成年斑马鱼的游泳耐力和游泳行为评估
Summary
成年斑马鱼能够在脊髓损伤后恢复功能，是阐明神经再生先天机制的首要模型系统。在这里，我们将游泳耐力和游泳行为测定描述为脊髓再生的功能读数。
Abstract
由于其著名的再生能力，成年斑马鱼是询问先天脊髓再生机制的首要脊椎动物模型。在完全横切脊髓后，斑马鱼将神经胶质桥和轴突延伸到切断的组织上，再生病变近端的神经元，并在受伤后8周内恢复游泳能力。因此，游泳功能的恢复是功能性脊髓修复的中心读数。在这里，我们描述了一组行为测定，以量化封闭游泳隧道内的斑马鱼运动能力。这些方法的目标是提供成年斑马鱼游泳耐力和游泳行为的可量化测量。为了保持游泳耐力，斑马鱼要承受不断增加的水流速度，直到筋疲力尽，并报告疲惫的时间。对于游泳行为评估，斑马鱼受到低流速的影响，并使用鱼的背视图捕获游泳视频。活动百分比、爆发频率和逆流而上的时间提供了游泳行为的可量化读数。我们量化了野生型斑马鱼在受伤前和脊髓切除后的游泳耐力和游泳行为。我们发现斑马鱼在脊髓横断后失去游泳功能，并在受伤后2至6周内逐渐恢复游泳能力。本研究中描述的方法可以应用于成年斑马鱼的神经行为，肌肉骨骼，骨骼肌再生和神经再生研究。
Introduction
成年斑马鱼主要用于研究神经肌肉和肌肉骨骼发育的机制以及疾病建模1，2，3。斑马鱼能够有效、自发地修复多种组织，包括大脑、脊髓和骨骼肌4，5，6，7。再生神经肌肉组织和模拟疾病的非凡能力正在吸引越来越多的科学界参与成年斑马鱼研究1，2，3。然而，虽然斑马鱼幼虫的运动和游泳行为测定是可用的和标准化的，但越来越需要在成鱼中开发类似的方案8，9，10，11。本研究的目的是描述量化成年斑马鱼的游泳耐力和游泳行为的方案。我们在脊髓再生研究的背景下提出了这些方案。然而，这里描述的行为方案同样适用于神经和肌肉再生，神经肌肉和肌肉骨骼发育的研究，以及神经肌肉和肌肉骨骼疾病建模。
斑马鱼在脊髓切除术完全后8周内逆转瘫痪。与再生能力差的哺乳动物不同，斑马鱼表现出功能性脊髓修复所需的促再生免疫，神经元和神经胶质损伤反应12，13，14。功能性脊髓修复的最终读数是病变组织在受伤后恢复其功能的能力。评估啮齿动物功能再生的一套标准化方法包括运动，运动，感觉和感觉运动测试15，16，17。在小鼠脊髓损伤中广泛使用的测试包括运动巴索小鼠量表（BMS），前肢运动测试，触觉感觉测试和网格行走感觉运动测试15，17。与哺乳动物或幼虫斑马鱼系统相比，成年斑马鱼的行为测试不太发达，但非常需要适应组织再生和疾病建模群落不断增长的需求。
完全脊髓横断导致损伤部位尾部完全瘫痪。受伤后不久，瘫痪的动物活动较少，并尽可能避免游泳。为了补偿游泳能力的损失，瘫痪的动物通过过度使用胸鳍（位于病变的喙）来表现出短暂而频繁的爆发。这种补偿性游泳策略会导致快速疲惫和游泳能力下降。随着斑马鱼脊髓的再生，动物在病变尾部恢复了平滑的振荡游泳功能，从而增加了游泳耐力并改善了游泳行为参数。在这里，我们描述了量化斑马鱼在增加水流速度下的游泳耐力和在低水流速度下的游泳行为的方法。
Protocol
Representative Results
Discussion
成年斑马鱼是一种流行的脊椎动物系统，用于模拟人类疾病和研究组织再生机制。CRISPR / Cas9基因组编辑彻底改变了用于斑马鱼疾病建模的反向遗传研究;然而，成年斑马鱼的大规模遗传学受到生物学和技术挑战的阻碍，包括成年斑马鱼组织无法获得高通量表型。鉴于成年斑马鱼的复杂解剖结构，需要长时间的组织学处理来获得和分析组织结构。本研究中描述的游泳耐力和游泳行为测定可用于在组?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢华盛顿大学斑马鱼共享资源对动物的护理。这项研究得到了NIH的支持（R01 NS113915至M.H.M）。
Materials
| AutoSwim software | Loligo Systems | MI10000 | Optional – for Automatic control of current velocity |
| Customized lid | Loligo Systems | MI10001 | This customized lid is used for swim endurance |
| DAQ-BT | Loligo Systems | SW10600 | Optional – for Automatic control of current velocity |
| Eheim pump | Loligo Systems | PU10160 | 20 L/min. This pump is placed in theflow-through tank. |
| Fiji | Fiji | Freely available through Image J (Fiji) | Specific script available at https://github.com/MokalledLab/SwimBehavior |
| Flowtherm | Loligo Systems | AC10000 | Handheld digital flow meter – for calibration |
| High Speed Camera | Loligo Systems | VE10380 | USB 3.0 color video camera (4MP) |
| IR light panel | Loligo Systems | VE10775 | 450 x 210 mm, placed under the swim tunnel chamber |
| Monofocal lens | Loligo Systems | VE10388 | 25mm manual lens |
| PVC Tubing | VWR | 60985-534 | 5/16 x 7/16" Wall thickness: 1/16" |
| R Studio | R Studio | Freely available. Version 3.6 with extra packages. | Specific script available at https://github.com/MokalledLab/SwimBehavior |
| Swim tunnel respirometer | Loligo Systems | SW10060 | 5L (120V/60Hz). The system includes the swim chamber, motor, manual control of water current velocity, 1 pump placed inside the chamber, standard swim tunnel lid for swim behavior, and modified swim tunnel lid for calibration |
| uEye Cockpit | IDS | Freely available software to control camera parameters | Alternative cameras and accompanying softwares could be used |
| Vane wheel flow probe | Loligo Systems | AC10002 | Digital flow probe – for calibration |
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