As zebrafish emerge as a species of choice for the investigation of biological processes, a number of experimental protocols are being developed to study their social behaviour. While live stimuli may elicit varying response in focal subjects owing to idiosyncrasies, tiredness and circadian rhythms, video stimuli suffer from the absence of physical input and rely only on two-dimensional projections. Robotics has been recently proposed as an alternative approach to generate physical, customizable, effective and consistent stimuli for behavioural phenotyping. Here, we contribute to this field of investigation through a novel four-degree-of-freedom robotics-based platform to manoeuvre a biologically inspired three-dimensionally printed replica. The platform enables three-dimensional motions as well as body oscillations to mimic zebrafish locomotion. In a series of experiments, we demonstrate the differential role of the visual stimuli associated with the biologically inspired replica and its three-dimensional motion. Three-dimensional tracking and information-theoretic tools are complemented to quantify the interaction between zebrafish and the robotic stimulus. Live subjects displayed a robust attraction towards the moving replica, and such attraction was lost when controlling for its visual appearance or motion. This effort is expected to aid zebrafish behavioural phenotyping, by offering a novel approach to generate physical stimuli moving in three dimensions.
Robotics is continuously being integrated in animal behaviour studies to create customizable, controllable, and repeatable stimuli. However, few systems have capitalized on recent breakthroughs in computer vision and real-time control to enable a two-way interaction between the animal and the robot. Here, we present a “closed-loop control” system to investigate the behaviour of zebrafish, a popular animal model in preclinical studies. The system allows for actuating a biologically-inspired 3D-printed replica in a 3D workspace, in response to the behaviour of a zebrafish. We demonstrate the role of closed-loop control in modulating the response of zebrafish, across a range of behavioural and information-theoretic measures. Our results suggest that closed-loop control could enhance the degree of biomimicry of the replica, by increasing the attraction of live subjects and their interaction with the stimulus. Interactive experiments hold promise to advance our understanding of zebrafish, offering new means for high throughput behavioural phenotyping.
The study of zebrafish behavior represents a cornerstone upon which basic researchers promise to advance knowledge in life sciences. Although zebrafish swim in a three-dimensional (3D) space, their behavior in the lab is almost exclusively scored in two dimensions, whereby zebrafish are recorded using a single camera providing 2D videos. Whether this dimensional reduction preserves the reliability of data has not been addressed. Here we show that, compared to a 3D observation, 2D data are flawed by over-reporting and under-reporting of locomotory differences. Specifically, we first reconstructed 3D trajectories through the integration of synchronous information derived from two cameras, and then compared them with the original 2D views in classical experimental paradigms assessing shoaling tendency, fear, anxiety, and general locomotion. Our results suggest that traditional behavioral scoring of individual zebrafish performed in 2D may undermine data integrity, thereby requiring a general reconsideration of scoring zebrafish behavior to incorporate a 3D approach. We then demonstrate that, compared to 2D, a 3D approach requires a reduced number of subjects to achieve the same degree of validity. We anticipate these findings to largely benefit animal welfare by reducing the number of experimental subjects, without affecting statistical power.
Zebrafish has recently become a species of choice in a number of preclinical studies to examine whether and how psychoactive compounds modulate individual and social behaviors. Here, we sought to contribute an improved understanding of the effects of caffeine, a well-known anxiogenic compound, on the swimming activity and the collective response of zebrafish. To investigate how the social environment influences individual response to acute caffeine treatment, we measured the behavior of a caffeine-treated subject swimming in isolation or in the presence of a group of untreated conspecifics. The experimental paradigm used a recently developed automatic tracking system to extract individual trajectories of zebrafish and preserve their identities over time. Our results indicate that caffeine reduces the swimming activity of fish tested in isolation, and that this effect is mitigated by the presence of untreated conspecifics. The collective response is also influenced by caffeine exposure at low doses, whereby caffeine-treated subjects may act as group leaders by taking anticipatory turning manoeuvres that are followed by the rest of the group. While anxiogenic effects of caffeine could explain the reduction in the swimming activity, the observed variation in the collective response is likely related to the interplay between anxiogenic and psychostimulant properties of caffeine.
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