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This modification refers to the protocol used at LaNeC, and involves tank modifications as well as addition of behavioral endpoints. These additions make the test more sensitive and refine the different variables controlling behavior in the light/dark preference test, justifying the decreased throughput.

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1. Room illumination is a critical variable, as it can alter fish behavior and the aversiveness of the white compartment. Before each trial, light levels should be measured; variations above or below 2 SDs from the average should be considered as too discrepant for an experiment. Ideally, lighting above the tank should be on the range of 500-600 lux.
2. Use Gaussian white noise throughout the experiment to cover unwarranted noise in the experimental room.
3. Bring the animal in a temporary transport recipient into the behavioral test room.
4. Transfer the animal to the central compartment of the tank with a net. Allow the animal to acclimate to the tank for 3 min
5. Remove the sliding doors, allowing the fish to swim freely in the apparatus for 15 min. Video recording is recommended.
6. Transfer the animal to a discard tank with a net.
7. Videos should be analyzed blindly (i.e., the experimenter should not know the treatment/genotype of the animal) by two experienced researchers. Variables to be recorded (and ZBC references) are:
   1. Time spent in the white compartment (ZBC 1.137)
   2. Latency to enter the white compartment
   3. Number of entries in the white compartment
   4. Duration of entries in the white compartment
   5. "Risk assessment" events (a fast (<1 s) entry in the white compartment followed by re-entry in the black compartment, or a partial entry in the white compartment (i.e., the pectoral fin does not cross the midline))
   6. Duration and number of erratic swimming (ZBC 1.51): a zig-zag, fast, unpredictable course of swimming of short duration.
   7. Duration and number of freezing events (ZBC 1.68): complete cessation of movements except eye and opercular movements.
   8. Time on thigmotaxis (ZBC 1.173): swimming in a distance of 2 cm or less from the nearest wall.

References

Kalueff et al., 2013 (ZBC; doi: 10.1089/zeb.2012.0861); Maximino et al., 2010 (doi: 10.1038/nprot.2009.225); Maximino et al., 2014 (doi: 10.1371/journal.pone.0103943)