Development and validation of a post-traumatic stress disorder model in zebrafish

MSc (Pharmacology), North-West University, Potchefstroom CampusPost-traumatic stress disorder (PTSD) is a severely debilitating chronic mental disorder brought about by encountering an actual or perceived, life-threatening or traumatic experience or series of events, like violence or combat. Specifically, PTSD is intrinsically associated with dysregulated fear conditioning, a form of Pavlovian conditioning where a neutral conditioned stimulus is paired with an aversive unconditioned stimulus. Lifetime prevalence of PTSD differs across regions of the world and is higher in countries emerging from conflict, with a reported lifetime PTSD prevalence of 2.3% in South Africa. Moreover, the pathophysiology of PTSD development is largely unknown and requires further examination. These hindrances necessitate innovative experimental apperception so that novel treatment strategies may be developed. One approach is to extend the range of translational model species used in PTSD research as to enable analysis of overlapping behavioural phenotypes. Because zebrafish exhibit evolutionary conserved homologies in neuronal circuitry and mediator systems with humans and rodents, this species has emerged as a useful model in translational research of complex neuropsychiatric disorders such as PTSD. Congruently, the advantages of using zebrafish in pre-clinical neuropsychiatric research also include homologous gene sequences and vertebrate-specific physiological processes, like organogenesis, shared with mammals. A commonly used animal model of PTSD is the predator exposure model (PEM), in which predator-related cues (visual or chemical) serve as warnings to animals about potential threats in the surrounding area. To this end, exposure to a pheromone-like exudate, conspecific alarm substance (CAS), mediates significant anti-predatory responses in zebrafish. CAS is produced by specialized epidermal club cells in the zebrafish skin whereupon it is secreted into the water consequent to skin damage. Released CAS is detected through olfaction by neighbouring zebrafish and alarm reactions are elicited. Accordingly, CAS-exposed zebrafish display exacerbated anxiety and fear behaviours and, thus, CAS may be useful in the analysis of fear and anxiety in zebrafish. In this study we aimed to develop a novel translational model of PTSD in zebrafish, in order to expand the PTSD research capabilities at the North West University (NWU). The primary objectives of the study were based on assessing the anxiety-like behavioural responses in zebrafish following CAS exposure. Furthermore, building on evidence in stress-restress models of PTSD in rodents, we aimed to explore the effect of re-experience in zebrafish. This would inform on whether CAS is capable of evoking a sustained anxiogenic response up to 2 days post stress exposure, with or without a visual reminder, thus emulating fear conditioned learning seen in clinical PTSD. In this regard, CAS was paired with contextual reminders to induce fear conditioning and perpetuated fear-like behaviour in the absence of the original stressor. Finally, we aimed to confer face (behavioural) validity to the model for future construct (biological) and predictive (treatment response) validity testing and subsequent application in pre-clinical drug screening initiatives at the NWU. A total of 72 zebrafish were employed in this study, of which 32 zebrafish were used as CAS donors. The remaining 40 fish were randomly divided into 4 groups (n = 10 per group), viz. ‘vehicle/no cue’, ‘CAS/no cue’, ‘vehicle/cue’ and ‘CAS/cue’. Zebrafish behaviour in the test tanks was recorded during both conditioning and re-experience. For conditioning on day 1, ensuing a 1-h habituation period, two groups were exposed to the vehicle (distilled water), and two groups were exposed to CAS. The exposure lasted for 6 min, with groups ‘vehicle/no cue’ and ‘CAS/no cue’ exposed in the absence of a specific visual cue, while groups ‘vehicle/cue’ and ‘CAS/cue’ were exposed in the presence of the visual cues (black and white stripes). For re-experience on day 2, zebrafish were returned to the test tanks for 6 min, groups ‘vehicle/no cue’ and ‘CAS/no cue’ in the absence of the visual cue, and groups ‘vehicle/cue’ and ‘CAS/cue’ in the presence of the visual cue, thereby allowing the conditioned stimulus to be assessed. Zebrafish were then captured and euthanised. The behavioural video recordings were analysed using EthoVision XT 14 tracking software to virtually divide the test tanks into 2 equal horizontal sections (bottom and top zones) and measuring frequency (number of entries) and total duration (s) in the top of the test tank. By that the position within the test tank was considered as a general index of anxiety with geotaxis indicative of greater anxiety. Further, total duration of immobility (s) and mean meandering (degrees/cm – degree of turning over distance travelled) were also scored as measurements of anxiety. CAS exposure significantly reduced frequency and time spent in the top zone, while immobility and meandering significantly increased following CAS exposure. Overall, this illustrates that CAS-exposed zebrafish displayed definite anxiety-like behaviour immediately after CAS exposure compared to non-exposed zebrafish. Thereon, the observed anxiety-like behaviour was sustained on day 2 under both cued and non-cued conditions. The current project establishes that CAS exposure evokes fear- and anxiety-like behaviour in zebrafish, not only during initial exposure, but also thereafter when zebrafish are presented with a contextual reminder in the absence of CAS. This ‘re-experiencing’ phenomenon is characteristic of PTSD-like fear conditioning and confirms the face validity of CAS ± contextual reminder as an effective stressor ± re-experience model of PTSD in zebrafish. That said, anxiety responses were independent of time and cue, implying that further development is required to firmly validate the perpetuation of behavioural fear responses over time using this protocol. Additionally, for future exploratory research to be meaningful, it is imperative that this model be assessed with respectMaster