The small teleost zebrafish (Danio rerio) has become a critically important laboratory animal in biomedicine. One of their key practical advantages, the convenient method of small-molecule administration via water immersion, has certain problems with dosing precision and drug delivery. Here, we present a simple protocol for the intranasal delivery of neuroactive drugs in adult zebrafish using arecoline and nicotine, two well-studied reference neuroactive drugs chosen for the proof of concept. Adult fish received 1 μL water solution of arecoline (1 and 10 mg/mL) or nicotine tartrate (5 and 10 mg/mL) or the same volume of drug-free water (control) into both nostrils, followed by behavioral testing in the novel tank test 5 min later. Mass spectrometry analyses confirmed that both drugs rapidly reached the zebrafish brain following intranasal administration. Intranasally administered arecoline (10 mg/mL) and nicotine (5 and 10 mg/mL) demonstrated overt behavioral profiles, evoking characteristic anxiolytic-like effects in zebrafish similar to those observed here for a standard 20-min water immersion method (10 mg/L arecoline or 30 mg/L nicotine). Overall, we showed that neuroactive drugs can be delivered to adult zebrafish intranasally to exert central effects, which may (at least for some drugs) occur faster and can need smaller drug quantities than for the water immersion delivery.

Tyramine, β-phenylethylamine, octopamine and other trace amines are endogenous substances recently recognized as important novel neurotransmitters in the brain. Trace amines act via multiple selective trace amine-associated receptors (TAARs) of the G protein-coupled receptor family. TAARs are expressed in various brain regions and modulate neurotransmission, neuronal excitability, adult neurogenesis, cognition, mood, locomotor activity and olfaction. Disrupted trace amine circuits have been implicated in various clinical neuropsychiatric disorders, including schizophrenia, Parkinson's disease, addiction, depression and anxiety. Dysregulated TAAR signaling has been linked in rodents to altered dopamine and serotonin neurotransmission, known to be associated with these psychiatric conditions. Complementing rodent genetic and pharmacological evidence, zebrafish (Danio rerio) are rapidly becoming a novel powerful model system in translational neuropharmacology research. Here, we review trace amine/TAAR neurobiology in zebrafish and discuss their developing translational utility as pharmacological and genetic models for unraveling the role of trace amines in CNS processes and brain disorders.

Zebrafish (Danio rerio) represents a complementary pre-clinical model in stress and anxiety research. Conspecific alarm substance (CAS), an alarm pheromone secreted by injured fish, acts as a warning signal and modulates fear responses. Given their schooling nature and that injury precedes CAS release, varying fresh CAS concentrations extracted from different numbers of CAS-donating zebrafish may uniquely influence trauma-related behaviours. Thus, we investigated the behaviour of juvenile and adult zebrafish exposed to traumatic stress protocols, in the presence of CAS extracted from varying numbers of donating zebrafish. Juveniles were assessed for anxiety and boldness in the light-dark and open field tests (LDT and OFT), while adults were assessed in the novel tank test (NTT) and novel OFT (nOFT). We found that (1) trauma minimally impacted juvenile behaviour regardless of donor-derived CAS concentrations, (2) trauma-exposed adults displayed reduced exploration and heightened risk-taking behaviours in the NTT and nOFT compared to control-exposed fish, (3) NTT and nOFT freezing behaviours were distinctly emulated in adult fish and (4) post-trauma behaviour in adults was influenced by the number of donors. Therefore, CAS concentration as determined by donor number has age-related effects on anxiety- and risk-taking behaviours in trauma-exposed zebrafish, a valuable finding for studies utilising fresh CAS as a stress trigger. While we did not directly investigate CAS concentration through serial dilution, our data are of significant translational and ethological relevance, highlighting the importance of in-house method standardization in stress-related studies utilizing fresh CAS as an alarm cue.

Traumatic stress exposure increases noradrenaline (NA) release, which contributes to anxiety and impaired risk-appraisal. Guanfacine, a selective alpha-2A adrenergic receptor agonist, has been used to treat stress-related disorders characterised by impaired prefrontal cortex function. By acting on both presynaptic inhibitory autoreceptors and postsynaptic heteroreceptors, guanfacine attenuates stress reactivity and enhances cognition. However, its effectiveness in treating trauma-related anxiety and risk-taking behaviour remains unclear. Leveraging the advantages of zebrafish (Danio rerio ) as a sensitive and efficient preclinical model which is ideal for stress research, we explored the impact of traumatic stress exposure combined with varying concentrations of guanfacine in adult zebrafish. Zebrafish were evaluated for trauma-related anxiety using both the novel tank test (NTT) and a novel version of the open-field test (nOFT), the latter which was also used to investigate risk-taking behaviour. We found that (1) traumatic stress exposure led to heightened risk-taking behaviour in the nOFT, and (2) low-to-moderate concentrations of guanfacine (3-20 µg/L) attenuated anxiety-like, but not risk-taking behaviour, with the highest concentration (40 µg/L), showing no effect. These results highlight the complex role of NA in modulating dysregulated behaviours during traumatic events and indicate the potential of guanfacine for improving trauma-related anxiety and risk-taking behaviour.

Despite years of progress in biotechnology, altering the genetic makeup of many plant species, especially their plastids, remains challenging. The existence of a cell wall poses a significant obstacle to the effectual transportation of biomolecules. Developing efficient methods to introduce genes into plant cells and organelles without causing harm is an ongoing area of research. Traditional approaches like Agrobacterium-mediated transformation, biolistic particle delivery, electroporation and polyethylene glycol (PEG) transformation have shown some success but come with limitations like laborious, time-consuming and causing tissue damage. For instance, the Agrobacterium method can be applied only to the restricted host range, while PEG transformation and biolistic particle delivery are not very efficient. In contrast, nanotechnology made an appearance in the field of genetic engineering. Nanoparticles act as delivery vehicles for many cargos in animals. However, in plants, the application of nanocarriers for the delivery of biomolecules is still in its infant stage. Nonetheless, it holds immense potential for the future of plant biotechnology and genome editing.

Studies on memory consolidation and reconsolidation, memory loss, and the associated biochemical mechanisms have garnered interest in the past decades due to knowledge of memory performance-affecting factors such as stress, emotions, sleep, age, several neurological diseases, drugs, and chemical pollutants. Memory research has been using animal models, with increased interest in the zebrafish model. This freshwater fish species shows a wide range of behaviors relevant to memory research such as social behavior, aggression, and predator avoidance; however, few studies have investigated the duration of long-term memory. Hence, we designed an experiment to test memory duration by exposing zebrafish to avoidance conditioning using electroshock as the aversive stimulus. Zebrafish were trained to avoid the black side of a black-and-white tank and subsequently tested for aversive memory at 24 h, 48 h, 72 h, 96 h, 168 h, and 240 h. At the 72 h-interval, another zebrafish group was trained and exposed to MK-801(NMDAr antagonist) and then tested. The fish retained memories of the task and avoided the black side of the tank for up to 7 days. At 10 days post-training, the animals could no longer retrieve the aversive memory. Zebrafish treated with MK-801 did not retrieve memory. Knowledge of memory and of long-term memory duration is crucial for optimizing the zebrafish model for use in research investigating cognitive impairments such as memory loss and its ramifications. Additionally, identifying a long-term aversive memory lasting up to 7 days in zebrafish enables further research into the neuronal changes underlying this persistence. Such in-depth investigation could bring valuable insights into memory mechanisms and facilitate targeted interventions for memory-related conditions.

Chronic stress-related brain disorders are widespread and debilitating, and often cause lasting neurobehavioral deficits. Minocycline, a common antibiotic and an established inhibitor of microglia, emerges as potential treatment of these disorders. The zebrafish (Danio rerio) is an important emerging model organism in translational neuroscience and stress research. Here, we evaluated the potential of minocycline to correct microglia-mediated behavioral, genomic and neuroimmune responses induced by chronic unpredictable stress (CUS) in adult zebrafish. We demonstrated that CUS evoked overt behavioral deficits in the novel tank, light-dark box and shoaling tests, paralleled by elevated stress hormones (CRH, ACTH and cortisol), and upregulated brain expression of the 'neurotoxic M1' microglia-specific biomarker gene (MHC-2) and pro-inflammatory cytokine genes (IL-1β, IL-6 and IFN-γ). CUS also elevated peripheral (whole-body) pro-inflammatory (IL-1β, IFN-γ) and lowered anti-inflammatory cytokines (IL-4 and IL-10), as well as reduced whole-brain serotonin, dopamine and norepinephrine levels, and increased brain dopamine and serotonin turnover. In contrast, minocycline attenuated most of these effects, also reducing CUS-elevated peripheral levels of IL-6 and IFN-γ. Collectively, this implicates microglia in zebrafish responses to chronic stress, and suggests glial pathways as potential evolutionarily conserved drug targets for treating stress-evoked neuropathogenesis. Our findings also support the growing translational value of zebrafish models for understanding complex molecular mechanisms of brain pathogenesis and its therapy.

With increasing evidence showing the connections between the microbiome, neurophysiology, and behavior, our research endeavors to investigate whether the consumption of a combination of two postbiotics with antioxidant effects can affect behavior regulation in model species. Here, we worked with a combination (1:1 ratio) of heat-treated Bifidobacterium longum subsp. longum ES1 (CECT7347) and Lacticaseibacillus rhamnosus BPL15 (CECT8361) as a dietary supplement. To examine the potential benefit of using this formulation to alleviate anxiety-like behavior, we employed two model species, Caenorhabditis elegans and adult Danio rerio. In C. elegans, the postbiotic supplementation reduced the anxiety-related behavior analyzed by means of the octanol avoidance test. In zebrafish, the novel tank test indicated a different swimming pattern 2 and 4 months after the animals were fed with the postbiotic combination. While fish did not exhibit any variance in their locomotion parameters such as pace and speed, they showed a statistically significant preference to spend more time in the upper zone of the water tank, a behavior that is correlated with a lower anxiety-like behavior in these species. Our aim with this study is to present evidence that can be used to develop whole-cell postbiotic-based novel and innovative dietary supplements for anxiety-related conditions.

Background/Objectives: Ongoing challenges in epilepsy therapy warrant research on alternative treatments that offer improved efficacy and reduced side effects. Designed to enhance mitochondrial targeting and increase bioavailability, mitocurcumin (MitoCur) was evaluated for the first time as an antiepileptic agent, with curcumin (Cur) and sodium valproate (VPA), a standard antiepileptic drug, included for comparison. This study investigated the effects on seizure onset, severity, and progression in a zebrafish model of pentylenetetrazole (PTZ)-induced seizures and measured the concentrations of the compounds in brain tissue. Methods: Zebrafish were pre-treated with MitoCur and Cur (both at 0.25 and 0.5 µM doses) and VPA (0.25 and 0.5 mM) and observed for four minutes to establish baseline locomotor behavior. Subsequently, the animals were exposed to a 5 mM PTZ solution for 10 min, during which seizure progression was observed and scored as follows: 1-increased swimming; 2-burst swimming, left and right movements; 3-circular movements; 4-clonic seizure-like behavior; 5-loss of body posture. The studied compounds were quantified in brain tissue through HPLC and LC-MS. Results: Compared to the control group, all treatments reduced the distance moved and the average velocity, without significant differences between compounds or doses. During PTZ exposure, seizure latencies revealed that all treatments effectively delayed seizure onset up to score 4, demonstrating efficacy in managing moderate seizure activity. Notably, MitoCur also provided significant protection against the most severe seizure score (score 5). Brain tissue uptake analysis indicated that MitoCur achieved higher concentrations in the brain compared to Cur, at both doses. Conclusions: These results highlight the potential of MitoCur as a candidate for seizure management.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and subsequent depletion of dopamine in the striatum. Solanesol, an alcohol that acts as a precursor to coenzyme Q10, possesses potential applications in managing neurological disorders with antioxidant, anti-inflammatory, and neuromodulatory potential. In this study, a zebrafish model was employed to investigate the effects of solanesol in tramadol induced PD like symptoms. Zebrafish were administered tramadol injections (50 mg/kg) over a 20-day period. Solanesol was administered at doses of 25, 50, and 100 mg/kg, three hours prior to tramadol administration from day 11 to day 20. Behavioral tests assessing motor coordination were conducted on a weekly basis using open field and novel diving tank apparatus. On day 21, the zebrafish were euthanized, and brain tissues were examined for markers of oxidative stress, inflammation, and neurotransmitters level. Chronic tramadol treatment resulted in motor impairment, reduced antioxidant enzyme levels, enhanced release of proinflammatory cytokines in the striatum, and disrupted neurotransmitter balance. However, solanesol administration mitigated these effects and exhibited a neuroprotective effect against neurodegenerative alterations in the zebrafish model of PD. This was evident through improvements in behavior, modulation of biochemical markers, attenuation of neuroinflammation, restoration of neurotransmitters level, and enhancement of mitochondrial activity. The histopathological study also confirmed that solanesol dose dependently restored neuronal cell density which confirmed its neuroprotective potential. Further investigations are required to elucidate the underlying mechanisms of solanesol neuroprotective effects and evaluate its efficacy in human patients.

Though commonly used to model affective disorders, zebrafish display notable differences in terms of the structure and function of the brain serotonin system, including responses to pharmacological interventions, as compared to mammals. For example, elevation of brain serotonin following acute administration of serotonin reuptake inhibitors (SRIs) generally has anxiogenic effects, both in the clinical situation and in rodent models of anxiety, but previous research has indicated the opposite in zebrafish. However, several issues remain unresolved. We conducted a systematic review of SRI effects in zebrafish models of anxiety and, on the basis of these results, performed a series of experiments further investigating the influence of serotonin-releasing agents on anxiety-like behaviour in zebrafish, with sex-segregated wild-type animals being administered either escitalopram, or the serotonin releaser fenfluramine, in the light-dark test. In the systematic review, we find that the available literature indicates an anxiolytic-like effect of SRIs in the novel-tank diving test. Regarding the light-dark test, most studies reported no behavioural effects of SRIs, although the few that did generally saw anxiolytic-like responses. In the experimental studies, consistent anxiolytic-like effects were observed with neither sex nor habituation influencing treatment response. We find that the general effect of acute SRI administration in zebrafish indeed appears to be anxiolytic-like, indicating, at least partly, differences in the functioning of the serotonin system as compared to mammals and that caution is advised when using zebrafish to model affective disorders.

This study investigated the effects of anxiogenic and anxiolytic drugs on zebrafish (Danio rerio) behaviour using a modified novel tank dive test with higher walls and a narrower depth. Zebrafish were administered chondroitin sulfate, beta-carboline, delta-9-tetrahydrocannabinol (THC), ethanol, and beta-caryophyllene, and their behaviours were evaluated for geotaxis, swimming velocity, and immobility. Both anxiogenic and anxiolytic compounds generally increased bottom-dwelling behaviour, suggesting that the tank's modified dimensions significantly influence zebrafish responses. EC50 values for ethanol showed a lower threshold for velocity reduction compared to zone preference. Chondroitin sulfate uniquely caused a sex-specific increase in male swimming velocity, whereas no other sex-differences were observed with any compound. Interestingly, the presence of drug-treated fish did not alter the behaviour of observer fish, suggesting limited social buffering effects. The findings underscore the complexity of zebrafish behavioural phenotypes and highlight the need for considering tank dimensions and multiple behavioural parameters to accurately assess the effects of anxiety-modulating drugs. This study demonstrates the utility of the modified novel tank dive test in providing nuanced insights into the behavioural effects of different pharmacological agents in zebrafish.

This paper introduces the Fish 3D Locomotion app (F3LA), a Python-based, Graphical User Interface (GUI)-equipped tool designed to automate behavioral endpoint extraction in zebrafish locomotion assays. Building on our previous work, which utilized a specialized aquatic tank with a mirror and a single camera for fish movement tracking in three dimensions, F3LA significantly enhances data processing efficiency. Its accuracy was tested by reanalyzing and comprehensively comparing the calculated data with the previously published data from prior publications. From the comparison results, 90% of endpoints showed a similar statistical difference result. These minor differences were due to the different starting points for the dataset and updated calculation formulas that are implemented in F3LA. In addition, shoaling area or shoaling volume calculations are also included in F3LA as a new feature that can serve as sensitive indicators of social cohesion, group dynamics, or stress responses, offering insights into neuropsychological conditions or the effects of pharmacological interventions. Furthermore, F3LA offers a marked improvement over manual operations, being at least five times faster, while maintaining consistent accuracy as it reduces human-induced errors, ensuring a higher degree of reliability in the results. Finally, the potency of F3LA was tested to evaluate the toxicities of 14 rare earth elements (REEs) to the adult zebrafish behaviors. Based on the results, our findings suggested that each tested REE altered fish behaviors in different patterns and magnitudes to each other. However, among the tested light rare earth elements (LREEs), neodymium was demonstrated to cause more relatively severe behavior alterations than other LREEs, indicated by the statistically higher value of entropy (0.2695 ± 0.04977 (mean with a standard deviation)) than the control group (0.2352 ± 0.05896). Meanwhile, in terms of heavy rare earth elements (HREEs), erbium seemed to lead to more distinct behavior toxicities than other HREEs, which was shown by the statistically lower level of fractal dimension (2.022 ± 0.3412) than the untreated group (2.255 ± 0.1661). Taken together, F3LA's development marks a significant advance in high-throughput toxicological and pharmacological assessments in zebrafish, leveraging three-dimensional locomotion data for a more comprehensive analysis of fish behavior performance, providing a significant contribution to research in various fields.

Betulinic acid (BA) is a natural triterpenoid extracted from Bacopa monnieri. BA has been reported to be used as a neuroprotective agent, but their molecular mechanisms are still unknown. Therefore, in this study, we attempted to investigate the precise mechanism of BA for its protective effect against Titanium dioxide nanoparticles (TiO2NP) induced neurotoxicity in zebrafish. Hence, our study observation showed that 10 µg/ml dose of TiO2NP caused a rigorous behavioral deficit in zebrafish. Further, biochemical analysis revealed TiO2NP significantly decreased GSH, and SOD, and increased MDA, AChE, TNF-α, IL-1β, and IL-6 levels, suggesting it triggers oxidative stress and neuroinflammation. However, BA at doses of 2.5,5,10 mg/kg improved behavioral as well as biochemical changes in zebrafish brain. Moreover, BA also significantly raised the levels of DA, NE, 5-HT, and GABA and decreased glutamate levels in TiO2NP-treated zebrafish brain. Our histopathological analysis proved that TiO2NP causes morphological changes in the brain. These changes were expressed by increasing pyknotic neurons, which were dose-dependently reduced by Betulinic acid. Likewise, BA upregulated the levels of NRF-2 and HO-1, which can reduce oxidative stress and neuroinflammation. Thus, our study provides evidence for the molecular mechanism behind the neuroprotective effect of Betulinic acid. Rendering to the findings, we can consider BA as a suitable applicant for the treatment of AD-like symptoms.

1-octyl-3-methylimidazolium bromide ([C8mim]Br), one of the ionic liquids (ILs), has been used in various fields as an alternative green solvent of conventional organic solvents. Increased application and stabilization of imidazole ring structure lead to its release into the aquatic environment and long-term retention. Structure-activity relationship consideration suggested that ILs may be acetylcholinesterase inhibitors; however, neurotoxicity in vivo, especially the underlying mechanisms is rarely studied. In this study, the zebrafish were exposed to 2.5-10 mg/L [C8mim]Br for 28 days to comprehensively evaluate the neurotoxicity of ILs on adult zebrafish from the behavioral profiles and neurotransmitter systems for the first time. The results indicate that zebrafish exhibit suppressed spatial working memory and anxious behaviors. To assess the potential neurotoxic mechanisms underlying the behavioral responses of zebrafish, we measured the levels of neurotransmitters and precursors, key enzyme activities, and expression levels of relevant genes. Nissl staining showed significant neural cell death in zebrafish after 28-day [C8mim]Br exposure, with corresponding decreases in the levels of neurotransmitters (acetylcholine, glutamate, 5-hydroxytryptophan, gamma-aminobutyric acid, dopamine, and norepinephrine). Furthermore, these results were associated with mRNA expression levels of the disrupted neurotransmitter key genes (th, tph2, mao, slc6a3, ache, gad67). Overall, our study determined that [C8mim]Br caused potential mental disorders like anxiety and memory deterioration in zebrafish by impairing neurotransmitter systems, providing recommendations for the industrial production and application of [C8mim]Br.

Research using zebrafish (Danio rerio) has begun to provide novel information in many fields, including the behavioral pharmacology of drug use and misuse. There have been limited studies on the effects of methamphetamine in adult zebrafish and the parameters of exposure (dose, test session length) have not been well-documented. Behavior following drug exposure is generally measured during relatively short sessions (6-10 min is common) in a novel tank environment. Many procedural variables (isolation, netting, novel tank) elicit anxiety-like behavior that is most apparent during the initial portion of a test session. This anxiety-like behavior might mask the initial effects of methamphetamine. During longer test sessions, these anxiety-like responses would be expected to habituate and drug effects should become more apparent. To test this idea, we measured several locomotor activity responses for 50-min following a range of methamphetamine doses (0.1-3.0 mg/L via immersion in methamphetamine solution). Methamphetamine failed to alter swimming velocity, distance travelled, or freezing time. In contrast, methamphetamine produced a dose-dependent decrease in time spent in the bottom of the tank, an increase in the number of visits to the top of the tank, and an increase in the number of transitions along the sides of the tank. The effects of methamphetamine were apparent 10-20 min following exposure and generally persisted throughout the session. These findings indicate that longer test sessions are required to measure methamphetamine-induced changes in behavior in zebrafish, as has been shown in other laboratory animals. The results also suggest that anxiety-like responses associated with various procedural aspects (netting, isolation, novel test apparatus) likely interfere with the ability to observe many behavioral effects of methamphetamine in zebrafish. Based on the current results, habituation to testing procedures to reduce anxiety-like behaviors is recommended in determining the effects of methamphetamine in zebrafish.

Indomethacin (INDO) is a widely utilized non-steroidal anti-inflammatory drug (NSAID) with recognized effect on the central nervous system. Although previous reports demonstrate that prolonged treatment with indomethacin can lead to behavioral alterations such as anxiety disorder, the biochemical effect exerted by this drug on the brain are not fully understood.

The aim of present study was to evaluate if anxiety-like behavior elicited by indomethacin is mediated by brains oxidative stress as well as if alpha-tocopherol, a potent antioxidant, is able to prevent the behavioral and biochemical alterations induced by indomethacin treatment.

Zebrafish were utilized as experimental model and subdivided into control, INDO 1 mg/Kg, INDO 2 mg/Kg, INDO 3 g/Kg, α-TP 2 mg/Kg, α-TP 2 mg/Kg + INDO 1 mg/Kg and α-TP + INDO 2 mg/Kg groups. Vertical distributions elicited by novelty and brain oxidative stress were utilized to determinate behavioral and biochemical alterations elicited by indomethacin treatment, respectively.

Our results showed that treatment with indomethacin 3 mg/kg induces animal death. No changes in animal survival were observed in animals treated with lower doses of indomethacin. Indomethacin induced significant anxiogenic-like behavior as well as intense oxidative stress in zebrafish brain. Treatment with alpha-tocopherol was able to prevent anxiety-like behavior and brain oxidative stress induced by indomethacin.

Data presented in current study demonstrated for the first time that indomethacin induces anxiety-like behavior mediated by brain oxidative stress in zebrafish as well as that pre-treatment with alpha-tocopherol is able to prevent these collateral effects.

Histamine receptors mediate important physiological processes and take part in the pathophysiology of different brain disorders. Histamine receptor 1 (HRH1) is involved in the development of neurotransmitter systems, and its role in neurogenesis has been proposed. Altered HRH1 binding and expression have been detected in the brains of patients with schizophrenia, depression, and autism. Our goal was to assess the role of hrh1 in zebrafish development and neurotransmitter system regulation through the characterization of hrh1-/- fish generated by the CRISPR/Cas9 system. Quantitative PCR, in situ hybridization, and immunocytochemistry were used to study neurotransmitter systems and genes essential for brain development. Additionally, we wanted to reveal the role of this histamine receptor in larval and adult fish behavior using several quantitative behavioral methods including locomotion, thigmotaxis, dark flash and startle response, novel tank diving, and shoaling behavior. Hrh1-/- larvae displayed normal behavior in comparison with hrh1+/+ siblings. Interestingly, a transient abnormal expression of important neurodevelopmental markers was evident in these larvae, as well as a reduction in the number of tyrosine hydroxylase 1 (Th1)-positive cells, th1 mRNA, and hypocretin (hcrt)-positive cells. These abnormalities were not detected in adulthood. In summary, we verified that zebrafish lacking hrh1 present deficits in the dopaminergic and hypocretin systems during early development, but those are compensated by the time fish reach adulthood. However, impaired sociability and anxious-like behavior, along with downregulation of choline O-acetyltransferase a and LIM homeodomain transcription factor Islet1, were displayed by adult fish.

Pseudospondias microcarpa is used in ethnomedicine to manage central nervous system diseases. The hydroethanolic extract (PME) from the leaves of the plant has shown anxiolytic-like properties in mice anxiety models. However, its effects in chronic anxiety models and possible mechanism(s) of action were not studied. Therefore, the current study evaluated the anxiolytic-like mechanisms of PME in zebrafish models of anxiety. The zebrafish light dark test (LDT) and novel tank test (NTT) were employed to assess the anxiolytic-like effects of PME (0.1, 0.3, 1.0 mg mL-1), fluoxetine (3 × 10-5 mg mL-1) and diazepam (1.5 × 10-7 mg mL-1). The chronic unpredictable stress (CUS) test was used to further evaluate the extract's anxiolytic-like properties. The potential mechanisms of anxiolytic action of the extract was evaluated after pre-treated with flumazenil, granisetron, methysergide, or pizotifen, all at 1 × 10-3 mg mL-1. The extract significantly decreased anxiety behaviours in the NT and LD tests. These observed effects of the extract were however counteracted by flumazenil, granisetron, methysergide and pizotifen pre-treatment. In addition, PME treatment significantly reversed CUS-induced anxiety behaviours in zebrafish. Results show that PME possesses anxiolytic-like effects possibly through interaction with serotonergic and gamma-aminobutyric acid mediated pathways.

Glabridin is extracted from the roots of Glycyrrhiza glabra, which has anti-oxidative and anti-inflammatory properties. We investigated the neuroprotective potential of Glabridin against the learning and memory deficit by triggering NRF2/HO-1 signaling in Titanium dioxide nanoparticles (TiO2NP) treated zebrafish. Our study suggests that Glabridin at doses of 12.5, 25, and 50 mg/kg/day for 7 days improved memory and lowered anxiety in the novel object recognition test, T-maze, and novel diving tank respectively. Biochemical analysis showed that Glabridin treatment in TiO2NP-exposed zebrafish enhanced GSH, CAT, SOD, and GPx activity and reduced MDA levels; inhibited proinflammatory mediators, namely, TNF-α, IL-1β, and IL-6. In histopathological evaluation, Glabridin significantly reduced pycnotic neurons in TiO2NP-treated zebrafish brains. Furthermore, Glabridin upregulated NRF2 and HO-1 levels, which leads to a decline in oxidative stress and neuroinflammation and were reversed by ML385 treatment. ML385 as a probe molecule that specifically inhibit NRF2 and prevents its downstream gene expression. Thus, these considerable outcomes provide new insights into the neuroprotective effect of glabridin.

In this review, we discuss the possible utility of zebrafish in research on psilocybin, a psychedelic drug whose recreational use as well as possible clinical application are gaining increasing interest. First, we review behavioral tests with zebrafish, focussing on anxiety and social behavior, which have particular relevance in the context of psilocybin research. Next, we briefly consider methods of genetic manipulations with which psilocybin's phenotypical effects and underlying mechanisms may be investigated in zebrafish. We briefly review the known mechanisms of psilocybin, and also discuss what we know about its safety and toxicity profile. Last, we discuss examples of how psilocybin may be employed for testing treatment efficacy in preclinical research for affective disorders in zebrafish. We conclude that zebrafish has a promising future in preclinical research on psychedelic drugs.

Trauma-related psychopathology transpires in some individuals after exposure to a life-threatening event. While aberrant adrenergic processes may contribute to this, a clear understanding of how said processes influence trauma-related conditions, remain inadequate. Here, we aimed to develop and describe a novel zebrafish (Danio rerio) model of life-threatening trauma-induced anxiety that may be representative of trauma related anxiety, and to evaluate the impact of stress-paired epinephrine (EPI) exposure in the model system. Four groups of zebrafish were each exposed to different and unique stress-related paradigms, i.e., i) a sham (trauma free), ii) high-intensity trauma (triple hit; THIT), iii) high-intensity trauma in the presence of EPI exposure (EHIT), and iv) EPI exposure on its own, all applied in the presence of a color context. Novel tank anxiety was subsequently assessed at 1, 4, 7 and 14 days after the traumatic event. The present results demonstrate that 1) through day 14, THIT or EPI exposure alone induced persistent anxiety-like behavior, 2) EHIT blunted the delayed anxiety-like sequalae associated with severe trauma, 3) exposure to a trauma-paired color context prior to anxiety testing bolstered the subsequent anxiety-like behavior of THIT, but not EHIT -exposed fish, and 4) despite this, THIT- and EPI-exposed fish showed a lesser degree of contextual avoidance behavior compared to sham- or EHIT-exposed fish. These results indicate that the stressors induced long-lasting anxiety-like behavior reminiscent of post trauma anxiety, while EPI displays complex interactions with the stressor, including a buffering effect to subsequent exposure of a trauma-paired cue.

Decabromodiphenyl-ether (BDE-209) is a persistent organic pollutant ubiquitously found in marine environments worldwide. Even though this emerging chemical contaminant is described as highly toxic, bioaccumulative and biomagnifiable, limited studies have addressed the ecotoxicological implications associated with its exposure in non-target marine organisms, particularly from a behavioural standpoint. Alongside, seawater acidification and warming have been intensifying their impacts on marine ecosystems over the years, compromising species welfare and survival. BDE-209 exposure as well as seawater acidification and warming are known to affect fish behaviour, but information regarding their interactive effects is not available. In this study, long-term effects of BDE-209 contamination, seawater acidification and warming were studied on different behavioural traits of Diplodus sargus juveniles. Our results showed that D. sargus exhibited a marked sensitivity in all the behaviour responses after dietary exposure to BDE-209. Fish exposed to BDE-209 alone revealed lower awareness of a risky situation, increased activity, less time spent within the shoal, and reversed lateralization when compared to fish from the Control treatment. However, when acidification and/or warming were added to the equation, behavioural patterns were overall altered. Fish exposed to acidification alone exhibited increased anxiety, being less active, spending more time within the shoal, while presenting a reversed lateralization. Finally, fish exposed to warming alone were more anxious and spent more time within the shoal compared to those of the Control treatment. These novel findings not only confirm the neurotoxicological attributes of brominated flame retardants (like BDE-209), but also highlight the relevance of accounting for the effects of abiotic variables (e.g. pH and seawater temperature) when investigating the impacts of environmental contaminants on marine life.

Isoliquiritigenin, a flavonoid compound, exhibits a variety of pharmacological properties, including anti-inflammatory, anti-oxidative, anti-microbial, anti-viral, and anti-tumor effects. In the past few years, the consumption of isoliquiritigenin-containing dietary supplements has increased due to their health benefits. Although the neuroprotective effects of isoliquiritigenin have been well-investigated, these studies were performed in cells and adult animals. The potential effects of isoliquiritigenin on the development, especially the neurodevelopment, of certain populations, such as zebrafish larvae, have not been investigated. In this study, zebrafish larvae were employed as a model to investigate the effects of isoliquiritigenin on development and neurodevelopment. Zebrafish embryos treated with high concentrations of isoliquiritigenin (10 and 15 μM) exhibited high rates of mortality, hatching, and malformation, indicating that isoliquiritigenin can affect zebrafish development. In addition, isoliquiritigenin impeded the development of central nervous system regions and the length of dopaminergic neurons located in midbrains and thalami of transgenic zebrafish larvae. The locomotor ability of zebrafish larvae exposed to high concentrations of isoliquiritigenin was negatively affected. The total distance and the average velocity significantly decreased, and anxiety-related behaviors were observed under light-dark challenge. Furthermore, the levels of gap43, tuba1b, mbp, hcrt, vmat2, and pomc, which mediate neurodevelopment, neurotoxicity, and anxiety were significantly decreased in zebrafish larvae exposed to isoliquiritigenin. These results indicate that isoliquiritigenin can disrupt the development of dopaminergic neurons and the function of the central nervous system in zebrafish, causing anxiety-like symptoms.

Antiepileptic drugs (AEDs) are globally prescribed to treat epilepsy and many other psychiatric disorders in humans. Their high consumption, low metabolic rate in the human body and low efficiency of wastewater treatment plants (WWTPs) in eliminating these chemicals results in the frequent occurrence of these pharmaceutical drugs in aquatic systems. Therefore, aquatic organisms, including ecologically and economically important teleost fishes, may be inadvertently exposed to these chemicals. Due to their physiological similarity with humans, fishes may be particularly vulnerable to AEDs. Almost all AED drugs are detectable in natural aquatic ecosystems, but diazepam (DZP) and carbamazepine (CBZ) are among the most widely detected AEDs to date. Recent studies suggest that these drugs have a substantial capacity to induce neurotoxicity and behavioral abnormality in fishes. Here we review the current state of knowledge regarding the potential mode of action of DZP and CBZ as well as that of some other AEDs on teleosts and put observable behavioral effects into a mechanistic context. We find that following their intended mode of action in humans, AEDs also disrupt the GABAergic, glutamatergic and serotonergic systems as well as parasympathetic neurotransmitters in fishes. Moreover, AEDs have non-specific modes of action in teleosts ranging from estrogenic activity to oxidative stress. These physiological changes are often accompanied by dose-dependent disruptions of anxiety, locomotor activity, social behaviors, food uptake, and learning and memory, but DZP and CBZ consistently induced anxiolytic effects. Thereby, AED exposure severely compromises individual fitness across teleost fish species, which may lead to population and ecosystem impairment. We also showcase promising avenues for future research by highlighting where we lack data when it comes to effects of certain AEDs, AED concentrations and behavioral endpoints.

Cocaine (COC) use concerns are on the increase for both authorities and civil society. Despite this, it is important to investigate COC effects or those of its main metabolite, belzoylecgonine (BE), in consolidated aquatic model organisms, such as the zebrafish (Danio rerio). This (mini) review consists in an assessment regarding toxicological studies carried out employing zebrafish (embryos, larvae or adults) exposed to COC and/or BE indexed at the SCOPUS and Web of Science databases. Ten different endpoints were analyzed in both embryos and larvae, whereas only four were analyzed in adults. Of the 23 studies, only five investigated COC and/or BE effects following an environmental approach when exposing zebrafish, while most (18 studies) analyzed COC effects under a drug of abuse approach. Cocaine exposure was noted as altering the expression of several genes, such as those linked to COC transport proteins, dopamine receptors, SP substance production, the tachykinin system, and the tyrosine hydroxylase enzyme. BE exposure resulted in more oxidative and proteomic effects than COC in embryos. Cocaine abstinence resulted in hyperactivity associated with stereotypy in adult fish, in addition to reduced responses to visual stimuli to red light and neuronal development pattern alterations. Cocaine was noted as accumulating in zebrafish eyes, possibly due to melanin binding, and causing dose-response cardiac effects in both embryos and adults. Despite the different effects addressed by our survey, we emphasize the lack of COC and BE exposure assessments in zebrafish employing an environmental point of view.

The zebrafish (Danio rerio) is a valuable animal model rapidly becoming more commonly used in pharmaceutical studies. Due to its low-cost maintenance and high breeding potential, the zebrafish is a suitable substitute for most adult rodents (mice and rats) in neuroscience research. It is widely used in various anxiety models. This species has been used to develop a conceptual framework for anxiety behavior studies with broad applications in the laboratory, including the study of herbal and chemical drugs. This review discusses the latest studies of anxiety-related behavior in the zebrafish model.

The prevalence of anxiety is a significant public health problem, being the 24th leading cause of disability in individuals affected by this disorder. In this context, chalcones, a flavonoid subclass obtained from natural or synthetic sources, interact with central nervous system (CNS) receptors at the same binding site as benzodiazepines, the primary drugs used in the treatment of anxiety. Thus, our study investigates the anxiolytic effect of synthetic chalcones derived from the natural product 2-hydroxy-3,4,6-trimethoxyacetophenone isolated from Croton anisodontus Müll.Arg. in modulating anxiolytic activity via GABAergic and serotoninergic neurotransmission in an adult zebrafish model. Chalcones 1 and 2 were non-toxic to adult zebrafish and showed anxiolytic activity via GABAA receptors. Chalcone 2 also had its anxiolytic action reversed by the antagonist granisetron, indicating the participation of serotonergic receptors 5HTR3A/3B in the anxiolytic effect. In addition, molecular docking results showed that chalcones have a higher affinity for the GABAA receptor than DZP and binding in the same region of the DZP binding site, indicating a similar effect to the drug. Furthermore, the interaction of chalcones with GABAA and 5-HT3A receptors demonstrates the anxiolytic effect potential of these molecules.Communicated by Ramaswamy H. Sarma.

Current methods for associative conditioning in animals involve human intervention that is labor intensive, stressful to animals, and introduces experimenter bias in the data. Here, we describe a simple apparatus and a flexible, microcontroller-based conditioning paradigm that minimizes human intervention. Our methodology exploits directed movement towards a target that depends on spatial working memory, including processing of sensory inputs, motivational drive, and attentional mechanisms. Within a stimulus-driven conditioning paradigm designed to train zebrafish, we present a localized pulse of light via LEDs and/or sounds via an underwater transducer. A webcam placed below a glass tank records fish-swimming behavior. For classical conditioning, animals simply associate a sound or light with an unconditioned stimulus, such as a small food reward presented at a fixed location, and swim towards that location to obtain a few grains of food dispensed automatically via a sensor-triggered, stepper motor. During operant conditioning, a fish must first approach a proximity sensor at a remote location and then swim to the reward location. For both types of conditioning, a timing-gated interrupt activates stepper motors via custom software embedded within a microcontroller (Arduino). "Ardulink", a Java facility, implements Arduino-computer communication protocols. In this way, a Java-based user interface running on a host computer can provide full experimental control. Alternatively, a similar level of control is achieved via an Arduino script communicating with an event-driven application controller running on the host computer. Either approach can enable precise, multi-day scheduling of training, including timing, location, and intensity of stimulus parameters; and the feeder. Learning can be tracked by monitoring turning, location, response times, and directional swimming of individual fish. This facilitates the comparison of performance within and across a cohort of animals. Our scheduling and control software and apparatus ("NemoTrainer") can be used to study multiple aspects of species-specific behaviors as well as the effects on them of various interventions.

Huntington's disease (HD) is an inherited neurodegenerative disease. 3-Nitropropionic acid (3-NP) causes increased reactive oxygen species production and neuroinflammation. Centella asiatica (CA) is a strong antioxidant. The aim of this study is to investigate the effect of hydroalcoholic extract of C. asiatica (HA-CA) on 3-NP-induced HD in adult zebrafish. Adult zebrafish (∼5-6 months old) weighing 470 to 530 mg was used and treated with 3-NP (5 mg/kg intraperitoneal [i.p.]). The animals received HA-CA (80 and 100 mg/L) daily for up to 28 days in water. Tetrabenazine (3 mg/kg i.p.) was used as a standard drug. We have done an open field test (for locomotor activity), a novel tank diving test (for anxiety), and a light and dark tank test (for memory), followed by biochemical analysis (acetyl-cholinesterase [AchEs], nitrite, lipid peroxidation [LPO], and glutathione [GSH]) and histopathology to further confirm memory dysfunctions. 3-NP-treated zebrafish exhibit reductions in body weight, progressive neuronal damage, cognition, and locomotor activity. The HA-CA group significantly reduced the 3-NP-induced increase in LPO, AchEs, and nitrite levels while decreasing GSH levels. Oral administration of HA-CA (80 or 100 mg/L) significantly reduces 3-NP-induced changes in body weight and behaviors, in addition to neuroinflammation in the brain by lowering tumor necrosis factor-α and interleukin-1β levels. Moreover, HA-CA significantly decreases the 3-NP-induced neuronal damage in the brain. HA-CA ameliorates neurotoxicity and neurobehavioral deficits in 3-NP-induced HD-like symptoms in adult zebrafish.

The anxiety disorders are the most prevalent mental health condition, and anxiety is considered the sixth cause of disability surpassing diabetes mellitus, chronic obstructive pulmonary disease, and osteoarthritis. Besides, the COVID-19 pandemic provided an increase in the number of psychiatric diseases diagnosis in all social layers around the world. About 55%-94% of patients diagnosed with anxiety disorders are treated with benzodiazepines, meanwhile benzodiazepines can promote several adverse effects. In this way, alternative therapies, such as essential oils may offer significant benefits in the treatment of patients with anxiety disorders. However, the anxiolytic effect of these essential oils must be proper evaluated appropriate as well as the suitable dosage and side effect need further research.

The aim was to evaluate the anxiolytic effect of Roman chamomile (Anthemis nobilis L.) and tangerine (Citrus reticulata Blanco) essential oils using the light-dark test in adult zebrafish (Danio rerio).

Both essential oils were analyzed by GC-MS and the major compounds were identified. The anxiolytic effect was evaluated by light-dark test in adult zebrafish.

The results showed that roman chamomile essential oil has anxiolytic effect in adult zebrafish, whereas tangerine essential oil tends to reduce anxiety The major compounds of tangerine essential oil were limonene and γ-terpinene, and the major compounds of roman chamomile were pentadecyl-3-methyl-2-butenoate, hexadecyl-3-methyl-2-butenoate, 1-piperidinol and trans-1-ethyl-3-methyl-cyclopentane.

The present study demonstrated that this anxiolytic effect may be attributed to the synergistic effect of the compounds present in roman chamomile essential oil, particularly the major compounds. The roman chamomile essential oil at the highest concentration showed anxiolytic effect. The tangerine essential oil showed a tendency to reduce anxiety, but it was not statistically significative. In addition, roman chamomile and tangerine essential oils did not cause cause alteration in locomotion activity and exploratory ability of the fish.

Channelopathies are a large group of systemic disorders whose pathogenesis is associated with dysfunctional ion channels. Aberrant transmembrane transport of K⁺, Na⁺, Ca²⁺ and Cl^- by these channels in the brain induces central nervous system (CNS) channelopathies, most commonly including epilepsy, but also migraine, as well as various movement and psychiatric disorders. Animal models are a useful tool for studying pathogenesis of a wide range of brain disorders, including channelopathies. Complementing multiple well-established rodent models, the zebrafish (Danio rerio) has become a popular translational model organism for neurobiology, psychopharmacology and toxicology research, and for probing mechanisms underlying CNS pathogenesis. Here, we discuss current prospects and challenges of developing genetic, pharmacological and other experimental models of major CNS channelopathies based on zebrafish.

The consumption of progestins has increased considerably in recent decades, as has their disposal into the environment. These substances can negatively affect the reproduction, physiology, and behavior of non-target organisms, such as fish. We aimed to evaluate the effects of exposure to environmentally relevant concentrations of levonorgestrel-control birth based (1.3, 13.3, 133, and 1330 ng/L) on the development and behavior of zebrafish (Danio rerio) in terms of mortality, hatching, spontaneous movement, and larval and adult behavioral tests. Exposure caused anxiogenic-like behavior in larvae, which persisted in adults, as demonstrated by the light-dark test. In contrast, it caused anxiolytic-like behavior in the novel tank test. There was a high mortality rate at all tested concentrations and increases in the hormone cortisol at 13.3 ng/L that affected the sex ratio. These changes may lead to an ecological imbalance, emphasizing the risk of early exposure to progestins in the environment.

Baicalein is a flavonoid mainly obtained from plants with wide range of biological activities, including neuroprotection. An acute and unexpected chronic stress (UCS) protocol has recently been adapted to zebrafish, a popular vertebrate model in brain research. The present study was aimed to evaluate baicalein's anti-anxiety potential in a zebrafish model by induction, which included neuropharmacological evaluation to determine behavioural parameters in the novel tank diving test (NTDT) and light-dark preference test (LDPT). The toxicity was also assessed using the brine shrimp lethality assay, and the 50% lethal concentration (LC50) was determined. The animals were then stressed for 7 days before being treated with different doses of baicalein (1 and 2 mg/L) for another 7 days in UCS condition. Due to acute stress and UCS, the frequency of entries and time spent in the 1) top region and 2) light area of the novel tank reduced significantly, indicating the existence of elevated anxiety levels. The biological activity of baicalein was demonstrated by its high LC50 values (1,000 μg/ml). Additionally, baicalein administration increased the frequency of entries and duration spent in the light region, indicating a significant decrease in anxiety levels. Overall, the present results showed that baicalein has a therapeutic advantage in reversing the detrimental consequences of UCS and acute stress, making it is a promising lead molecule for new drug design, development, and therapy for stress.

Zebrafish (Danio rerio) share a considerable amount of biological similarity with mammals, including identical or homologous gene expression pathways, neurotransmitters, hormones, and cellular receptors. Zebrafish also display complex social behaviors like shoaling and schooling, making them an attractive model for investigating normal social behavior as well as exploring impaired social function conditions such as autism spectrum disorders. Newly-formed and established shoals exhibit distinct behavior patterns and inter-member interactions that can convey the group's social stability. We used a three-chamber open-swim preference test to determine whether individual zebrafish show a preference for an established shoal over a newly-formed shoal. Results indicated that both sexes maintained greater proximity to arena zones nearest to the established shoal stimulus. In addition, we report the novel application of Shannon entropy to discover sex differences in systematicity of responses not revealed by unit-based measurements; male subjects spent more time investigating between the two shoals than female subjects. This novel technique using established versus newly-formed shoals can be used in future studies testing transgenics and pharmacological treatments that mimic autism spectrum disorder and other disorders that affect social interaction.

In an effort to find alternatives to study in vivo the so-called New Psychoactive Substances (NPS), the present work was undertaken to investigate the use of zebrafish larvae as animal model in pharmaco-toxicology, providing behavioural and metabolism information. For this purpose, fentanyl, the progenitor of an extremely dangerous group of NPS, was administered at different doses to zebrafish larvae (1, 10, 50, 100 µM) in comparison to mice (0.1, 1, 6, 15 mg/kg), as a well-established animal model. A behavioural assay was performed at the time of the peak effect of fentanyl, showing that the results in larvae are consistent with those observed in mice. On the other hand, several morphological abnormalities (namely yolk sac edema, abnormal pericardial edema, jaw defect and spinal curvature) were found in larvae mostly at high fentanyl doses (50, 100 µM). Larva extract and mice urine were analyzed by using liquid chromatography coupled to high resolution mass spectrometry to identify the metabolic pathways of fentanyl. The main metabolites detected were norfentanyl and hydroxyfentanyl in both the tested models. In conclusion, the present study provides evidence that fentanyl effects on zebrafish larvae and metabolism are similar to rodents and consequently support the hypothesis of using zebrafish larvae as a suitable rapid screening tool to investigate new drugs, and particularly NPS.

Structure-based virtual screening of the Enamine database of 1.7 million compounds followed by WaterMap calculations (a molecular dynamics simulation-based method) was applied to identify novel AChE inhibitors. The inhibitory potency of 29 selected compounds against electric eel (ee) AChE was determined using the Ellman's method. Three compounds were found active (success rate 10%). For the most potent compound (~40% of inhibition at 10 μM), 20 derivatives were discovered based on the Enamine similarity search. Finally, five compounds were found promising  (IC 50 ranged from 6.3 µM to 17.5 µM) inhibitors of AChE. The performed similarity and fragment analysis confirmed significant structural novelty of novel AChE inhibitors. Toxicity/safety of selected compounds was determined in zebrafish model.

Antidepressant (AD) drugs are widely prescribed for the treatment of psychiatric disorders, including depression and anxiety disorders. The continuous use of ADs causes significant quantities of these bioactive chemicals to enter the aquatic ecosystems mainly through wastewater effluent discharge. This may result in many aquatic organisms being inadvertently affected by these drugs. Fluoxetine (FLX) and venlafaxine (VEN) are currently among the most widely detected ADs in aquatic systems. A growing body of experimental evidence demonstrates that FLX and VEN have a substantial capacity to induce neurotoxicity and cause behavioral dysfunctions in a wide range of teleost species. At the same time, these studies often report seemingly contradictory results that are confounding in nature. Hence, we clearly require comprehensive reviews that attempt to find overarching patterns and establish possible causes for these variable results. This review aims to explore the current state of knowledge regarding the neurobehavioral effects of FLX and VEN on fishes. This study also discusses the potential mechanistic linkage between the neurotoxicity of ADs and behavioral dysfunction and identifies key knowledge gaps and areas for future research.

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.

Autism spectrum disorder (ASD) refers to a complicated range of childhood neurodevelopmental disorders which can occur via genetic or non-genetic factors. Clinically, ASD is associated with problems in relationships, social interactions, and behaviors that pose many challenges for children with ASD and their families. Due to the complexity, heterogeneity, and association of symptoms with some neuropsychiatric disorders such as ADHD, anxiety, and sleep disorders, clinical trials have not yielded reliable results and there still remain challenges in drug discovery and development pipeline for ASD patients. One of the main steps in promoting lead compounds to the suitable drug for commercialization is preclinical animal testing, in which the efficacy and toxicity of candidate drugs are examined in vivo. In recent years, zebrafish have been able to attract the attention of many researchers in the field of neurological disorders such as ASD due to their outstanding features. The presence of orthologous genes for ASD modeling, the anatomical similarities of parts of the brain, and similar neurotransmitter systems between zebrafish and humans are some of the main reasons why scientists draw attention to zebrafish as a prominent animal model in preclinical studies to discover highly effective treatment approaches for the ASD through genetic and non-genetic modeling methods.

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.

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.

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.

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.