Behavioural and physiological responses to a conditioning protocol for adult zebrafish, Danio rerio, held in groups

The current study developed and evaluated a conditioning protocol for zebrafish which can be used in groups of fish instead of isolated individuals. The protocol consisted of the application of a neutral conditioned stimulus (CS), i.e. a novel object in the tank, followed by an aversive, stressful unconditioned stimulus (US), i.e. chasing, in groups of fish in experimental tanks. This training protocol was applied to fish once per day for one- or three- consecutive days. The following day fish were tested for the display of behavioural, i.e. bottom dwelling, and neuroendocrine, i.e. cortisol and stress-related gene expression, conditioned responses. Results showed that one-day of conditioning was not adequate to produce conditioned responses. Repetition of training sessions for three days, however, led to the display of conditioned responses upon exposure to the conditioning stimulus alone. Specifically, when exposed to the CS, paired fish showed increased bottom dwelling, as well as elevated trunk cortisol concentration and c-fos gene expression in the brain. Data suggest that the applied CS can be perceived by fish as a signal to prepare anticipatory behavioural and neuroendocrine responses to cope with the expected aversive event. Finally, this study is one of the few to test associative learning in groups of zebrafish, thus avoiding training and testing fish individually which can be stressful and impede normal behaviour.