在完整的成年斑马鱼大脑电生理记录
Summary
本文介绍了如何在成年斑马鱼可以被固定，气管插管，并用于体内电生理实验，让录音和操作在一个完整的动物的神经活动。
Abstract
此前，在成年斑马鱼的电生理研究一直局限于切片制剂或以眼罩准备和electrorentinogram录音。本文介绍了如何在成年斑马鱼可以被固定，气管插管，并用于体内电生理实验，允许记录神经活动。成年的固定化，需要提供溶解氧的鳃代替口腔和鳃盖运动的机构。用我们的技术，将动物固定，灌注水栖息地满足这一要求。根据三卡因甲磺酸盐进行开颅手术（MS-222，三卡因）麻醉提供接入到大脑。主电极被定位在开颅窗口内记录细胞外脑部活动。通过使用多管灌注系统，多种药理化合物可以施用给成年鱼和任何改动的神经活动可以观察到。该方法不仅可用于观测到进行关于变化的神经活性，而且它也允许幼虫和成虫斑马鱼之间作出比较。这使研究人员能够识别在神经活动的变化，由于在不同的人生阶段引入各种化合物的能力。
Introduction
在这篇文章中，协议描述为获得在成年斑马鱼的神经活动体内录音。细胞外记录方法被使用，以提供神经组织的一个小的区域内的电活动的电压测量。调查此方法涉及监测在表现动物1有大量的细胞。此前，记录片已在成人和幼虫进行，因为有眼杯的筹备和视网膜电录音。这些实验基本上都被执行细节的各种感觉系统2-5的生理反应。直到最近，完整的大脑准备工作仅是可利用的与斑马鱼幼虫3,6,7，其中呼吸和氧的扩散可能发生通过皮肤进行电。我们准备允许成年斑马鱼的原生神经活动，而动物保持完全清醒，并意识到o到被测量f及其周边地区。
斑马鱼（ 斑马鱼 ）当前播放的一个典范遗传，毒理学，药理学和病理生理学研究3的基础性作用。斑马鱼已经获得了神经科学领域内的知名度，因为它们与哺乳动物在遗传，神经和内分泌水平8有着广泛的同源性。在过去的十年中，标准神经解剖学和免疫组化技术已被用来确定不同的神经递质3,8,13的分布和斑马鱼神经系统9-12的具体特征的组织。最近，研究人员已经将工作重点转向功能研究14,15，其中许多集中在行为过程16-19和感觉系统2,13,20的电生理特性。少数这些研究都集中在了Adul的特定区域的电活动吨斑马鱼脑部21-23，但没有进行使用体内的方法。
这个协议可以适于自发和诱发活性的斑马鱼神经系统来描述在特定脑区活动模式中的电生理学研究。使用这种技术的允许的年轻幼虫阶段和成人的神经活动之间作出比较。此外，我们的协议允许遗传或药理改变之间的比较。连同其他的方法，如基因工程或药理学试验，这种方法提供了用于神经元沟通和可塑性的完好成年动物以及潜在的应用，如研究迟发性癫痫或神经变性过程的功能分析的新可能性。
Protocol
所有的实验过程中严格按照健康指南的国家机构实验动物的护理和使用进行并遵循协议＃A2011 09-003，这是审查，批准和格鲁吉亚机构动物护理和使用的监督大学委员会。 1。设备安装 对于开颅手术灌注系统 成年的固定化就必须插管系统，提供溶氧鱼。各种各样的系统都可以使用，但由60毫升注射器的一个简单的重力系统被使用。提高压头到一个高度?…
Representative Results
此协议已被用于测量体内成年斑马鱼的神经活动。这些电生理记录一致地和可重复性地获得的。 图5示出的成年斑马鱼的神经活性的天然的和诱导的变化有代表性的例子，当戊四氮（PTZ），公共chemoconvulsant 6,7,25,26，在插管被引入设置。 成年斑马鱼的天然神经活动的每个实验（ 图5A）进行了监测。但一直在观察到这种行为是自发的，小?…
Discussion
此协议已被用于测量体内成年斑马鱼的神经活动。通过练习，神经活动可以持续观察，虽然记录的活动的特征（振幅和事件的形状）可以鱼个体之间有所不同。细胞外记录技术的运用可以解释这一观察。该方法提供同时监测大量的区域1内的细胞，所以变化的主电极的定位，可以在观察到的活性中发挥作用。主电极的深度也改变被测量的区域。如果电极的尖端的区域内放置过深，所?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作是由美国国立卫生研究院/ NINDS格兰特R01NS070159（以TMD，JDL和ATS）的支持。
Materials
| 70% Ethanol | Decon Laboratories | 2750HC | Dilute 100% to 70% with DI water |
| 2 M Potassium Chloride | J.T. Baker | ||
| 2 M Sodium Chloride | J.T. Baker | 3624-05 | |
| 0.4% Tris-Buffered Tricaine | Sigma-Aldrich | E10521 | pH 7.2-7.4; stored at -20 oC |
| Pancuronium Bromide | Sigma-Aldrich | P1918 | Diluted to 1 μg/μl in 1x phosphate buffered saline |
| Habitat water | pH 7.0-7.4, conductivity of 400-450 μS; maintained by Instant Ocean and Sodium Bicarbonate | ||
| Pentylenetetrazol | Sigma-Aldrich | P6500 | Diluted to 300 mM in 1x phosphate buffered saline |
| Nanofil syringe | World Precision Instruments, Inc. | 06A | |
| 34 G Beveled needle | World Precision Instruments, Inc. | NF34BV | |
| Sponge | Small pore and chemical-free | ||
| Foam-backed fine sand paper | 5 x 5 cm2 is large enough | ||
| 9 V Battery | |||
| Wires with alligator clips | Need 2 | ||
| 37 cm x 42 cm Kimwipe | Kimberly-Clark Professional | TW31KEM | |
| 11 cm x 21 cm Kimwipe | Kimberly-Clark Professional | TW31KWP | |
| 1/8 in diameter tube | |||
| 1 cm diameter tube | |||
| 1 mm diameter tube | |||
| Reducing valve with female Luer lock cap and silicone ferrule | Qosina | 51505 | |
| Microscope (Leica MZ APO) | Another microscope can be used | ||
| Vanna scissors | Roboz Surgical Instruments Co., Inc. | 15018-10 | |
| 60 ml Luer lock syringe tubes | Becton, Dickinson and Company | 309653 | |
| 3-way Stopcocks with Luer connections | |||
| 1-way Stopcock with Luer connection | |||
| Fisherbrand 100 mm x 15 mm Petri dish | Fisher Scientific | NC9299146 | |
| Fisherbrand 60 mm x 15 mm Petri dish | Fisher Scientific | S67961 | |
| 4 in Borosilicate capillary tube | World Precision Instruments | TW100F-4 | Can contain a filament to aid in filling with solution |
| P-97 Flaming/Brown Micropipette Puller | Sutter Instrument Co. | ||
| Digidata 1440 | Molecular Devices | ||
| Axon Aloclamp 900A | Molecular Devices | ||
| Axoclamp software | Molecular Devices | ||
| HS-9Ax 1U headstage | Molecular Devices | ||
| 0.010 in Silver wire | A-M Systems, Inc. | ||
| Q-series electrode holder | Warner Instruments | QSW-A10P | |
| 10 ml Luer lock syringe | |||
| 1 mm x 15 in Tubing | Connect Luer lock syringe to Q-series electrode holder | ||
| Micromanipulator | Warner Instruments | Need 2 | |
| Microsoft-based PC | Dell | ||
| Faraday Cage | |||
| Air Table | |||
| Dissecting Microscope |
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Citer Cet Article
Johnston, L., Ball, R. E., Acuff, S., Gaudet, J., Sornborger, A., Lauderdale, J. D. Electrophysiological Recording in the Brain of Intact Adult Zebrafish. J. Vis. Exp. (81), e51065, doi:10.3791/51065 (2013).