This protocol allows exploration on how the internal state or personality of a zebrafish influences the orientation response to cues coming from the environment such as water currents or magnetic fields. This technique is used to observe how both internal factors and environmental cues impact to the zebrafish orientation. However, it can also be applied to a broad variety of experimental designs.
This method could provide insight into behavioral ecology of sensor biology research. And is species flexible so it's suitable for any aquatic animal that actively swims. We advise that, upon performing in the experiments, you become familiar with the behavior of the animal you intend to use and with how to minimize animal stress.
For personality selection in zebrafish collect individual fish using a 250 milliliter beaker in a net to move them into a transfer tank and transfer the fish to the dark side of the personality selection tank. After one hour of acclimatization open the sliding door and allow the individuals you exit from the hole to explore the bright side of the tank for 10 minutes. At the end of the exploration period, gently collect the fish in two separated containers, collecting the proactive fish from the bright side and after additional five minutes the reactive fish from the dark box.
To set up a magnetic field with one dimensional magnetic field manipulation, keep the tunnel open and switch on power unit and magnetometer. Place a magnetic probe connected to a gas Tesla meter inside the tunnel and verify the voltage necessary to obtain the chosen magnetic field value along the major axis of the tunnel. Then disconnect the probe and connect the flow tunnel to the swimming apparatus.
To set up a magnetic field with three dimensional magnetic field manipulation, turn on the computer, digital to analog converter in coil drivers and set the chosen magnetic field on the x, y, z axes. Then place the tunnel in the center of the Helmholtz pairs set. When the magnetic field that choice has been set up, select one to five fish for the rheotactic test.
Transfer the fish in the tunnel, set the flow rate in the tunnel to 4.5 liters per minute and turn on the pump. Be sure to close the outlet valve before feeling the tunnel with water and transfer the fish and open it before setting the floor right. After the animals have acclimated there in the swimming tunnel for an hour, start recording the behavior of the fish within the tunnel and begin the step wise increasing the flow rate according to the chosen experimental protocol.
The same swimming tunnel with the coil removed, can be placed in the center of the Helmholtz pairs set. At the end of the rheotaxis experiment, take photos of individual fish in amorphous metric chamber and open the photos in image J.Note 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.
Next, click analyze and set scale. To set the scale of the image in centimeters, using the camera scale as reference and with the segmented line tool selected, draw a line from the snout tip to the coddled pentacle to measure standard length or to the tail tip for the total length. Then click Analyze and measure and record the linear length of the animal in centimeters.
To track the zebrafish activity within the tunnel, open the appropriate video analysis and modeling software program. Click File, Import and video and open one of the videos and click coordinate system to set the length units to centimeters and the time units, to seconds. Click coordinate axes and set the reference system to track the position of the fish over time with the x axis along the tunnel, setting the origin at the low corner of the wall at the water outlet.
Click track new in calibration to set the scale and track new in point to mass, to start tracking one fish at a time. Advanced the video manually at half second, five frame intervals and mark the time and position of the animal at each upstream downstream turn and at each downstream upstream turn. At the end of each tracking session select the X and time values from the table.
Right click on the data and click copy data in full precision. Then save the time values and x values of all of the turning positions on a template spreadsheet file. To calculate the total upstream and downstream time, as well as the values of the rheotactic index and percentages for each full step.
In this representative experiment, after a one hour acclimation period in the tunnel as demonstrated, one show at a time was video recorded while swimming in the tunnel in the water current was accelerated with a stepwise increase in the flow rate for seven steps. During these trials, the magnetic field was controlled in one dimension. During the tracked time, the terms of each fish at each flow rate were highlighted and used as reference points for calculating the rheotactic index of each fish at each full speed.
When the rheotactic index is higher than 50%and indicates that the animal had a positive rheotactic response corresponding to a prevalence of upstream swimming, a rheotactic index not significantly different from 50%indicates an absence of a rheotactic response. Arc sign transformation of the average rheotactic index percent values of all five fish in this experiment demonstrated a sigmoidal increase in the rheotactic index value when the water speed increases, allowing quantification of the zebrafish rheotaxis using a simple mathematical method. From these curves, it is visible that under magnetic fields of different directions, the rheotactic responsive of proactive fish is not affected and the response of the two experimental groups is represented by the same curve.
On the other hand, reactive fish change the rheotactic orientation according to the magnetic field and the response is represented by to significantly different curves. Remember that this is a behavioral experiment and the fish behavior can be affected by stress. Handling and transferring fish can be tricky.
So, train yourself before the experiments. This rheotactic magnetic technique has allowed us to further explore the effects of personality on magnetal sensitivity in zebrafish and the other aquatic vertebrates and invertebrates.
