Schematic diagram showing the experimental CPP procedure.

On day one (pretest), each zebrafish was tested for baseline preference for each side of the tank (light brown or white). On days two, three and four, zebrafish were individually restricted first to the brown side for 20 min and consecutively to their non-preferred side (white side), where they were exposed to nicotine (nicotine-paired group) or saline (control group) for 20 min. For the counterbalanced nicotine-unpaired control, animals were restricted to the brown or white compartment for 20 min and then exposed to nicotine alternatively in the brown (days 1 and 3) and the white (day 2) compartment also for 20 min. On day five, the place preference of each zebrafish was measured by determining the percentage of time spent on each side of the tank over a 5 min test period, following a 5 min interval for habituation. Behavioral analysis was performed during the five days as indicated by crosses.