斑马鱼幼虫的前脑的电生理记录
Summary
外场电位记录在幼虫斑马鱼的前脑的一个简单的方法来描述。该方法提供了一个强大的<em在体内</em>读出癫痫样活动。这种技术可以使用转基因斑马鱼幼虫携带癫痫相关基因或癫痫发作诱发的惊厥药给药。
Abstract
癫痫会影响近3万人在美国和世界各地多达50万人。定义为发生自发的无端发作，癫痫，可以获取到大脑或基因突变的结果的一种侮辱。在动物模型癫痫发作的努力，主要用于收购侮辱（惊厥药，刺激或脑损伤）和遗传操作（反义击倒，同源重组或转基因）在啮齿类动物。斑马鱼是脊椎动物模型系统1-3，可以提供一个有价值的替代老鼠癫痫研究。斑马鱼被广泛应用于脊椎动物的遗传学研究或发展，对哺乳动物的遗传相似性，表现出高度表达的同系物〜85％的已知的人类单基因遗传性癫痫突变。由于它们的小尺寸（长4-6毫米），斑马鱼幼虫可以保持低的流体体积为100微升，在早期的发展和阿拉进行期间，在多井板。试剂可直接加入到该溶液中，在胚胎发育，简化给药，从而实现快速的在体内筛选试验化合物4。可用于合成寡核苷酸（吗啉代），致突变，锌指核酸酶和转基因方法，以迅速产生基因敲除或突变的斑马鱼5-7。这些属性提供了前所未有的斑马鱼研究的统计动力分析优势的研究啮齿动物的神经系统疾病，如癫痫。 ，因为癫痫研究的“金标准”是描述监测和分析的异常放电起源于一个中央的大脑结构（ 即癫痫发作），斑马鱼幼虫的大脑活动的方法，有效地记录在这里。该方法是一种适应传统的细胞外记录技术，并允许稳定的长期监测在完整的斑马鱼幼虫的大脑活动。小号充足的记录上所显示的急性发作引起的惊厥药和自发发作在转基因鱼浴应用。
Protocol
Representative Results
Discussion
这里介绍的方法使细胞外记录大脑活动的一个非常敏感和快速的分析。这些录音是类似的脑电图（EEG）监测常用的评估存在的异常放电（ 即癫痫发作）在啮齿类动物模型癫痫11和患者12。细胞外记录可以结合药理的操作，如下所示。也可用于这些类型的记录，以评估潜在的癫痫表型的转基因斑马鱼。确定，在ENU的突变屏幕（见斑马鱼资源中心， <a href="http://zebrafish.org/zirc/home…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者想，感谢他们及早建立斑马鱼在实验室的彼得·卡斯特罗和马修Dinday。这项工作是由美国国立卫生EUREKA（＃R01NS079214-01）资助。
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| Agarose low melting | Fisher-Scientific | BP1360-100 | Dissolve in embryo media at 1.2% |
| Recording media | Fisher-Scientific | BP3581, P330-3, BP410-1, BP214-500, D16-1, C77-500 | 1 mM NaCl, 2.9 mM KCl, 10 mM HEPES, 1.2 mM MgCl2, 10 mM Dextrose, 2.1 mM CaCl2 pH to approximately 7.3 with 1 N NaOH |
| Tricaine | Argent Labs | MS-222 | 0.02% |
| α-bungarotoxin | Tocris Bioscience | 2133 | 1 mg/ml |
| Capillary glass tubing | Warner Instruments | G120TF-3 | Pull to a resistance of 2 -7 MΩ |
| Patch clamp amplifier | Warner Instruments | PC-505B | We use a Warner amplifier in current-clamp mode; Gain set at 2 mV/pA and Bessel filter set at 2K. Comparable models can be used according to manufacturer’s instructions. |
| Filter/amplifier | Cygnus Technology | FLA-01 | We use a Cygnus pre-amplifier; Gain set at 10-20; Cut-off frequency set at 1-2K; Notch filter IN. Comparable models can be used according to manufacturer’s instructions. |
| Axon A/D board and Axoscope software | Molecular Devices | Axon Digidata 1320A; Axoscope 8.2 | Data is collected in Axoscope using gap-free acquisition mode; sampling at 10 kHz. Comparable models and programs can be used according to manufacturer’s instructions. |
| Egg water | Instant Ocean | 3 g Instant Ocean sea salt, 2 ml 0.1% methylene blue in 10 ml deionized water |
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