Experimental design scheme. During the study, 3-week-old zebrafish were treated with the tested drugs and then subjected to behavioural procedures to assess their anxiety and social behaviours. Following the behavioural tests, the fish were euthanised and their tissues were used for molecular studies to evaluate changes in gene expression, including serotonergic receptors and transporter, oxytocin receptors and vasopressin receptor genes, the oxytocin level as well as alterations in the ERK1/2 and AKT signalling pathways. Zebrafish were treated with a compound of interest (COI) for 60 min and then behavioural tests were performed (a). To evaluate anxiety behaviour in fish, the novel environment exploration test, which is based on thigmotaxis – a natural tendency of fish to stay close to the periphery of a new environment, was performed. During the procedure, the first 6 min were designated as a light period, followed by 4 min of darkness. The findings indicate that the transition from light to dark did not affect the distance untreated fish moved. (b) To determine whether fish exhibit a preference for light or dark conditions, a light–dark preference test was conducted. The test utilised a chamber with two zones, one light and one dark, and lasted for 8 min. The findings indicate that the untreated control fish did not display a preference for either zone (c). To evaluate social behaviour in fish, a three-chambered setup with fish present as social stimuli was applied. In the tested arena, there were two distinguishable zones: the social zone, located in front of the chamber containing familiar fish stimulus, and the non-social zone, located in front of the empty chamber. The first 15 min of the experiment was for the acclimation phase without the stimulus fish, followed by 15 min of the social cue phase with the stimulus fish present. The findings indicate that fish tend to exhibit a preference for conspecifics (d). Data were analysed using t-test (b, d). Mann–Whitney test was used for datasets that failed normality testing or had significantly different variances (c). The confidence limit of *p < 0.05 was considered statistically significant. n = 30 for anxiety behaviour assessment; n = 24 for light–dark preference assessment; n = 14 for social behaviour assessment. COI: compound of interest.

Diazepam elicits anxiolytic activity in 3-week-old fish. To validate the methodology used in further studies, we decided to test diazepam (10 μM) in the novel environment exploration test under light and dark conditions. Diazepam was able to increase distance moved (a) and time spent (b) in the inner zone by fish, and the effects were not ascribed to changes in locomotion (c) under light conditions. Similar effects were observed under dark conditions; diazepam increased distance moved (d) and time spent (e) in the inner zone by fish without affecting their overall movement (f). This confirms the anxiolytic activity of diazepam and the reliability of our method. Data were analysed using the Mann–Whitney test. The confidence limit of *p < 0.05 was considered statistically significant, n = 54–102.

MDMA and oxytocin receptor agonists show anxiolytic activity in fish. The novel environment exploration was conducted to measure anxiety levels in fish exposed to MDMA and oxytocin receptor agonist – WAY-267464 in both light and dark conditions. The study found that MDMA exhibited an inverted-U-shaped effect on anxiety behaviour. The lowest tested dose appeared to reduce the distance moved by fish in the inner zone, while with increasing concentration, the effect was the opposite before it diminished once again. Under light, a dose of 2.5 μM increased the distance moved significantly, whereas 100 μM reduced the distance moved in the inner zone (a). The same result was also observed when the time spent in the inner zone by fish was considered (b). Starting from the dose of 5 μM, MDMA inhibited fish locomotion (c). Under dark conditions, MDMA only reduced the distance moved (d) and time spent (e) in the inner zone at the dose of 100 μM and inhibited locomotion from the dose of 5 μM (f). Interestingly, WAY-267464 had no effect on anxiety behaviour under light conditions (g–i), however, was able to increase distance moved (j) and time spent (k) by fish in the inner zone under dark conditions. The highest dose tested of 200 μM resulted in reduced fish locomotion (l). Data were analysed using a one-way analysis of variance followed by Dunnett’s post hoc test (k). Kruskal–Wallis test with Dunn’s post hoc was used for datasets that failed normality testing or had significantly different variances (a–j, l). The confidence limit of *p < 0.05 was considered statistically significant, n = 29–66.

Oxytocin receptor antagonists had no effect on the anxiety levels of fish. To better understand how the oxytocin system affects anxiety-related behaviour, the oxytocin receptor antagonist – L-368,899 – was tested on fish. The results showed that the compound did not have any impact on the distance moved (a) or time spent (b) by fish in the inner zone, nor did it affect their movement (c) under light conditions. A lack of effects was also observed under dark conditions (d–f). Data were analysed using Kruskal–Wallis test with Dunn’s post hoc, n = 30–36.

Lighting conditions are crucial to reveal anxiety behaviours in fish. A study was conducted to examine the relationship between lighting conditions and anxiety-like behaviour in fish. The light–dark preference test was used to evaluate the effects of MDMA and oxytocin receptor agonist, WAY-267464. The results showed that MDMA administered at doses of 1.0 and 2.5 μM increased the time spent in the dark zone (a). At a dose of 2.5 μM, it also significantly reduced the time it took for the fish to cross into the dark zone (b). The compound had no effect on the frequency of crossing into the dark zone (c) or locomotion (d), indicating that the observed effects were not due to changes in overall movement. Previously shown to be anxiolytic – WAY-267464 – there was no change in the time spent in the light or dark zone in comparison with the control group (e), and even increased the time required to first move into the dark zone (f). The compound did not affect the frequency of crossing into the dark zone (g) or locomotion (h). Data were analysed using a one-way analysis of variance followed by Dunnett’s post hoc test (e, f). Kruskal–Wallis test with Dunn’s post hoc was used for datasets that failed normality testing or had significantly different variances (a–d, g, h). The confidence limit of *p < 0.05 was considered statistically significant, n = 14–24.

MDMA and oxytocin receptor agonists produce prosocial effects in fish. To assess how 3-week-old fish respond to synthetic (MDMA) and endogenous (WAY-267464) empathogens, a three-chambered setup was utilised. Results showed that MDMA at the lower tested dose of 0.5 μM significantly increased the social preference index (a) and the doses of 1.0 and 5.0 μM increased the percentage of entries into the social zone (b), indicating its prosocial activity. The effect was not due to changes in fish locomotion (c). Similarly, WAY-267464, an oxytocin receptor agonist, was able to promote prosocial behaviour, as shown by an increase in social preference index (d) and an increase in the percentage of entries into the social zone (e) at the highest tested dose of 100 μM. The compound had no effect on overall fish movement (f). By contrast, the oxytocin receptor antagonist, L-368,899, had no effect on the social preference index (g) nor entries to the social zone (h) and locomotion of fish (i). Data were analysed using a one-way analysis of variance followed by Dunnett’s post hoc test (a–d, f–h). Kruskal–Wallis test with Dunn’s post hoc was used for datasets that failed normality testing or had significantly different variances (e, i). The confidence limit of *p < 0.05 was considered statistically significant, n = 11–34.

The expression of serotonergic receptor and transporter, oxytocin receptor, and vasopressin receptor genes is influenced by the use of MDMA and oxytocin receptor ligands. To unravel the mechanisms behind the behavioural effects, the changes in gene expression related to the serotonergic system, oxytocin and vasopressin receptors after exposure to MDMA, WAY-267464 and L-368,899 were studied. After the behavioural tests, the fish were euthanised, mRNA was isolated and a real-time PCR technique was utilised (a). The expression values between the reference sample and the test sample were converted to log2 (fold change, FC) and presented as a heat map (b). Our findings indicate that MDMA decreased the expression of the coding serotonin transporter (scl6a4), increased the expression of oxytocin receptors (oxtra and oxtrb) and dramatically reduced the expression of gene coding vasopressin receptor (avpr1aa). WAY-267464, oxytocin receptor agonists, increased expression of the gene coding the serotonin 5-HT1B receptor (htr1b) whereas reducing the gene coding the serotonin 5-HT2B (htr2b) and scl6a4. However, it had no effect on genes coding oxytocin receptors and vasopressin receptor. L-368,899, oxytocin receptor antagonist, increased expression of htr1b, scl6a4 and oxtra genes. The expression level of the oxtra gene was not assessed at the lowest MDMA dose of 0.5 μM (indicated by the grey field). n = 3, MDMA had no effect on the release of oxytocin. To determine whether MDMA led to the release of oxytocin in zebrafish, the ELISA test was conducted (c). According to the results, MDMA did not affect the oxytocin level in fish (d). Data were analysed using a one-way analysis of variance followed by Dunnett’s post hoc test, n = 4. COI: compound of interest; FC: fold change.

MDMA suppresses AKT phosphorylation. Next, we determined whether the AKT or ERK1/2 signalling pathways played a role in the behavioural effects of MDMA. Following the behavioural tests, the fish were euthanised, and protein levels were normalised before utilising the western blotting technique (a). The results indicate that a dose of 0.5 μM of MDMA decreased AKT phosphorylation (b), while neither of the tested doses had an impact on ERK1/2 phosphorylation (c). Data were analysed using a t-test (b: 0.5, 1.0 μM, c: 0.5, 1.0 μM). Mann–Whitney test was used for datasets that failed normality testing or had significantly different variances (b: 2.5 μM, c: 2.5 μM). The confidence limit of *p < 0.05 was considered statistically significant, n = 6–8. COI: compound of interest.

Oxytocin receptor agonist increases ERK1/2 phosphorylation. The compound WAY-267464 did not impact AKT phosphorylation (a), but when used at a dosage of 50 μM, it was found to enhance ERK1/2 phosphorylation (b). Data were analysed using a t-test (a: 100 μM, b: 50 μM). Mann–Whitney test was used for datasets that failed normality testing or had significantly different variances (a: 50 μM, b: 100 μM). The confidence limit of *p < 0.05 was considered statistically significant, n = 5–7.