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Capable of functional recovery after spinal cord injury, adult zebrafish is a premier model system to elucidate innate mechanisms of neural regeneration. Here, we describe swim endurance and swim behavior assays as functional readouts of spinal cord regeneration.
Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. Following complete transection of their spinal cord, zebrafish extend glial and axonal bridges across severed tissue, regenerate neurons proximal to the lesion, and regain their swim capacities within 8 weeks of injury. Recovery of swim function is thus a central readout for functional spinal cord repair. Here, we describe a set of behavioral assays to quantify zebrafish motor capacity inside an enclosed swim tunnel. The goal of these methods is to provide quantifiable measurements of swim endurance and swim behavior in adult zebrafish. For swim endurance, zebrafish are subjected to a constantly increasing water current velocity until exhaustion, and time at exhaustion is reported. For swim behavior assessment, zebrafish are subjected to low current velocities and swim videos are captured with a dorsal view of the fish. Percent activity, burst frequency, and time spent against the water current provide quantifiable readouts of swim behavior. We quantified swim endurance and swim behavior in wild-type zebrafish before injury and after spinal cord transection. We found that zebrafish lose swim function after spinal cord transection and gradually regain that capacity between 2 and 6 weeks post-injury. The methods described in this study could be applied to neurobehavioral, musculoskeletal, skeletal muscle regeneration, and neural regeneration studies in adult zebrafish.
Adult zebrafish are eminently used to investigate mechanisms of neuromuscular and musculoskeletal development and disease modeling1,2,3. Zebrafish are capable of efficient, spontaneous repair of multiple tissues, including the brain, spinal cord, and skeletal muscle4,5,6,7. The remarkable capacity to regenerate neuromuscular tissues and model diseases is attracting a growing scientific community into adult zebrafish research1....
Adult zebrafish of the Ekkwill and AB strains were maintained at the Washington University Zebrafish Core Facility. All animal experiments were performed in compliance with IACUC institutional animal protocols.
NOTE: An example of the experimental setup is shown in Figure 1A. The calibration lid (customized), swim endurance lid (customized), and swim behavior lid (standard, enclosed tunnel lid) are shown in Figure 1B. The experimental.......
We set up the swim tunnel as described in section 1 of this protocol (Figure 1). We assessed the swim endurance (section 2 of this protocol) as well as swim behavior (sections 3 and 4 of this protocol) of adult zebrafish at baseline and after spinal cord injury (Figure 2).
For establishing baseline motor function, we examined the swim endurance of wild-type zebrafish under increasing water current velocities (Figu.......
Adult zebrafish are a popular vertebrate system for modeling human diseases and studying mechanisms of tissue regeneration. CRISPR/Cas9 genome editing has revolutionized reverse genetic studies for modeling disease in zebrafish; however, large-scale genetics in adult zebrafish has been hindered by biological and technical challenges, including the unavailability of adult zebrafish tissues to high-throughput phenotyping. Given the complex anatomy of adult zebrafish, prolonged histological processing is required to obtain .......
We thank the Washington University Zebrafish Shared Resource for animal care. This research was supported by the NIH (R01 NS113915 to M.H.M.).
....Name | Company | Catalog Number | Comments |
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"Â 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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