞之间的通讯。理解由这些电信号编码的信息是了解神经系统在生理和疾病状态下工作的方式的关键。
为了研究大多数非侵入性生理条件下的细胞电学性质，最近开发出几种遗传编码电压指标1 。与前几代光电压传感器（主要是电压敏感染料） 2相反，GEVIs允许完整神经系统的体内分析，以及它们的表达可以限于特定的细胞类型或种群。
斑马鱼胚胎是体内 “底物”，可以利用GEVIs的巨大潜力。事实上，由于其光学透明度及其简化且进化上保守的神经系统，斑马鱼模型允许直接识别和操纵网络中的每个蜂窝组件。事实上，使用基于FRET的GEVI美人鱼3导致在肌萎缩性侧索硬化（ALS）斑马鱼模型中鉴定脊髓运动神经元行为的先兆症状变化4 。
以下体内协议描述了如何以神经元特异性方式监测表达美人鱼的完整斑马鱼胚胎中脊髓运动神经元的电学性质。此外，它展示了药理学诱导的chan具有这种电性质的ges可能与胚胎自发盘旋的频率的变化相关联，这种变化的运动活动表征斑马鱼在非常早期的发育阶段的运动行为。
Protocol
Representative Results
Discussion
这里提出的方案允许我们探讨斑马鱼胚胎脊髓运动神经元的电性质与自发卷取行为之间的关联，最早的定型运动活动，出现在17 hpf的胚胎发育，并持续到24 hpf 10 。
我们的方法为研究人员提供了研究完整胚胎神经系统的工具，充分保护了开发功能网络中细胞间相互作用的复杂性。斑马鱼胚胎的体内成像通过将其浸入低熔点琼脂糖中简单地固定来进行。?…
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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