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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 research1,2,3. However, while assays of locomotion and swim behavior are available and standardized for larval zebrafish, there is a growing need to develop analogous protocols in adult fish8,9,10,11. The goal of this study is to describe protocols to quantify swim endurance and swim behavior in adult zebrafish. We present these protocols in the context of spinal cord regeneration research. However, the behavioral protocols described here are equally applicable to studies of neural and muscle regeneration, neuromuscular and musculoskeletal development, as well as neuromuscular and musculoskeletal disease modeling.
Zebrafish reverse paralysis within 8 weeks of complete spinal cord transection. Unlike poorly regenerative mammals, zebrafish display pro-regenerative immune, neuronal, and glial injury responses that are required for functional spinal cord repair12,13,14. An ultimate readout of functional spinal cord repair is the ability of the lesioned tissue to regain its function after injury. A suite of standardized methods to assess functional regeneration in rodents include locomotor, motor, sensory, and sensorimotor tests15,16,17. Widely used tests in mouse spinal cord injury include the locomotor Basso Mouse Scale (BMS), forelimb motor tests, tactile sensory tests, and grid walking sensorimotor tests15,17. In contrast with mammalian or larval zebrafish systems, behavioral tests in adult zebrafish are less developed, yet much needed to accommodate the growing needs of the tissue regeneration and disease modeling communities.
Complete spinal cord transections result in complete paralysis caudal to the injury site. Shortly after the injury, paralyzed animals are less active and avoid swimming as much as possible. To compensate for lost swim capacity, paralyzed animals display short, frequent bursts by overusing their pectoral fins, which lie rostral to the lesion. This compensatory swim strategy results in rapid exhaustion and lower swim capacity. As the zebrafish spinal cord regenerates, animals regain a smooth oscillatory swim function caudal to the lesion, allowing for increased swim endurance and improved swim behavior parameters. Here, we describe methods to quantify zebrafish swim endurance at increasing water current velocities and swim behavior at low current velocities.
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 workflow is presented in Figure 2.
1. Swim tunnel preparation and calibration
2. Assessment of swim endurance
NOTE: Experimental groups are divided into groups of 10 or fewer animals for swim endurance.
3. Capturing videos for swim behavior assay
NOTE: Only up to five animals can be tracked at a time. If experimental groups are larger than five animals, multiple videos can be taken for each group, where the first video tracks five or fewer animals and the second video tracks the other five or fewer animals. For longitudinal studies that aim to track individual animals over time, fish can be individually housed and tracked across multiple time points. All scripts for tracking and analyzing are available via GitHub (see Table of Materials).
4. Analyzing movies for swim behavior assessment
NOTE: Movie recording and analysis can be completed on separate days.
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 ...
The authors have no conflicts of interest.
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" 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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