Zebrafish exhibit associative learning for an aversive robotic stimulus

Zebrafish have quickly emerged as a species of choice in preclinical research, holding promise to advance the field of behavioral pharmacology through high-throughput experiments. Besides biological and heuristic considerations, zebrafish also constitute a fundamental tool that fosters the replacement of mammals with less sentient experimental subjects. Notwithstanding these features, experimental paradigms to investigate emotional and cognitive domains in zebrafish are still limited. Studies on emotional memories have provided sound methodologies to investigate fear conditioning in zebrafish, but these protocols may still benefit from a reconsideration of the independent variables adopted to elicit aversion. Here, we designed a fear-conditioning paradigm in which wild-type zebrafish were familiarized over six training sessions with an empty compartment and a fear-eliciting one. The fearful stimulus was represented by three zebrafish replicas exhibiting a fully synchronized and polarized motion as they were maneuvered along 3D trajectories by a robotic platform. When allowed to freely swim between the two compartments in the absence of the robotic stimulus (test session), zebrafish displayed a marked avoidance of the stimulus-paired one. To investigate whether fear conditioning was modulated by psychoactive compounds, two groups of zebrafish were administered ethanol (0.25% and 1.00%, ethanol/water, by volume) a few minutes before the test session. We observed that ethanol administration abolished the conditioned avoidance of the stimulus-paired compartment. Ultimately, this study confirms that robotic stimuli may be used in the design of fear-conditioning paradigms, which are sensitive to pharmacological manipulations.