We thank the Washington University Zebrafish Shared Resource for animal care. This research was supported by the NIH (R01 NS113915 to M.H.M.).

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...

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The authors have no conflicts of interest.

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 ...

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.

<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

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.

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...

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

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. ...

Zebrafish offer a valuable neural regeneration model because of their ability to recover from spinal cord injury. This method describes swim endurance and swim behavior as readouts of functional spinal cord repair. This technique provides reliable and quantifiable assessment of swim endurance and swim behavior in adult zebrafish.These methods are equally applicable to studies of neuromuscular and musculoskeletal development, disease, and regeneration. To assess swim endurance, open the flow velocity control software. Click on the box labeled Experiment and uncheck Uswim and Uwater.Then change the flow speed in the Uwater box on the bottom left to adjust water current velocities. To begin an automated protocol, click on the Start logging box. In the dialogue window that opens, choose Automated from the dropdown list.To open a previously saved protocol file, click on the file icon next to the protocol file. Next, set up the output file by clicking on the file icon next to the log file. In the file explorer window that opens, name the output file and save it in the desired location.Then, set up a split lap timer window to ensure simultaneous access to the flow velocity control software and timer windows on the computer screen. Next, set up a fish collection tank to house the exhausted fish after their removal from the swim tunnel. Fill the collection tank and a long polyvinyl chloride tube with zebrafish system water.Place one end of the prefilled tube in the collection tank and another in the buffer tank, ensuring that water can freely flow from the buffer tank into the collection tank. Clamp the upper end of the tube with a binder clip to prevent water flow, and use the binder clip to control the outflow of water as needed. Close the swim tunnel with the swim endurance lid before placing one group of fish inside the swim tunnel.To acclimate the fish to the swim tunnel and flow direction, start the split lap timer and adjust the current velocity to zero centimeters per second for the first five minutes, nine centimeters per second for the next five minutes, and 10 centimeters per second for the following five minutes. Following the acclimation, open the protocol and start the automated flow velocity control program, which will increase the water current velocity by two centimeters per second every minute. Monitor the fish for exhaustion.Exhausted fish will be pushed toward the back end of the swim tunnel. To ensure a fish is exhausted, gently tap the back end of the tunnel or create a shadow over that area to stimulate the fish to swim. Exhausted fish did not respond to the startle stimulus and lay flat at the back end of the tunnel.When a fish is exhausted, unclamp the fish collection tube. Open the swim tunnel window and collect the fish in the collection tank. Record the time at exhaustion using the split lap timer.After all the fish are exhausted and collected in the collection tank, click on the Emergency Stop button on the flow velocity control software and stop the timer. To capture movies for the swim behavior assay, place a group of fish in the swim tunnel and close the tunnel using a standard, fully-enclosed lid. Then, open a new recording window and name the file.Do not click Record yet. Before beginning a new experiment, place a paper towel or piece of fabric on the side of the swim tunnel to ensure all behaviors are due to fish swimming, and not due to a startle response caused by movement in the environment. After ensuring that the water is calm and no ripples are moving across the frame, click on Record in the camera software window to start recording the movie file.Then click on Start in the flow velocity control software to begin the protocol, which will continue uninterrupted. Watch the movie to ensure that no frames are dropped, there are no bubbles in the field-of-view, and all fish are recorded. Once the movie recording is completed, click on Emergency Stop to end the flow velocity control protocol and check that the data output file is saved automatically.Then, close the recording window to save the movie file. After the recording, remove the lid. Carefully retrieve the fish and return them to their tank.To analyze the captured movies, open the tracking_v2. ijm script in Fiji and click on Run to begin the program. In the popup window, choose the folder containing the swim behavior movies to track and click on Open.Look for frame one of the first movie, a dialogue box, and the region-of-interest, or ROI manager, that will come up. Follow the directions given in the dialogue box and create an ROI at the bottom of the swim tunnel chamber. Then click on OK and ensure that no black corners are seen.The threshold window will open along with an edited thresholded frame one. Change the color scheme from black and white to red and adjust the max value until frame one only shows the fish in red and nothing else. Record the threshold and click OK in the dialogue box.For aligning, assembling, and acquire descriptive statistics, open the SwimBehavior_v7-3. R script in RStudio and click on Source in the top right corner of the script section. In a new window that opens, choose the folder containing the _raw.csv files generated by Fiji and click Open. The program will run automatically. In the popup dialogue box, confirm the number of fish in each movie.Click on Yes if the given numbers are correct or no if the numbers are incorrect. Once the files are aligned, check for a newly-generated _aligned. csv file.After ensuring that the program combines the data, runs statistics, and plots output graphs, check for the analysis files generated in a new folder labeled Results within the parent folder containing the _raw. csv and _aligned. csv files.Swim endurance assessed at two, four, and six weeks post-injury showed a 60%loss of swim endurance capacity at two weeks. The regenerating fish gradually regained swim endurance at four and six weeks post-injury. In the swim behavior assay, controls swam steadily in the front part of the swim tunnel chamber corresponding to an elevated Y position.In contrast, at two weeks post-injury, injured fish could not maintain steady swim capacity against the current. Consequently, their swim tracks are more irregular, with an overall decrease in Y position. Y position increased at four and six weeks post-injury, indicating that regenerating animals gradually regained their ability to swim.Furthermore, relative to uninjured controls, lesioned animals at two weeks post-injury were markedly less active, stalled in the rear quadrant of the swim tunnel, and lost their ability to swim against low-current velocities. Consistent with their innate ability to achieve functional recovery, lesioned animals gradually normalized swim behavior parameters at four and six weeks post-injury. The use of the flow velocity control software in this protocol is optional.The alternative is to manually control the water current motor. The assays described in this study can be used to pre-screen for neural, muscular, or skeletal phenotypes. The tissue of interest can then be harvested for histological or molecular examination.Our lab and others have used this protocol to identify genes that are required for innate neural repair and factors that are sufficient to enhance neural regeneration.

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Neuroscience

3.0K visualizações.  Washington University School of Medicine. Os zebrafish oferecem um modelo valioso de regeneração neural devido à sua capacidade de se recuperar da lesão medular. Este método descreve a resistência à natação e o comportamento de natação como leituras de reparação funcional da medula espinhal. Esta técnica fornece avaliação confiável e quantificável da resistência à natação e comportamento de natação em zebrafish adultos.Esses métodos são igualmente aplicáveis aos estudos de desenvolvimento neuromuscul...