Assessment of Swim Endurance and Swim Behavior in Adult Zebrafish

Summary

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.

Abstract

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.

Introduction

Adult zebrafish are eminently used to investigate mechanisms of neuromuscular and musculoskeletal development and disease modeling^1,2,3. Zebrafish are capable of efficient, spontaneous repair of multiple tissues, including the brain, spinal cord, and skeletal muscle^4,5,6,7. The remarkable capacity to regenerate neuromuscular tissues and model diseases is attracting a growing scientific community into adult zebrafish research¹....

Protocol

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

Results

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.......

Discussion

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 .......

Materials

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