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

Overview

This video describes the rheotaxis assay in zebrafish. The method involves the measurement of orientation behavior of zebrafish in response to different water flow rates under the influence of different magnetic fields.

Protocol

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.

Representative Results

Figure 1: Setup for magnetic field control. (A) Rendering of the swimming tunnel with a solenoid for the induction of a static, horizontal magnetic field within the tunnel. The solenoid (0.83 turns/cm) is connected to a power unit and it generates fields in the range of ±250 µT (intensity range that includes the earth’s magnetic field range). On the right-hand side, a photo of the solenoid tunnel connected to the swimming apparatus is shown. The tunnel is made of acrylic and it has two perforated acrylic plates placed at the water inlet, which guarantee the flow to be close to laminar. (B) Diagram and photo of the three orthogonal Helmholtz pairs set for the control of the magnetic field in the geomagnetic range of intensities. The magnetic field probe, the CPU, the digital-to-analog converter, and the coil drivers used to close the loop are also shown. Each pair of coils is composed of two circular coils with a radius (r) of 30 cm and N = 50 turns of AWG-14 copper wires. A three-axes magnetometer (sensor) with selectable scale (± 88 µT to ± 810 µT) is placed close to the center of the coil set. The sensor range is set to values ranging to ±130 µT. These values were also used for the measurements described in the representative results (in these conditions, the nominal sensor resolution is about 0.1 µT). The intensity and the direction of the magnetic field are controlled with a digital feedback system. The sensor measures the three components of the magnetic field vector (the three axes), and the corresponding error signals are extracted. Then, the correction signals are generated by a simple integrator filter. The digital correction signals are converted to voltage by a digital-to-analog converter and amplified by a suitable coil driver. These last signals are used to drive the Helmholtz pairs. The sampling frequency is fixed to 5 Hz and the unity gain frequency of the loops is about 0.16 Hz. Once the currents in the Helmholtz pairs of the coils are set, the total magnetic field varies less than 2% from its mean intensity value in the central cubic volume (with edge [L] = 10 cm) of the coils. During the measurements, the magnetic field rms is less than 0.2 µT. In both the setups (panels A and B) a static electric field is generated by the current in the coils producing the magnetic field. The intensity of the electric field is about 0.4 V/m when the maximum current is applied; this value is negligible compared to natural or artificial static fields present in the environment whose intensity is of the order of 1 kV/m.

Figure 2: Diagram of the flow rates used during the tests to determine the rheotactic threshold of zebrafish. The flow during the 1 h acclimation period was enough to guarantee an adequate oxygen supply to the animals. It can be assumed that, with this design, oxygen supply is never a limit, even in the first 10 min step with flow 0. Indeed, with an oxygen content of water at 27 °C of about 7.9 mg/L and an animal oxygen consumption of 1 mg/h.g (an excess approximation for zebrafish oxygen consumption both under routine conditions and at low-speed swimming), it is possible to calculate that, in the absence of flow, the Po₂ in the flume will not decrease more than 2% per animal, remaining well above the critical Po₂ (about 40 torr for zebrafish).

Materials

Name	Company	Catalog Number	Comments
9500 G meter	FWBell	N/A	Gaussmeter, DC-10 kHz; probe resolution: 0.01 μT
AD5755-1	Analog Devices	EVAL-AD5755SDZ	Quad Channel, 16-bit, Digital to Analog Converter
ALR3003D	ELC	3.76024E+12	DC Double Regulated power supply
BeagleBone  Black	Beagleboard.org	N/A	Single Board Computer
Coil driver	Home made	N/A	Amplifier based on commercial OP (OPA544 by TI)
Helmholtz pairs	Home made	N/A	Coils made with standard AWG-14 wire
HMC588L	Honeywell	900405	Rev E Digital three-axis magnetometer
MO99-2506	FWBell	129966	Single axis magnetic probe
Swimming apparatus	M2M Engineering Custom Scientific Equipment	N/A	Swimming apparatus composed by peristaltic pump and SMC Flow switch flowmeter with digital feedback
TECO 278	TECO	N/A	Thermo-cryostat