Zebrafish Rheotaxis Assay: A Method for Measuring Orientation Behavior in Response to Different Water Flow Rates

1.Set up of the Magnetic Field with the One-dimensional Magnetic Field Manipulation

1. Switch on the Power unit (Figure 1A).
2. Place the coiled tunnel in the location where the rheotactic protocol will be performed (section 3) but keep it disconnected from the swimming apparatus (Figure 1A). Place a magnetic probe connected with a Gauss/Teslameter inside the tunnel and verify which voltage is necessary to obtain the chosen magnetic field value along the major axis of the tunnel.
   NOTE: Because of the magnetic properties of a solenoid, the field is reasonably uniform inside the tunnel; this can be checked by slowly moving the probe both horizontally and vertically.
3. Disconnect the probe and connect the flow tunnel to the swimming apparatus.
4. Start with the rheotactic protocol (section 3).

2. Set Up of the Magnetic Field with the Three-dimensional Magnetic Field Manipulation

1. Switch on the CPU, DAC, and coil drivers (Figure 1B).
2. Set the chosen magnetic field on each one of the three axes (x, y, and z).
3. Place the tunnel in the center of the Helmholtz pairs set.
4. Start with the rheotactic protocol (section 3).

3. Test of the Zebrafish Rheotaxis in the Flow Chamber

1. Transfer one to five fish to the flow tunnel using a 2 L tank with the sides and the bottom obscured.
2. Turn on the pump and set the flow rate in the tunnel to 1.7 cm/s.
   NOTE: This slow-moving water will keep the water in the tunnel oxygenated and it will facilitate animal recovery.
3. Let the animals acclimate to the swimming tunnel for 1 h.
4. Start the video recording of the behavior of the fish in the tunnel.
   NOTE: We used a camera (e.g., Yi 4K Action) with remote control (e.g., Bluetooth) and saved the video as .mpg (30 frames/s).
5. Start the stepwise increase of the flow rate according to the chosen experimental protocol (1.3 cm/s in this study; Figure 2).
   NOTE: For this protocol, we used low flow rates which, for zebrafish, range from 0 to 2.8 BL (body lengths)/s. These flow speeds are in the lower range of flow rates that induce continuous oriented swimming in zebrafish (3%–15% of critical swimming speed [U_crit]). The use of low flow rates (following Brett’s protocol) is linked to the specific behavioral characteristics of this species in the presence of water currents. Zebrafish tend to swim along the major axis of the chamber, turning frequently, even in the presence of water flow, and tend to swim both upstream and downstream. This behavior is affected by the water flow rate, disappearing at relatively high speeds (>8 BL/s), when the animals continuously swim facing upstream (full positive rheotactic response). Vertical and transversal displacements are very rare.
6. Perform morphometry of the animals (sex and total length [TL], fork length [FL], or BL) on pictures of fish in a morphometric chamber​.
   1. Select the appropriate picture.
   2. Open the picture in ImageJ.
   3. Take note of the sex of the animal (male zebrafish are slender and tend to be yellowish, while females are more rounded and tend to have blue and white colorings).
   4. Click Analyze > Set Scale and set the scale of the image in centimeters, using the whole horizontal length of the tunnel as reference.
   5. Click Analyze > Measure and record the linear length of the animal.
   6. Calculate its body weight (BW).
      NOTE: BW is calculated from sex-FL-BW relationships previously built in the lab or from metadata. The whole procedure avoids manipulation stress on the animals.