The endocannabinoid gene faah2a modulates stress-associated behavior in zebrafish

Abstract

The ability to orchestrate appropriate physiological and behavioral responses to stress is important for survival, and is often dysfunctional in neuropsychiatric disorders that account for leading causes of global disability burden. Numerous studies have shown that the endocannabinoid neurotransmitter system is able to regulate stress responses and could serve as a therapeutic target for the management of these disorders. We used quantitative reverse transcriptase-polymerase chain reactions to show that genes encoding enzymes that synthesize (abhd4, gde1, napepld), enzymes that degrade (faah, faah2a, faah2b), and receptors that bind (cnr1, cnr2, gpr55-like) endocannabinoids are expressed in zebrafish (Danio rerio). These genes are conserved in many other vertebrates, including humans, but fatty acid amide hydrolase 2 has been lost in mice and rats. We engineered transcription activator-like effector nucleases to create zebrafish with mutations in cnr1 and faah2a to test the role of these genes in modulating stress-associated behavior. We showed that disruption of cnr1 potentiated locomotor responses to hyperosmotic stress. The increased response to stress was consistent with rodent literature and served to validate the use of zebrafish in this field. Moreover, we showed for the first time that disruption of faah2a attenuated the locomotor responses to hyperosmotic stress. This later finding suggests that FAAH2 may be an important mediator of stress responses in non-rodent vertebrates. Accordingly, FAAH and FAAH2 modulators could provide distinct therapeutic options for stress-aggravated disorders.

Novel zebrafish behavioral assay to identify modifiers of the rapid, nongenomic stress response

“…Some of these findings have been replicated in animal models, yet it needs to be investigated what the neuronal changes observed in vitro mean in the context of adaptive SR. Our assay system and what we are learning from modeling stress in zebrafish will be a useful platform to screen for such neural pathways and molecular components. In fact, we have identified several genetic loci that alter the rapid locomotor response to salt challenge including novel genes discovered in a pilot screen of random insertional mutants and targeted knockouts of cannabinoid receptor 1 ( cnr1 ) or fatty acid amide hydrolase 2a ( faah2a ) …”

“…Zebrafish, a freshwater teleost, depend primarily on cortisol signaling for osmoregulation . We previously reported that WT larval zebrafish (4 dpf) display increased frequencies of locomotion (number of movement/min) in response to hyperosmotic stress and that knockouts of endocannabinoid signaling genes cnr1 and faah2a and several novel genetic loci discovered in a pilot forward genetic screen show altered locomotor response to hyperosmotic stress . Others have observed that larval zebrafish swim away from an area with an increased osmolarity …”

“…40,41 We previously reported that WT larval zebrafish (4 dpf ) display increased frequencies of locomotion (number of movement/min) in response to hyperosmotic stress 28 and that knockouts of endocannabinoid signaling genes cnr1 and faah2a and several novel genetic loci discovered in a pilot forward genetic screen show altered locomotor response to hyperosmotic stress. 42,43 Others have observed that larval zebrafish swim away from an area with an increased osmolarity. 44 A noxious stimulant assay, using cinnamon oil (7.4 μg/mL), has been used as a control paradigm to show that changes in locomotion are not due to a simple loss of locomotor capacity ( Figure 1C).…”

“…In fact, we have identified several genetic loci that alter the rapid locomotor response to salt challenge including novel genes discovered in a pilot screen of random insertional mutants 43 and targeted knockouts of cannabinoid receptor 1 (cnr1) or fatty acid amide hydrolase 2a (faah2a). 42…”

Countering the Modern Metabolic Disease Rampage With Ancestral Endocannabinoid System Alignment

“…From the evolutionary standpoint, it is likely that the motivation and reward aspects of the central eCB/CB1R system co-evolved with the energy homeostatic aspect in order to coordinate feelings of pleasure with the successful acquisition of nourishment. As the search and pursuit of energy would have also entailed significant exposure to threats and stressors, the central eCB/CB1R system of zebrafish also influences the stress response and habituation to various stressors (124). In mammalian organisms, the eCBs of the pre-frontal cortex play a significant role in coping, resiliency, and termination of stress-activated glucocorticoid release induced by the hypothalamic-pituitary-adrenal (HPA) axis (125, 126).…”