3.1. Effect of serotonergic psychedelic administrations on the immobility time in the FST in mice
To examine the dose-dependency of the antidepressant-like effects of serotonergic psychedelics, male C57BL/6J mice were subjected to the FST 24 h after administration of psilocin (1.5, 2.0, and 4.0 mg/kg), DOI (0.05, 0.1, 0.25, 1.0, and 2.0 mg/kg), or TCB-2 (0.5, 1.0, and 5.0 mg/kg). Mice that received psilocin (1.5 mg/kg), DOI (0.1 and 0.25 mg/kg), or TCB-2 (5.0 mg/kg) had significantly decreased immobility time in the FST compared with vehicle-treated control mice. Other tested doses of psilocin, DOI, and TCB-2 had no significant effect on the immobility time in the FST (Kruskal–Wallis nonparametric ANOVA followed by Bonferroni’s test; psilocin: H(3) = 6.80, p = 0.033, Fig. 1a; DOI: H(6)=16.06, p = 0.0067, Fig. 1b; TCB-2: H(4) = 8.26, p = 0.035, Fig. 1c). In contrast, acute treatment with the antidepressants fluoxetine, a selective serotonin reuptake inhibitor, and imipramine, a tricyclic antidepressant, did not affect immobility time in the FST 24 h after treatment (Kruskal–Wallis nonparametric ANOVA followed by Bonferroni’s test; H(3) = 2.73, p = 0.26, Fig. 1d). These results suggest that psychedelics have antidepressant-like effects at optimal concentration in mice. As the optimal concentrations, psilocin, DOI, and TCB-2 were henceforth used at 1.5 mg/kg, 0.1 mg/kg, and 5.0 mg/kg, respectively, unless otherwise indicated.
3.2. Role of 5-HT2A in the antidepressant-like effect of serotonergic psychedelics in mice
To confirm the involvement of 5-HT2A in the antidepressant effects of psilocin, DOI, and TCB-2, we assessed the effect of pretreatment with the 5-HT2A antagonist volinanserin on the decreased immobility time in the FST and TST in mice treated with psychedelics. As depicted in Fig. 1, psilocin (1.5 mg/kg), DOI (0.1 mg/kg), and TCB-2 (5.0 mg/kg) decreased the immobility time in the FST and TST, and pretreatment with volinanserin prevented this effect of serotonergic psychedelics in the FST (Kruskal–Wallis nonparametric ANOVA followed by Bonferroni’s test; psilocin: H(3) = 7.11, p = 0.029, Fig. 2a; DOI: H(4)=11.22, p = 0.011, Fig. 2b; TCB-2: H(3) = 15.68, p = 0.00040, Fig. 2c) and TST (Kruskal–Wallis nonparametric ANOVA followed by Bonferroni’s test; psilocin: H(3) = 7.27, p = 0.026, Fig. 2d; DOI: H(4)=9.95, p = 0.019, Fig. 2e; TCB-2: H(3) = 11.06, p = 0.0040, Fig. 2f).
Next, we investigated the contribution of 5-HT2C to the antidepressant-like effects of psilocin and DOI because serotonergic psychedelics have a modest affinity for 5-HT2C (Table 1). We tested the effect of pretreatment with the 5-HT2C antagonist SB242084 on the decreased immobility time in the FST of mice treated with psilocin or DOI.As shown in Figs. 1 and 2, the administration of psilocin or DOI significantly decreased the immobility time in the FST 24 h after treatment, which was not influenced by pretreatment with SB242084 (Kruskal–Wallis nonparametric ANOVA followed by Bonferroni’s test; psilocin: H(4) = 15.21, p = 0.0016, Supplemental Fig. 1a; DOI: H(3)=7.87, p = 0.020, Supplemental Fig. 1b), suggesting that serotonergic psychedelics have an antidepressant-like effect through 5-HT2A, rather than 5-HT2C.
To rule out the potential effect of volinanserin alone, mice were subjected to the FST and TST 24 h after the administration of a single dose of volinanserin. There was no significant difference in the immobility time in the FST (Fig. 2b) and TST (Fig. 2e) between vehicle-treated control and volinanserin-treated mice.
3.3. Role of 5-HT2A in the anxiolytic-like effect of serotonergic psychedelics in mice
Next, we examined the involvement of 5-HT2A in the anxiolytic-like effects of serotonergic psychedelics. The NSFT has been used to test anxiety-like behaviors in rodents. The latency to consume pellets was measured as an indicator of anxiety-like behavior in a novel environment after fasting for 24 h. Mice were subjected to the NSFT 24 h after treatment with psilocin, DOI, or TCB-2. Psilocin treatment significantly reduced the latency to feed (Fig. 3a), which was not attenuated by pretreatment with volinanserin (Kruskal–Wallis nonparametric ANOVA followed by Bonferroni’s test; H(6) = 21.70, p = 0.00090; Fig. 3a). In contrast, DOI or TCB-2 administration did not affect the latency to feed in the NSFT (Fig. 3a). These results suggest that psilocin exerts an anxiolytic effect independent of 5-HT2A.
Psilocin and DOI did not affect feeding count and time 24 h after psychedelic administration. However, mice treated with TCB-2 showed a significant increase in feeding count and time compared to vehicle-treated control mice (p < 0.001, Fig. 3b; p < 0.0001, Fig. 3c) (Kruskal–Wallis nonparametric ANOVA followed by Bonferroni’s test; feeding count: H(6) = 14.83, p = 0.011, Fig. 3b; feeding time: H(6) = 17.69, p = 0.0034, Fig. 3c). Further studies are needed to explain why TCB-2 affects feeding count and time in this test.
3.4. Effect of serotonergic psychedelic administrations on social behavior in the three-chamber sociability test in mice
In this study, we tested the effects of a single dose of serotonergic psychedelics on social behavior using the three-chamber sociability test (Fig. 4a). The mice were subjected to the test 24 h after treatment with psilocin, DOI, or TCB-2. In habituation, there were no statistically significant differences in the interaction time between empty cages among the groups (Wilcoxon matched-pairs signed-rank test, vehicle: W=-42.00, p = 0.25; psilocin: W=-7.00, p = 0.56; DOI: W=-12.00, p = 0.46; TCB-2: W=-10.00, p = 0.47; Fig. 4b). In the sociability session, mice treated with either the vehicle or serotonergic psychedelics showed an increase in the interaction time with the stranger1 mouse over the empty cage (Wilcoxon matched-pairs signed-rank test, vehicle: W = 120.00, p < 0.0001; psilocin: W = 21.00, p < 0.05; DOI: W=36.00, p < 0.01; TCB-2: W = 36.00, p < 0.01, Fig. 4c); while, no significant difference was noted in the interaction time with stranger1 among all groups (Kruskal–Wallis nonparametric ANOVA followed by Bonferroni’s test; H(4) = 5.29, p = 0.15, Fig. 4c). Similarly, there was no difference in the sociability index among the four groups (Kruskal–Wallis nonparametric ANOVA followed by Bonferroni’s test; H(4) = 2.19, p = 0.53, Fig. 4d). These results suggest that mice treated with or without psychedelics showed the same level of sociability. In the social novelty session, mice treated with the vehicle spent more time interacting with the stranger2 mouse than with the stranger1 mouse, whereas mice treated with serotonergic psychedelics showed no statistical difference (Wilcoxon matched-pairs signed-rank test, vehicle: W = 112.0, p < 0.001; psilocin: W = 17.00, p = 0.0938; DOI: W=22.00, p = 0.15; TCB-2: W = 18.00, p = 0.25; Fig. 4e). On the other hand, no differences were observed in the interaction time with stranger2 (Kruskal–Wallis nonparametric ANOVA followed by Bonferroni’s test; H(4) = 4.62, p = 0.20, Fig. 4e) and in the social novelty index (Kruskal–Wallis nonparametric ANOVA followed by Bonferroni’s test, H(4) = 2.68, p = 0.44, Fig. 4f) among the four groups during the social novelty session, indicating that even though the vehicle-treated control mice exhibited social novelty/discrimination, the mice treated with psychedelics did not show such behavior. A single dose of psychedelic acid may have negatively affected social novelty/discrimination under the present experimental conditions.
3.5. Effect of serotonergic psychedelic administrations on the locomotor activities and the HTR in mice
Spontaneous locomotor activity in mice was measured for 15 min 24 h after psilocin, DOI, or TCB-2 administration, and no significant differences were found among the groups (Kruskal–Wallis nonparametric ANOVA followed by Bonferroni’s test; H(4) = 5.01, p = 0.17, Fig. 5a). Next, to assess the expression of hallucination-like behaviors in mice following the administration of serotonergic psychedelics, we investigated HTR for 30 min either immediately or 24 h after treatment with serotonergic psychedelics. Mice showed significantly greater HTR immediately after treatment with psilocin (1.5 mg/kg), DOI (1.0 or 2.0 mg/kg), or TCB-2 (5.0 mg/kg) (Fig. 5b-d). Interestingly, the mice showed no HTR induction even immediately after treatment with DOI (0.1 mg/kg) (Fig. 5c). On the other hand, psilocin (1.5 mg/kg), DOI (0.1 mg/kg), or TCB-2 (5.0 mg/kg) did not affect the HTR in mice 24 h after treatment (Kruskal–Wallis nonparametric ANOVA followed by Bonferroni’s test; psilocin: H(4) = 26.71, p < 0.0001, Fig. 5b; DOI: H(5)=42.50, p < 0.0001, Fig. 5c; TCB-2: H(4) = 26.60, p < 0.0001, Fig. 5d).
3.6. Long-lasting antidepressant-like effect of serotonergic psychedelics in the FST in mice
A previous clinical study showed that psilocybin treatment had a long-lasting effect on MDD for 12 months (Gukasyan et al., 2022). In the present study, we evaluated the persistent antidepressant-like effects of serotonergic psychedelics in mice. To accomplish this, the mice were administered psilocin, DOI, or TCB-2, and one week later, they were subjected to the FST. A decreasing effect of psilocin on immobility time was observed compared to that in vehicle-treated control mice. In contrast, DOI and TCB-2 did not affect the immobility time a week after treatment (Kruskal–Wallis nonparametric ANOVA followed by Bonferroni’s test; H(4) = 9.23, p = 0.026, Fig. 6a). To further evaluate how long this effect of psilocin persisted, the FST was repeated every week for a month. As a result, psilocin significantly and persistently decreased immobility time for three weeks after administration (two-way ANOVA followed by Bonferroni’s test; FTreatment(1,38) = 25.36, p < 0.0001; FTime(2,38) = 2.92, p = 0.067; FTreatment x Time(2,38) = 0.035, p = 0.96, Fig. 6b), but the effect was not observed four weeks after psilocin administration [Mann–Whitney U test, U = 15, p = 0.15, median with interquartile range, immobility time (sec); vehicle: 564.75 (263.31-647.03), psilocin: 462.99 (175.87-495.61); data not shown]. These results suggested that psilocin, but not DOI or TCB-2, may have long-lasting antidepressant effects.