Name: assessment of swim endurance and swim behavior in adult zebrafish
Uploaded: 2021-11-12T13:00:00
Description: Watch this Scientific Journal Video about Assessment of Swim Endurance and Swim Behavior in Adult Zebrafish at JoVE.com

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）。

Ekkwill和AB品系的成年斑马鱼在华盛顿大学斑马鱼核心设施中饲养。所有动物实验均按照IACUC机构动物协议进行。
注：实验设置示例如图 1A所示。校准盖（定制）、游泳耐力盖（定制）和游泳行为盖（标准封闭式隧道盖）如图 1B所示。实验工作流程如图 2所示。
1. 游泳道准备和校准</str...

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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?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AutoSwim software</td>
			<td>Loligo Systems</td>
			<td>MI10000</td>
			<td>Optional - for Automatic control of current velocity</td>
		</tr>
		<tr>
			<td>Customized lid</td>
			<td>Loligo Systems</td>
			<td>MI10001</td>
			<td>This customized lid is used for swim endurance</td>
		</tr>
		<tr>
			<td>DAQ-BT</td>
			<td>Loligo Systems</td>
			<td>SW10600</td>
			<td>Optional - for Automatic control of current velocity</td>
		</tr>
		<tr>
			<td>Eheim pump</td>
			<td>Loligo Systems</td>
			<td>PU10160</td>
			<td>20 L/min. This pump is placed in theflow-through tank.</td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td>Fiji</td>
			<td>Freely available through Image J (Fiji)</td>
			<td>Specific script available at https://github.com/MokalledLab/SwimBehavior</td>
		</tr>
		<tr>
			<td>Flowtherm</td>
			<td>Loligo Systems</td>
			<td>AC10000</td>
			<td>Handheld digital flow meter - for calibration</td>
		</tr>
		<tr>
			<td>High Speed Camera</td>
			<td>Loligo Systems</td>
			<td>VE10380</td>
			<td>USB 3.0 color video camera (4MP)</td>
		</tr>
		<tr>
			<td>IR light panel</td>
			<td>Loligo Systems</td>
			<td>VE10775</td>
			<td>450 x 210 mm, placed under the swim tunnel&#160; chamber</td>
		</tr>
		<tr>
			<td>Monofocal lens</td>
			<td>Loligo Systems</td>
			<td>VE10388</td>
			<td>25mm manual lens</td>
		</tr>
		<tr>
			<td>PVC Tubing</td>
			<td>VWR</td>
			<td>60985-534</td>
			<td>5/16 x 7/16&#34;&#160; Wall thickness: 1/16&#34;</td>
		</tr>
		<tr>
			<td>R Studio</td>
			<td>R Studio</td>
			<td>Freely available. Version 3.6 with extra packages.</td>
			<td>Specific script available at https://github.com/MokalledLab/SwimBehavior</td>
		</tr>
		<tr>
			<td>Swim tunnel respirometer</td>
			<td>Loligo Systems</td>
			<td>SW10060</td>
			<td>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</td>
		</tr>
		<tr>
			<td>uEye Cockpit</td>
			<td>IDS</td>
			<td>Freely available software to control camera parameters</td>
			<td>Alternative cameras and accompanying softwares could be used</td>
		</tr>
		<tr>
			<td>Vane wheel flow probe</td>
			<td>Loligo Systems</td>
			<td>AC10002</td>
			<td>Digital flow probe - for calibration</td>
		</tr>
	</tbody>
</table>

assessment of swim endurance and swim behavior in adult zebrafish

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。

我们按照该协议的第1节（图1）中所述设置了游泳隧道。我们评估了成年斑马鱼在基线和脊髓损伤后的游泳耐力（本方案第2节）以及游泳行为（本方案的第3节和第4节）（图2）。
为了建立基线运动功能，我们检查了野生型斑马鱼在水流速度增加下的游泳耐力（图3A）。在该测定中，野生型斑马鱼游了41分钟才?...

Watch this Scientific Journal Video about Assessment of Swim Endurance and Swim Behavior in Adult Zebrafish at JoVE.com

Assessment of Swim Endurance and Swim Behavior in Adult Zebrafish

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。

成年斑马鱼的游泳耐力和游泳行为评估

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. ...

斑马鱼提供了一个有价值的神经再生模型，因为它们能够从脊髓损伤中恢复过来。该方法将游泳耐力和游泳行为描述为功能性脊髓修复的读数。该技术为成年斑马鱼的游泳耐力和游泳行为提供了可靠且可量化的评估。这些方法同样适用于神经肌肉和肌肉骨骼发育，疾病和再生的研究。要评估游泳耐力，请打开流速控制软件。点击标有实验的框，然后取消选中Uswim和Uwater。然后改变左下角Uwater箱中的流速以调整水流速度。若要开始自动协议，请单击“启动日志记录”框。在打开的对话窗口中，从下拉列表中选择“自动”。要打开以前保存的协议文件，请单击协议文件旁边的文件图标。接下来，通过单击日志文件旁边的文件图标来设置输出文件。在打开的文件资源管理器窗口中，命名输出文件并将其保存在所需位置。然后，设置一个分开的单圈计时器窗口，以确保同时访问计算机屏幕上的流速控制软件和计时器窗口。接下来，设置一个鱼收集池，以便在将枯竭的鱼从游泳隧道中移走后容纳它们。用斑马鱼系统水填充收集罐和长聚氯乙烯管。将预填充管的一端放在收集罐中，另一端放在缓冲罐中，确保水可以自由地从缓冲罐流入收集罐。用粘结夹夹住管的上端，以防止水流，并根据需要使用粘结夹控制水的流出。用游泳耐力盖关闭游泳隧道，然后将一组鱼放入游泳隧道内。要使鱼适应游泳隧道和流动方向，请启动分圈计时器，并将当前速度调整为前五分钟为零厘米/秒，接下来的五分钟为每秒9厘米，接下来的五分钟为每秒10厘米。适应后，打开协议并启动自动流速控制程序，这将使水流速度每分钟增加两厘米/秒。监测鱼的疲惫。精疲力竭的鱼将被推向游泳隧道的后端。为了确保鱼筋疲力尽，请轻轻敲击隧道的后端或在该区域上空形成阴影以刺激鱼游泳。精疲力竭的鱼对惊吓的刺激没有反应，平躺在隧道的后端。当鱼筋疲力尽时，松开鱼收集管。打开游泳隧道窗口，将鱼收集到收集池中。使用分圈计时器记录耗尽时的时间。将所有鱼排尽并收集到收集池中后，单击流速控制软件上的紧急停止按钮并停止计时器。要捕获游泳行为测定的影片，请将一组鱼放入游泳隧道中，并使用标准的全封闭盖子关闭隧道。然后，打开一个新的录制窗口并为该文件命名。不要单击“录制”。在开始新的实验之前，在游泳隧道的一侧放置一张纸巾或一块织物，以确保所有行为都是由于鱼的游泳，而不是由于环境中的运动引起的惊吓反应。确保水平静并且没有涟漪在画面上移动后，单击相机软件窗口中的“录制”以开始录制动画文件。然后单击流速控制软件中的“启动”以启动协议，该协议将继续不间断地进行。观看影片以确保没有帧掉落，视野中没有气泡，并且所有鱼都被记录下来。动画录制完成后，单击紧急停止以结束流速控制协议并检查数据输出文件是否自动保存。然后，关闭录制窗口以保存动画文件。录制后，取下盖子。小心地取回鱼并将它们放回水箱。要分析捕获的影片，请打开tracking_v2。斐济的ijm脚本，然后单击“运行”以开始程序。在弹出窗口中，选择要跟踪的包含游泳行为电影的文件夹，然后单击“打开”。查找第一部电影的第一帧，一个对话框，以及将出现的兴趣区域或ROI管理器。按照对话框中给出的说明进行操作，并在游泳隧道室的底部创建 ROI。然后单击“确定”，并确保看不到黑色角落。阈值窗口将与编辑的阈值框架一起打开。将配色方案从黑色和白色更改为红色，并调整最大值，直到第一帧仅显示红色的鱼，而没有其他内容。记录阈值，然后在对话框中单击“确定”。要对齐、组合和获取描述性统计量，请打开SwimBehavior_v7-3。RStudio 中的 R 脚本，然后单击脚本部分右上角的“源”。在打开的新窗口中，选择包含_raw的文件夹。由斐济生成的 csv 文件，然后单击打开。程序将自动运行。在弹出对话框中，确认每部电影中的鱼的数量。如果给定的数字正确，请单击“是”;如果数字不正确，请单击“否”。对齐文件后，检查新生成的_aligned。csv 文件。确保程序合并数据、运行统计数据并绘制输出图形后，在包含_raw的父文件夹中检查在标记为“结果”的新文件夹中生成的分析文件。csv 和 _aligned。csv 文件。在受伤后两周，四周和六周评估的游泳耐力显示，两周时游泳耐力能力损失了60%。再生鱼在受伤后四周和六周逐渐恢复游泳耐力。在游泳行为测定中，对照组在对应于升高的Y位置的游泳隧道室的前部稳定游动。相比之下，在受伤后两周，受伤的鱼无法保持稳定的游泳能力。因此，他们的游泳轨迹更加不规则，Y位置整体下降。Y位置在受伤后4周和6周增加，表明再生动物逐渐恢复了游泳能力。此外，相对于未受伤的对照组，受伤后两周的病变动物明显不那么活跃，停滞在游泳道的后象限，并失去了在低流速度下游泳的能力。与它们与生俱来的实现功能恢复的能力一致，病变动物在受伤后四周和六周逐渐使游泳行为参数正常化。在此协议中使用流速控制软件是可选的。另一种方法是手动控制水流电机。本研究中描述的测定可用于预先筛选神经，肌肉或骨骼表型。然后可以收获感兴趣的组织进行组织学或分子检查。我们的实验室和其他人已经使用该协议来鉴定先天神经修复所需的基因和足以增强神经再生的因素。

Research

JoVE Journal

Neuroscience

Studying the Integration of Adult-born Neurons

成人出生的神经元的整合研究

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the ...

科研

神经科学

The Mouse Forced Swim Test

小鼠强迫游泳实验

The forced swim test is a rodent behavioral test used for evaluation of antidepressant drugs, antidepressant efficacy of new compounds, and experimental ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

视网膜神经节细胞逆行标记的成年斑马鱼与荧光染料

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

在完整的成年斑马鱼大脑电生理记录

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

的自动分析<em&gt;℃。线虫</em&gt;游泳行为使用天书软件

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has ...

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

成年斑马鱼脑 Amyloid-β42毒性和神经的模型研究

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

成人脑切片长期抑郁症诱导评估

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

研究钝力损伤对成年斑马鱼影响的可扩展模型

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and ...

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

穿梭盒测定作为使用成人斑马鱼进行学习和记忆研究认知评估的关联学习工具

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and ...

Deep-Tissue Three-Photon Fluorescence Microscopy in Intact Mouse and Zebrafish Brain

完整小鼠和斑马鱼大脑的深组织三光子荧光显微镜

Multiphoton microscopy techniques, such as two-photon microscopy (2PM) and three-photon microscopy (3PM), are powerful tools for deep-tissue in vivo ...