Moderate early life stress improves adult zebrafish (Danio rerio) working memory but does not affect social and anxiety-like responses.

Early life stress (ELS) is defined as a short or chronic period of trauma, environmental or social deprivation, which can affect different neurochemical and behavioral patterns during adulthood. Zebrafish (Danio rerio) have been widely used as a model system to understand human neurodevelopmental disorders and display translationally relevant behavioral and stress-regulating systems. In this study, we aimed to investigate the effects of moderate ELS by exposing young animals (6-weeks postfertilization), for 3 consecutive days, to three stressors, and analyzing the impact of this on adult zebrafish behavior (16-week postfertilization). The ELS impact in adults was assessed through analysis of performance on tests of unconditioned memory (free movement pattern Y-maze test), exploratory and anxiety-related task (novel tank diving test), and social cohesion (shoaling test). Here, we show for the first time that moderate ELS increases the number of alternations in turn-direction compared to repetitions in the unconditioned Y-maze task, suggesting increased working memory, but has no effect on shoal cohesion, locomotor profile, or anxiety-like behavior. Overall, our data suggest that moderate ELS may be linked to adaptive flexibility which contributes to build "resilience" in adult zebrafish by improving working memory performance.

The Free-movement pattern Y-maze: A cross-species measure of working memory and executive function.

Numerous neurodegenerative and psychiatric disorders are associated with deficits in executive functions such as working memory and cognitive flexibility. Progress in developing effective treatments for disorders may benefit from targeting these cognitive impairments, the success of which is predicated on the development of animal models with validated behavioural assays. Zebrafish offer a promising model for studying complex brain disorders, but tasks assessing executive function are lacking. The Free-movement pattern (FMP) Y-maze combines aspects of the common Y-maze assay, which exploits the inherent motivation of an organism to explore an unknown environment, with analysis based on a series of sequential two-choice discriminations. We validate the task as a measure of working memory and executive function by comparing task performance parameters in adult zebrafish treated with a range of glutamatergic, cholinergic and dopaminergic drugs known to impair working memory and cognitive flexibility. We demonstrate the cross-species validity of the task by assessing performance parameters in adapted versions of the task for mice and Drosophila, and finally a virtual version in humans, and identify remarkable commonalities between vertebrate species' navigation of the maze. Together, our results demonstrate that the FMP Y-maze is a sensitive assay for assessing working memory and cognitive flexibility across species from invertebrates to humans, providing a simple and widely applicable behavioural assay with exceptional translational relevance.

Female adult zebrafish (Danio rerio) show higher levels of anxiety-like behavior than males, but do not differ in learning and memory capacity.

Zebrafish (Danio rerio) are widely used as a translational model for human neuropsychiatric conditions. Many studies have not considered sex differences in their analyses. Here, we studied sex differences of adult zebrafish in two behavioral domains: Anxiety and Memory. To assess whether sex influences anxiety-like responses, we used two different behavioral protocols, the novel tank diving task and the light-dark test. To assess sex differences in learning and memory tasks, we explored two memory domains, short-term spatial memory (free movement pattern Y-maze task) and short-term fear memory (Pavlovian fear-conditioning task). Although we did not find any significant difference in learning and memory tasks, female zebrafish showed robust increases in anxiety-like behavioral endpoints in both anxiety tests. Overall, our data suggest that zebrafish is a sensitive model to work with sex differences when modeling anxiety-related disorders and this should be an important factor to consider in different experimental designs.

Zebrafish models of impulsivity and impulse control disorders.

Impulse control disorders (ICDs) are characterized by generalized difficulty controlling emotions and behaviors. ICDs are a broad group of the central nervous system (CNS) disorders including conduct disorder, intermittent explosive, oppositional-defiant disorder, antisocial personality disorder, kleptomania, pyromania and other illnesses. Although they all share a common feature (aberrant impulsivity), their pathobiology is complex and poorly understood. There are also currently no ICD-specific therapies to treat these illnesses. Animal models are a valuable tool for studying ICD pathobiology and potential therapies. The zebrafish (Danio rerio) has become a useful model organism to study CNS disorders due to high genetic and physiological homology to mammals, and sensitivity to various pharmacological and genetic manipulations. Here, we summarize experimental models of impulsivity and ICD in zebrafish and highlight their growing translational significance. We also emphasize the need for further development of zebrafish ICD models to improve our understanding of their pathogenesis and to search for novel therapeutic treatments.

Taurine modulates the stress response in zebrafish.

The zebrafish (Danio rerio) is used as an emergent model organism to investigate the behavioral and physiological responses to stress. The anxiolytic-like effects of taurine in zebrafish support the existence of different mechanisms of action, which can play a role in preventing stress-related disorders (i.e., modulation of GABAA, strychnine-sensitive glycine, and NMDA receptors, as well as antioxidant properties). Herein, we investigate whether taurine modulates some behavioral and biochemical responses in zebrafish acutely submitted to chemical and mechanical stressors. We pretreated zebrafish for 1 h in beakers at 42, 150, and 400 mg/L taurine. Fish were later acutely exposed to a chemical stressor (conspecific alarm substance) or to a mechanical stressor (net chasing), which elicits escaping responses and aversive behaviors. Locomotion, exploration, and defensive-like behaviors were measured using the novel tank and the light-dark tests. Biochemical (brain oxidative stress-related parameters) and whole-body cortisol levels were also quantified. We showed that taurine prevents anxiety/fear-like behaviors and protein carbonylation and dampens the cortisol response following acute stress in zebrafish. In summary, our results demonstrate a protective role of taurine against stress-induced behavioral and biochemical changes, thereby reinforcing the growing utility of zebrafish models to investigate the neuroprotective actions of taurine in vertebrates.

Zebrafish (Danio rerio) behavioral laterality predicts increased short-term avoidance memory but not stress-reactivity responses.

Once considered a uniquely human attribute, behavioral laterality has proven to be ubiquitous among non-human animals, and is associated with several neurophenotypes in rodents and fishes. Zebrafish (Danio rerio) is a versatile vertebrate model system widely used in translational neuropsychiatric research owing to their highly conserved genetic homology, well-characterized physiological responses, and extensive behavioral repertoire. Although spontaneous left- and right-biased responses, and associated behavioral domains (e.g., stress reactivity, aggression, and learning), have previously been observed in other teleost species, no information relating to whether spontaneous motor left-right-bias responses of zebrafish predicts other behavioral domains has been described. Thus, we aimed to investigate the existence and incidence of natural left-right bias in adult zebrafish, exploiting an unconditioned continuous free movement pattern (FMP) Y-maze task, and to explore the relationship of biasedness on performance within different behavioral domains. This included learning about threat cues in a Pavlovian fear conditioning test, and locomotion and anxiety-related behavior in the novel tank diving test. Although laterality did not change locomotion or anxiety-related behaviors, we found that biased animals displayed a different search strategy in the Y-maze, making them easily discernable from their unbiased counterparts, and increased learning associated to fear cues. In conclusion, we showed, for the first time, that zebrafish exhibit a natural manifestation of motor behavioral lateralization which can influence aversive learning responses.

Negative impacts of social isolation on behavior and neuronal functions are recovered after short-term social reintroduction in zebrafish.

Recently, social isolation measures were crucial to prevent the spread of the coronavirus pandemic. However, the lack of social interactions affected the population mental health and may have long-term consequences on behavior and brain functions. Here, we evaluated the behavioral, physiological, and molecular effects of a social isolation (SI) in adult zebrafish, and whether the animals recover such changes after their reintroduction to the social environment. Fish were submitted to 12 days of SI, and then reintroduced to social context (SR). Behavioral analyses to evaluate locomotion, anxiety-like and social-related behaviors were performed after SI protocol, and 3 and 6 days after SR. Cortisol and transcript levels from genes involved in neuronal homeostasis (c-fos, egr, bdnf), and serotonergic (5-HT) and dopaminergic (DA) neurotransmission (thp, th) were also measured. SI altered social behaviors in zebrafish such as aggression, social preference, and shoaling. Fish submitted to SI also presented changes in the transcript levels of genes related to neural activity, and 5-HT/DA signaling. Interestingly, most of the behavioral and molecular changes induced by SI were not found again 6 days after SR. Thus, we highlight that SR of zebrafish to their conspecifics played a positive role in social behaviors and in the expression of genes involved in different neuronal signaling pathways that were altered after 12 days of SI. This study brings unprecedented data on the effects of SR in the recovery from SI neurobehavioral alterations, and reinforces the role of zebrafish as a translational model for understanding the neurobiological mechanisms adjacent to SI and resocialization.

Transgenerational effects of early-life stress on anxiety behavior in zebrafish (Danio rerio).

Early-life adversity impacts on anxiety-related behaviors in adulthood. The effects of such adversity not only affect the animal itself, but can be passed on transgenerationally. Pervasive effects of experimentally-induced early-life stress (ELS) have been documented in adult zebrafish but it is not clear if this can be passed on via the germline. Here, we investigated the effects of ELS across three generations, by analyzing the responses of adult animals exposed to ELS in two different anxiety-related tasks, as well as in social behavior, memory, and cognition. Animals exposed to ELS (at 7 days-post-fertilization) showed a marked attenuation of specific anxiety-related behaviors (F0) as adults, and these alterations were maintained across two subsequent generations (F1 and F2). This suggest zebrafish may be a useful model organism to study the transgenerational effects of ELS, and how this pertains to (for example) neuropsychiatric disorders. In addition, our data may naturally provoke questions regarding consideration of the environment of laboratory-housed zebrafish at early developmental stages. In particular, more work may be necessary to determine how different environmental stressors could affect data variability across laboratories.

Ontogeny of working memory and behavioural flexibility in the free movement pattern (FMP) Y-maze in zebrafish.

The acquisition of executive skills such as working memory, decision-making and adaptive responding occur at different stages of central nervous system development. Zebrafish (Danio rerio) are increasingly used in behavioural neuroscience for complex behavioural tasks, and there is a critical need to understand the ontogeny of their executive functions. Zebrafish across developmental stages (4, 7, 14, 30 and 90 days post fertilisation (dpf)), were assessed to track development of working memory (WM) and behavioural flexibility (BF) using the free movement pattern Y-maze (FMP Y-maze). Several differences in both WM and BF were identified during the transition from yolk-dependent to independent feeding. Specifically, WM is evident in all age groups, even from 4 dpf. However, BF is not developed until larvae start free feeding, and show significant improvement thereafter, with young adults (90 dpf) demonstrating the most well-defined BF. We demonstrate, for the first time, objective WM processes in 4 dpf zebrafish larvae. This suggests that those wishing to study WM in zebrafish may be able to do so from 4 dpf, thus drastically increasing throughput. In addition, we show that zebrafish follow distinct stages of cognitive development and age-related changes during the early developmental period. Finally, our findings indicate distinct WM and BF mechanisms, which may be useful to study for translational purposes.

adgrl3.1-deficient zebrafish show noradrenaline-mediated externalizing behaviors, and altered expression of externalizing disorder-candidate genes, suggesting functional targets for treatment.

Externalizing disorders (ED) are a cause of concern for public health, and their high heritability makes genetic risk factors a priority for research. Adhesion G-Protein-Coupled Receptor L3 (ADGRL3) is strongly linked to several EDs, and loss-of-function models have shown the impacts of this gene on several core ED-related behaviors. For example, adgrl3.1-/- zebrafish show high levels of hyperactivity. However, our understanding of the mechanisms by which this gene influences behavior is incomplete. Here we characterized, for the first time, externalizing behavioral phenotypes of adgrl3.1-/- zebrafish and found them to be highly impulsive, show risk-taking in a novel environment, have attentional deficits, and show high levels of hyperactivity. All of these phenotypes were rescued by atomoxetine, demonstrating noradrenergic mediation of the externalizing effects of adgrl3.1. Transcriptomic analyses of the brains of adgrl3.1-/- vs. wild-type fish revealed several differentially expressed genes and enriched gene clusters that were independent of noradrenergic manipulation. This suggests new putative functional pathways underlying ED-related behaviors, and potential targets for the treatment of ED.

The zebrafish (Danio rerio) anxiety test battery: comparison of behavioral responses in the novel tank diving and light-dark tasks following exposure to anxiogenic and anxiolytic compounds.

RATIONALE: Triangulation of approaches (i.e., using several tests of the same construct) can be extremely useful for increasing the robustness of the findings being widely used when working with behavioral testing, especially when using rodents as a translational model. Although zebrafish are widely used in neuropharmacology research due to their high-throughput screening potential for new therapeutic drugs, behavioral test battery effects following pharmacological manipulations are still unknown. METHODS: Here, we tested the effects of an anxiety test battery and test time following pharmacological manipulations in zebrafish by using two behavioral tasks: the novel tank diving task (NTT) and the light-dark test (LDT). Fluoxetine and conspecific alarm substance (CAS) were chosen to induce anxiolytic and anxiogenic-like behavior, respectively. RESULTS: For non-drug-treated animals, no differences were observed for testing order (NTT → LDT or LDT → NTT) and there was a strong correlation between performances on the two behavioral tasks. However, we found that during drug treatment, NTT/LDT responses are affected by the tested order depending on the test time being fluoxetine effects higher at the second behavioral task (6 min later) and CAS effects lower across time. CONCLUSIONS: Overall, our data supports the use of baseline behavior assessment using this anxiety test battery. However, when working with drug exposure, data analysis must carefully consider time-drug-response and data variability across behavioral tasks.

The larval diving response (LDR): Validation of an automated, high-throughput, ecologically relevant measure of anxiety-related behavior in larval zebrafish (Danio rerio).

BACKGROUND: Zebrafish are used in anxiety research as the species' naturalistic diving response to a new environment is a reliable and validated marker for anxiety-like behavior. One of the benefits of using zebrafish is the potential for high throughput drug screens in fish at the larval stage. However, at present, tests of anxiety in larvae and adults often measure different endpoints. NEW METHOD: Here, for the first time, we have adapted the novel tank diving response test for examining diving behavior in zebrafish larvae to assess anxiety-like behaviors at very early-stages (7 days-post-fertilization [dpf]). COMPARISON WITH EXISTING METHODS: Current methods to examine anxiety in larvae can show low reliability, and measure different endpoints as in adults, thus calling into question their translational relevance. RESULTS: We found that 7dpf zebrafish spent more time at the bottom of a small novel tank. We validated this as anxiety-like behaviors with diazepam reducing, and caffeine increasing the time spent in the bottom of the novel environment. CONCLUSIONS: This new automated and high-throughput screening tool has the potential use for screening of anxiogenic and anxiolytic compounds, and for studies aiming to better understand anxiety-like behaviors.

Modelling ADHD-Like Phenotypes in Zebrafish.

The use of multiple species to model complex human psychiatric disorders, such as ADHD, can give important insights into conserved evolutionary patterns underlying multidomain behaviors (e.g., locomotion, attention, and impulsivity). Here we discuss the advantages and challenges in modelling ADHD-like phenotypes in zebrafish (Danio rerio), a vertebrate species that has been widely used in neuroscience and behavior research. Moreover, multiple behavioral tasks can be used to model the core symptoms of ADHD and its comorbidities. We present a critical review of current ADHD studies in zebrafish, and how this species might be used to accelerate the discovery of new drug treatments for this disorder.

Using zebrafish (Danio rerio) models to understand the critical role of social interactions in mental health and wellbeing.

Social behavior represents a beneficial interaction between conspecifics that is critical for maintaining health and wellbeing. Dysfunctional or poor social interaction are associated with increased risk of physical (e.g., vascular) and psychiatric disorders (e.g., anxiety, depression, and substance abuse). Although the impact of negative and positive social interactions is well-studied, their underlying mechanisms remain poorly understood. Zebrafish have well-characterized social behavior phenotypes, high genetic homology with humans, relative experimental simplicity and the potential for high-throughput screens. Here, we discuss the use of zebrafish as a candidate model organism for studying the fundamental mechanisms underlying social interactions, as well as potential impacts of social isolation on human health and wellbeing. Overall, the growing utility of zebrafish models may improve our understanding of how the presence and absence of social interactions can differentially modulate various molecular and physiological biomarkers, as well as a wide range of other behaviors.

Tricaine Methanesulfonate (MS222) Has Short-Term Effects on Young Adult Zebrafish (Danio rerio) Working Memory and Cognitive Flexibility, but Not on Aging Fish.

Exposure to anesthetic drugs is common in biomedical sciences being part of routine procedures in different translational species, however its impacts on memory and cognition are still debated, having different impacts depending on drug and age. The zebrafish (Danio rerio) is a translational species widely used in behavioral neuroscience, where tricaine methanesulfonate (MS222) is the most acceptable and used drug when conducting routine procedures. Based on this, we investigated the effects of MS222 (100 mg/l) in young adults and aging zebrafish 1, 2, 3, and 7 days after exposure. Animals' were submitted to the anesthetic procedure until loss of body posture, slowing of opercular movements and lack of response to tail touch with a plastic pipette were achieved, then further left in the drug for 3 min. After that, animals (6 mpf vs. 24 mpf) were transferred to a recovery tank until fully recovered and transferred back to their housing system until further testing in the free movement pattern (FMP) Y-maze, which assesses zebrafish working memory and cognitive lexibility. Young animals had significant impairment in their working memory and cognitive flexibility 1 and 2 days after the exposure to MS222, being fully recovered by day 3 and with no effects 7 days post drug exposure. Increased repetitions were also observed for animals exposed to MS222 which could indicate increased stress-related response in animals up to 2 days after drug exposure. No drug effect was observed in aging animals besides their natural decreased alternations and working memory. Overall, behavioral experiments after routine procedures using MS222 should be performed with caution and need to be delayed, at least 3 days after exposure where working memory, cognitive flexibility, and repetitive behavior are back to normal.

The impact of water changes on stress and subject variation in a zebrafish (Danio rerio) anxiety-related task.

BACKGROUND: Zebrafish have been widely used to study anxiety-related phenotypes using the novel tank test (NTT). Although the NTT is well-characterized and commonly used by researchers, there is still a lack of information regarding how different experimental variables such as water quality can influence NTT performance. Zebrafish use different chemical cues and olfactory stimuli to communicate in water, so we predicted that water change frequency would affect cortisol, locomotion and anxiety-related parameters in the NTT. NEW METHODS: After extensive literature research, we found that only about 18% of papers using NTT report partial or complete water changes between subjects. Here, we tested multiple zebrafish in the NTT using the same water up to 9 consecutive times (with no water change) and analyzed cortisol levels, as a stressrelated marker. RESULTS: We found that when using the same water for more than 4 trials, data variability is increased and a higher number of extreme values is observed for the time spent in the top zone and immobility. Moreover, after 4 trials with no water change, increased cortisol levels are observed, indicating that animals show increased stressrelated responses with the lack of water changes. CONCLUSIONS: This study shows that lack of water change can significantly influence zebrafish stress-responses in the NTT. Altogether, behavioral experiments should avoid using the same water when testing multiple fish in the task, especially when looking at anxiety in the NTT.

The cognitive and behavioral effects of D-amphetamine and nicotine sensitization in adult zebrafish.

BACKGROUND: Zebrafish are growing in use as a model for understanding drug dependence and addiction. Sensitization paradigms have been a useful tool in identifying mechanisms involved in drug-induced behavioral and neurological changes, but in zebrafish have tended to focus on locomotor, rather than cognitive, endpoints. METHODS: Here, we used a novel method, the FMP Y-maze, which measures continuous performance through a series of repeated binary choices (L vs R), to establish a model for assessing parameters associated with psychostimulant-induced behavioral and cognitive sensitization in adult zebrafish. RESULTS: Repeat, intermittent exposure to d-amphetamine (AMPH) for 14 days increased alternations (LRLR) in the maze, suggesting improved working memory, which was enhanced further following drug challenge after a short withdrawal period, suggesting behavioral sensitization. However, this cognitive enhancement coincided with a reduction in the use of other exploration strategies, hypolocomotion, and inhibition of cognitive flexibility. Like AMPH, exposure to nicotine (NIC) increased alternations following drug challenge after chronic treatment. Repeat NIC exposure appeared to induce both cognitive and psychomotor sensitization, as evidenced by increased working memory performance (alternations) and locomotor activity, without negatively impacting other search strategies or cognitive flexibility. CONCLUSION: Chronic treatment with AMPH or NIC boosts cognitive performance in adult zebrafish. Cognitive sensitization occurred with both drugs, resulting in enhanced working memory; however, repeat AMPH exposure, following a withdrawal period, resulted in inhibited cognitive flexibility, an effect not evident with repeat NIC exposure. Cognitive and behavioral sensitization paradigms in zebrafish could serve as a useful tool for assessing cognitive states which result in cognitive enhancing or impairing effects of drugs.

Chronic unpredictable early-life stress (CUELS) protocol: Early-life stress changes anxiety levels of adult zebrafish.

Early-life stress can lead to two different behavioral responses: (1) increased susceptibility to psychiatric disorders or (2) resilience. Here, we created a chronic unpredictable early-life stress (CUELS) protocol to assess the effects of early experiences in adult zebrafish. Animals were exposed to mild stressors twice a day and the duration was varied between groups (0, 1, 3, 7 and 14 days of stress). The stressor consisted of light/dark cycle changes; social isolation; overcrowding; water changes; water cooling; mechanical stirring; water heating; and immersion in shallow water. Behavior was assessed at young stages (21 days post-fertilization - open field analysis) and adulthood (4-months-old - novel tank diving test, light/dark task, shoaling, free movement pattern Y-maze and Pavlovian fear conditioning). Cortisol levels were assessed to evaluate the impact of CUELS in the HPI axis. Zebrafish exposed to 7 days of CUELS showed a decreased anxiety-like phenotype in two behavioral tasks, presenting increased time spent in top and decreased time spent in the dark area. Animals exposed to 14 days of CUELS showed an opposite anxious phenotype compared to 3 and 7 days of CUELS. No significant changes were observed in memory and cognition, social behavior and cortisol levels. In general, 7 days of CUELS protocol decreased anxiety in young and adult zebrafish, and could be used to understand the mechanisms underlying early-life experiences-derived alterations in neural circuits of anxiety.

The effects of two stressors on working memory and cognitive flexibility in zebrafish (Danio rerio): The protective role of D1/D5 agonist on stress responses.

Acute stressors are recurrent in multiple species' lives and can facilitate or impair cognition. The use of zebrafish (Danio rerio) as a translational species to understand the mechanisms by which stress induces different behavioral phenotypes has been widely studied. Two acute stressors are recognized when using this species: (1) conspecific alarm substance (CAS); and (2) net chasing. Here, we tested if CAS or net chasing would affect working memory and cognitive flexibility by testing performance in the FMP Y-maze after exposure to stress. We observed that CAS altered zebrafish behavioral phenotypes by increasing repetitive behavior; meanwhile, animals showed different patterns of repetitive behavior when exposed to net chasing, depending on the chasing direction. Because D1 receptors were previously studied as a potential mechanism underlying stress responses in different species, here, we pretreated fish with a D1/D5 agonist (SKF-38393) to assess whether this system plays a role in repetitive behavior in the FMP Y-maze. The pretreatment with D1/D5 agonist significantly decreased repetitive behavior in CAS exposed animals, and cortisol levels for both stressed groups, suggesting that the dopaminergic system plays an important role in zebrafish stress-related responses.

The impact of chronic unpredictable early-life stress (CUELS) on boldness and stress-reactivity: Differential effects of stress duration and context of testing.

Early-life stress (ELS) has been shown to result in a diverse array of long-lasting impacts; for example, increasing vulnerability to disease or building 'resilience' in adulthood. Previously, zebrafish (Danio rerio) have been used to understand the mechanisms by which ELS induces different behavioral phenotypes in adults, with alterations in both learning and anxiety observed in exposed individuals. Here, we subjected zebrafish larvae to chronic unpredictable early-life stress (CUELS) for 7 or 14 days, to investigate the impact on boldness towards a new environment and novel object, and stress-reactivity. We observed that 7 days of CUELS resulted in increased time spent in the top of a novel tank (indicating boldness) but did not alter approach to a novel object. Although CUELS did not affect stress-reactivity in terms of cortisol levels, decreased anxiety-like response to conspecific alarm substance (CAS) was observed in both ELS groups (7 and 14 days of CUELS). Therefore, for the first time, we observe a potential negative effect of CUELS by dampening the behavioral stress response following exposure to CAS. Overall, these data support the use of zebrafish as a translational model to study the broad range of ELS-induced permanent changes in behavior. It could also be used to investigate the mechanisms underlying both the positive and the negative effects of early-life adversity.