Posttraumatic anxiety-like behaviour in zebrafish is dose-dependently attenuated by the alpha-2A receptor agonist, guanfacine.

The current study investigated the independent and combined effects of caffeine and taurine on anxiety-like behavior and neuroendocrine responses in adult zebrafish (Danio rerio). Caffeine (1,3,7-trimethylpurine-2,6-dione), the world’s most commonly used psychoactive drug, acts as an adenosine receptor blocker and a mild central nervous system stimulant. However, excessive use of caffeine is associated with heightened anxiety levels. Taurine (2-aminoethanesulfonic acid), a semi-essential amino acid synthesized within the human brain, has been hypothesized to play a role in regulating anxiolytic behavior. Caffeine and taurine are two common additives in energy drinks and are often found in high concentrations in these beverages. However, few studies have investigated the interaction of these two chemicals with regards to anxiety measures. A suitable vertebrate to examine anxiety-like behavior and physiological stress responses is the zebrafish, which has shown promise due to substantial physiological and genetic homology with humans. Anxiety-like behavior in zebrafish can be determined by analyzing habituation to novelty when fish are placed into a novel tank and scototaxis (light avoidance) behavior in the light-dark test. Stress-related neuroendocrine responses can be measured in zebrafish by analyzing whole-body cortisol levels. The goal of this study was to determine if exposure to caffeine, taurine, or a combination of the two compounds altered anxiety-like behavior and whole-body cortisol levels in zebrafish relative to control. Zebrafish were individually exposed to either caffeine (100 mg/L), taurine (400 mg/L), or both for 15 min. Zebrafish in the control group were handled in the same manner but were only exposed to system tank water. After treatment, fish were transferred to the novel tank test or the light-dark test. Behavior was tracked for the first 6 min in the novel tank and 15 min in the light-tark test. Fifteen min after introduction to the behavioral task, fish were euthanized for the analysis of whole-body cortisol levels. The results demonstrate that caffeine treatment decreased the amount of exploration in the top of the novel tank and increased scototaxis behavior in the light-dark test, which supports the established anxiogenic effect of acute exposure to caffeine. Taurine alone did not alter basal levels of anxiety-like behavioral responses nor ameliorated the anxiogenic effects of caffeine on behavior when the two compounds were administered concurrently. None of the drug treatments altered basal levels of whole-body cortisol. The current results of this study suggest that, at least at this dose and time of exposure, taurine does not mitigate the anxiety-producing effects of caffeine when administered in combination, such as with energy drink consumption.

Caffeine neuroprotects against dexamethasone-induced anxiety-like behaviour in the Zebrafish (Danio rerio)

Alkaloids are a structurally complex group of natural products that have a diverse range of biological activities and significant therapeutic applications. In this study, we examined the acute, anxiolytic-like effects of nicotinic acetylcholine receptor (nAChR)-activating alkaloids with reported neuropharmacological effects but whose effects on anxiety are less well understood. Because α4β2 nAChRs can regulate anxiety, we first demonstrated the functional activities of alkaloids on these receptors in vitro. Their effects on anxiety-like behavior in zebrafish were then examined using the zebrafish novel tank test (NTT). The NTT is a relatively high-throughput behavioral paradigm that takes advantage of the natural tendency of fish to dive down when stressed or anxious. We report for the first time that cotinine, anatabine, and methylanatabine may suppress this anxiety-driven zebrafish behavior after a single 20-min treatment. Effective concentrations of these alkaloids were well above the concentrations naturally found in plants and the concentrations needed to induce anxiolytic-like effect by nicotine. These alkaloids showed good receptor interactions at the α4β2 nAChR agonist site as demonstrated by in vitro binding and in silico docking model, although somewhat weaker than that for nicotine. Minimal or no significant effect of other compounds may have been due to low bioavailability of these compounds in the brain, which is supported by the in silico prediction of blood–brain barrier permeability. Taken together, our findings indicate that nicotine, although not risk-free, is the most potent anxiolytic-like alkaloid tested in this study, and other natural alkaloids may regulate anxiety as well.

Anxiety is a normal emotion representing a reaction to potential danger, whereas fear can be defined as a reaction to real, explicit danger. Anxiety-like behavior in animal models has been associated with differences in the serotonergic system. To understand the roles of the 5-HT1A receptor in zebrafish anxiety-like behavior and sociality. Adult zebrafish were treated with 8-OH-DPAT and subjected to the phototaxis (light-dark preference) assay, the novel tank test (NTT), or the social preference test. Separate cohorts were treated with increasing doses of 8-OH-DPAT, while 5-HT1A receptors were blocked with a silent dose of WAY 100635. 8-OH-DPAT (0.3 mg/kg) decreased anxiety-like behavior in the NTT, but increased it in the phototaxis (light-dark preference) assay, both considered assays for anxiety-like behavior for this species. The same dose decreased social approach in both the social investigation and social novelty phases of the social preference test. Blocking the 5-HT1A receptor with WAY 100635 (0.01 mg/kg) shifted the dose-response curve (0.03-3 mg/kg) for the NTT rightward. These effects suggest a participation of the 5-HT1A heteroreceptors in zebrafish anxiety and social preference, modulating anxiety in a test-dependent way and decreasing sociality. Thus, the study of this receptor is important for a better understanding of anxiety-like behavior in zebrafish and its relationship with similar phenomena in vertebrates.

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.

Monoamine oxidases (MAO) are a valuable class of mitochondrial enzymes with a critical role in neuromodulation. In this study, we investigated the effect of natural MAO inhibitors on novel environment-induced anxiety by using the zebrafish novel tank test (NTT). Because zebrafish spend more time at the bottom of the tank when they are anxious, anxiolytic compounds increase the time zebrafish spend at the top of the tank and vice versa. Using this paradigm, we found that harmane, norharmane, and 1,2,3,4-tetrahydroisoquinoline (TIQ) induce anxiolytic-like effects in zebrafish, causing them to spend more time at the top of the test tank and less time at the bottom. 2,3,6-trimethyl-1,4-naphtoquinone (TMN) induced an interesting mix of both anxiolytic- and anxiogenic-like effects during the first and second halves of the test, respectively. TIQ was unique in having no observable effect on general movement. Similarly, a reference MAO inhibitor clorgyline—but not pargyline—increased the time spent at the top in a concentration-dependent manner. We also demonstrated that the brain bioavailability of these compounds are high based on the ex vivo bioavailability assay and in silico prediction models, which support the notion that the observed effects on anxiety-like behavior in zebrafish were most likely due to the direct effect of these compounds in the brain. This study is the first investigation to demonstrate the anxiolytic-like effects of MAO inhibitors on novel environment-induced anxiety in zebrafish.

Anxiolytic-like effects of noribogaine in zebrafish

Interventions to the gut microbiome manipulate the gut–brain axis and could be useful in the treatment of anxiety and depression. In this study, we demonstrated that administration of the bacterium Paraburkholderia sabiae reduces anxiety-like behavior in adult zebrafish. P. sabiae administration increased the diversity of the zebrafish gut microbiome. Linear discriminant analysis Effect Size (LEfSe) analysis revealed that the populations of Actinomycetales including Nocardiaceae, Nocardia, Gordoniaceae, Gordonia, Nakamurellaceae, and Aeromonadaceae were reduced, whereas those of Rhizobiales including Xanthobacteraceae, Bradyrhizobiaceae, Rhodospirillaceae, and Pirellulaceae were increased in the gut microbiome. Functional analysis using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) predicted that P. sabiae administration altered taurine metabolism in the zebrafish gut, and we demonstrated that P. sabiae administration increased the taurine concentration in the brain. Since taurine functions as an antidepressant neurotransmitter in vertebrates, our results suggest that P. sabiae could improve anxiety-like behavior in zebrafish via the gut-brain axis.

The influence of exercise on anxiety-like behavior in zebrafish (Danio rerio)

The β-Carboline FG-7142 is a partial inverse agonist at the benzodiazepine allosteric site on the GABA-A receptor that induces anxiogenic, proconvulsant, and appetite-reducing effects in many species, including humans. Seizure-kindling effects have been well studied, however anxiogenic properties are relatively unexplored. This study aimed to investigate concentration-dependent effects of FG-7142 on anxiety-like behaviour and fear responses in zebrafish (Danio rerio) using the open-field test (OF) and novel object approach test (NOA). A U-shaped distribution was found with maximal responses in increased immobility and reduced distance moved at 10 µM in the NOA but not the OF. Follow up experiments demonstrated a lack of effect in repeated OF testing and no changes in opercular movements. Furthermore, the effect of FG-7142 was reversed with ethanol treatment. These results suggest that FG-7142 elicits a ‘freezing’ response in zebrafish via the introduction of novelty, suggesting fear-induction. These findings indicate that FG-7142 may act as an agent to promote acute fear responses in zebrafish.

Underwater noise pollution, driven by human activities, is an emerging environmental concern, yet its effects on fish behavior and physiology remain poorly understood. As a vertebrate model with conserved stress pathways, zebrafish (Danio rerio) is well-suited for investigating the mechanistic basis of such impacts. We hypothesized that daytime and nighttime noise exposure would differentially induce anxiety-like behavior and associated neuroendocrine disruptions in zebrafish, with effects varying by sex. To evaluate this hypothesis, adult zebrafish were exposed to anthropogenic noise (100-1000 Hz, 130 dB) for seven days, specifically during daytime (08:00-20:00) and nighttime (20:00-08:00) periods. Behavioral assays revealed that noise exposure delayed the first entry of females into the top zone during daytime, while both sexes exhibited prolonged bottom-dwelling and reduced exploratory behavior under nighttime noise. Physiological analyses showed elevated plasma cortisol levels in females, accompanied by up-regulated HPI-axis genes, whereas males displayed a non-significant cortisol increase. Neurotransmitter profiling indicated a sex-specific response to nighttime noise: In females, brain 5-hydroxytryptamine (5-HT) showed a non-significant increasing trend, whereas in males it was significantly elevated, while dopamine (DA) decreased in both sexes. Gene expression analysis further revealed disruptions in 5-HT and DA pathways. These findings demonstrate that underwater noise induces anxiety-like behavior in zebrafish by dysregulating endocrine and neurotransmitter systems, with nighttime noise exhibiting more pronounced effects, suggesting that chronic exposure to anthropogenic noise may impair natural behavior and stress regulation in aquatic species, particularly during nighttime periods.

Zebrafish (Danio rerio) are quickly becoming an important model organism in behavioural neuroscience and drug addiction research. Conditioned place preference studies show that drugs of abuse produce responses in zebrafish that are similar to mammalian animal models. Repeated administration of ethanol in zebrafish results in withdrawal-induced behavioural responses that vary with dose and exposure duration, requiring additional investigation. Here, we examine the effects of ethanol withdrawal on anxiety-like behaviours in adult zebrafish after a 21-day ethanol dosing schedule at either 0.4% or 0.8%. Anxiety-like behaviour was measured with the novel object approach test; this test involves placing a fish in a circular arena with a novel object in the centre and observing the amount of exploration of the object. We found increased anxiety-like behaviour during ethanol withdrawal. This study adds to the growing body of literature that validates the zebrafish as a model organism in the field of behavioural neuroscience and addiction.

Cannabis is a commonly used illicit substance, often consumed for its potential to promote relaxation and alleviate anxiety. This study employed the light-dark transition test to examine the effects of acute delta-9-tetrahydrocannabinol (THC) exposure on anxiety-like behavior in larval zebrafish. At 7 days post-fertilization, zebrafish larvae were exposed to one of several treatments: egg water (control), 1% dimethyl sulfoxide (DMSO, vehicle control), 100 mg/L caffeine in egg water, 10 mg/L alprazolam in DMSO, and 0.01 and 0.1 mg/L THC in DMSO for 2 hours. Their behavior was then recorded during alternating light and dark periods to measure the distance traveled in each condition. Both egg water and DMSO controls demonstrated significantly higher activity levels in the dark compared to the light (p = 0.003 and 0.011, respectively). THC exposure exhibited a biphasic effect in the dark period relative to the DMSO control: lower concentrations of THC (0.01 mg/L) increased distance traveled, whereas higher concentrations (0.1 mg/L) decreased it, though these effects were not statistically significant. Additionally, larvae exposed to 0.01 mg/L THC showed higher locomotion in both light and dark periods compared to those exposed to alprazolam, but this difference was not significant. These findings suggest that THC has both anxiolytic and anxiogenic effects in the light-dark transition assay, aligning with some existing research on THC's impact on anxiety. Further studies are needed to draw more definitive conclusions, but this research provides valuable insights into THC's effects on anxiety-like behavior.

Efavirenz (EFV) is a medication widely used for the treatment of HIV-positive patients. Several studies have demonstrated that the prolongate use of EFV can lead to the development of neurological diseases, such as panic syndrome, depression, and anxiety disorders. In this current study, we evaluate whether the ascorbic acid (AA) treatment can prevent anxiety-like behavior and brain oxidative stress induced by EFV treatment in zebrafish. Our data demonstrated that the EFV treatment induces anxiogenic-like behavior and intense lipid peroxidation in the zebrafish brain. The AA treatment was able to prevent both anxiogenic-like behavior and brain oxidative stress elicited by the EFV treatment. Therefore, our data provide robust evidence that the EFV induced anxiety-like behavior in zebrafish via a redox-dependent pathway and that AA treatment can minimize these adverse effects. Taken together, our preclinical study strongly suggests that the use of an AA-enriched diet can minimize the effects of EFV on the central nervous system (CNS) and improve the quality of life for patients undergoing EFV treatment.

Combinatory effects of low concentrations of 17α-etinylestradiol and citalopram on non-reproductive behavior in adult zebrafish (Danio rerio)