Exploring quadruple stress in zebrafish (Danio rerio) as a novel model of posttraumatic stress disorder

Abstract

Posttraumatic stress disorder (PTSD)1 is a severe and incapacitating mental disorder that manifests in some individuals following a personal encounter with or experiencing a perceived threat and/or witnessing/observing others encountering a traumatic life-threatening event, e.g. sexual and/or physical assault, war-related incidents, and natural disasters. What makes PTSD unique from other anxiety-related neuropsychiatric disorders is that its diagnosis is contingent on a specific traumatic experience. Two principal components contribute to the development of PTSD, namely 1) encountering a severe, life-threatening and traumatizing event and 2) having a genetic, clinical or familial predisposition to the disorder. PTSD is characterized by flashbacks, emotional outbursts (mainly anger), avoidance of contextual reminders of the original traumatic event, and sleep disturbances. Patients suffering from PTSD are often subject to disrupted personal and familial relationships which place further strain on their social, medical, and financial stability. Moreover, up to 75 % of all individuals will at some point in time be exposed to a traumatic life event that fits the prodromal picture of PTSD. However, only a select few of those, i.e. roughly 12 %, will progress to develop PTSD. This points to individual variances in the risk for and resilience against the development of PTSD. The Diagnostic and Statistical Manual of Mental Disorders, 5th edition (DSM-5)2 describes the symptoms of PTSD under four main categories, i.e. i) intrusion (reliving the traumatic event in flashbacks); ii) avoidance (suppression or attempted suppression of distressing internal reminders); iii) negative mood and cognition (being incapable of remembering important details of the event); and iv) arousal and reactivity (i.e. sleep disturbances, hypervigilance, and lack of concentration). Suffice to say that the diagnosis of PTSD is complex, in that symptom manifestation varies considerably between individuals. The symptomatic manifestation of PTSD varies in each individual and many patients experience only one of the four main symptoms. Our knowledge and understanding of the pathophysiology of PTSD is less than optimal. This may likely be due to the complicated nature of its aetiology, as this is intricately linked to events that are mostly unforeseen. However, there are some hypotheses that provide insight into the pathophysiological basis of the condition. These all point to dysfunctional processes underlying the manner in which the human body processes and mitigates the immediate and long-lasting psychobiological effects of acute fear-induced stress following exposure to life-threatening situations.

Norepinephrine (NE)1, being a key stress hormone that guide the mammalian and human response to both implicit and external stressors, has been the focus of many PTSD2 and other anxiety disorder studies, since it is intricately involved in the processing of emotional memories. Specifically, it has been shown that the adrenergic alpha (α)1/2 receptors influence processes of working memory, spatial learning, and fear appraisal, while beta (β)1/2 receptors play a role in the processing of auditory fear, spatial reference, and fear memory retrieval. Evidence points to PTSD patients often presenting with an increased level of sympathetic functioning, compared to healthy controls, indicating a clear role for inflated noradrenergic signaling in PTSD. Regardless, given the suboptimal response of PTSD to drugs that target processes, our understanding of the actions of NE in PTSD remains unclear. Across all fields of neuropsychiatric research, animal models of human illness have historically proven to be invaluable tools to divulge the etiopathological mechanisms underlying human disorders and to identify novel targets for pharmacological therapy. Most rodent models of PTSD, although well studied, deliver varying and incongruent results. Zebrafish recently gained interest in preclinical research since the species presents with a variety of attributes that allow for experimental manipulation and the accurate characterization of specific behavioral traits that provide a valuable background for the study of PTSD-like phenomena. Despite this, further validation of the species as an animal model system of relevance for human disorders is still required. Here, we attempted to develop a standardized preclinical zebrafish model of PTSD by investigating whether a life-threatening, severe stress paradigm, i.e., a high intensity trauma, that is aimed at combining various but replicable constructs of life-threatening trauma, will be adequate to induce sustained PTSD-like fear in a zebrafish model as defined at both 1-, 4-, 7- and 14-days post-trauma. Further, considering the current uncertainty regarding the role of epinephrine (EPI3) in the development and manifestation of trauma-related anxiety, we will further seek to divulge its effect on trauma-related behaviors which will tested against the behavior of subjects exposed to either a sham4 (trauma-free) protocol, trauma inflicted in the absence of EPI administration, i.e. triple high intensity trauma (THIT5), or EPI administration only.

Importantly, we show that 1) trauma-paired EPI1 exposure consistently blunted the delayed anxiety-like sequalae associated with severe trauma, but bolstered persistent neophobia-related anxiety when paired with novelty (exposure to a novel circumstance) in the absence of additional stressors, 2) exposure to a trauma-paired context prior to testing strengthened the subsequent anxiety-like behavior trauma-exposed fish in the absence of additional EPI, and 3) despite this, trauma exposed- and EPI-only fish showed a lesser degree of contextual avoidance behavior, compared to sham2- or EPI-free, trauma-exposed fish In conclusion, we show that zebrafish are able to contextualize a severe and realistic life-threatening stressor. We further show that the resultant post-traumatic anxiety, which is context-sensitive, manifests for at least two weeks after trauma exposure. Since the concepts of persistent and delayed post-trauma anxiety, as well as contextual relevance are pivotal diagnostic symptoms of clinical PTSD3, the present work extends current efforts in preclinical PTSD research by describing a readily reproducible zebrafish model framework in which the psychobiological mechanisms that may underlie the clinical condition, can be explored. Further, the present investigation also highlighted a dichotomous role for adrenergic processes in the acquisition and consolidation of fear memory, which needs continued investigation.