Ethopharmacological evaluation of antidepressant-like effect of serotonergic psychedelics in C57BL/6J male mice

Serotonergic psychedelics such as psilocybin, lysergic acid diethylamide, and DOI exert a hallucinatory effect through serotonin 5-HT 2A receptor (5-HT2A) activation. Recent studies have revealed that serotonergic psychedelics have therapeutic potential for neuropsychiatric disorders, including major depressive and anxiety-related disorders. However, the involvement of 5-HT2A in mediating the therapeutic effects of these drugs remains unclear. In this study, we ethopharmacologically analyzed the role of 5-HT2A in the occurrence of anxiolytic-and antidepressant-like effects of serotonergic psychedelics such as psilocin, an active metabolite of psilocybin, DOI, and TCB-2 in mice. Mice with acute intraperitoneal psychedelic treatment exhibited significantly shorter immobility times in the forced swimming test (FST) and tail-suspension test (TST) than vehicle-treated control mice 24 h post-treatment. These effects were eliminated by pretreatment with volinanserin, a 5-HT2A antagonist. Surprisingly, the decreasing immobility time in the FST in response to acute psilocin treatment was sustained for at least three weeks. In the novelty-suppressed feeding test (NSFT), the latency to feed, an indicator of anxiety-like behavior, was decreased by acute administration of psilocin; however, pretreatment with volinanserin did not diminish this effect. In contrast, DOI and TCB-2 did not affect the NSFT performance in mice. Furthermore, psilocin, DOI, and TCB-2 treatment did not affect the spontaneous locomotor activity or head-twitch response, a hallucination-like behavior in rodents. These results suggest that 5-HT2A contributes to the antidepressant effects of serotonergic psychedelics rather than an anxiolytic effects.


Introduction
Approximately 30-40% of patients with major depressive disorder (MDD) have treatment-resistant depression (TRD), de ned as the failure of two or more medication trials, such as tricyclic antidepressants and selective serotonin reuptake inhibitors (SSRIs). Patients with TRD are more likely to attempt suicide than treatment responders (Nelsen and Dunner, 1995). Additionally, existing antidepressants require chronic dosing for several weeks, leading to an increased risk of adverse effects such as serotonin syndrome; i.e., altered mental status, autonomic hyperactivity, and neuromuscular abnormalities (Wisniewski et al., 2009, Foong et al., 2018. In the last 20 years, the N-methyl-D-aspartate (NMDA) receptor antagonist ketamine has shown rapid and sustained antidepressant effects in patients and animal models of depression (Berman et al., 2000, Zhang andHashimoto, 2019). Further, Spravato® (esketamine, the S-(+) enantiomer of ketamine) nasal spray was approved for the treatment of TRD by the U.S. Food and Drug Administration (FDA) as well as the European Commission (Turner, 2019, Singh et al., 2020. Unfortunately, esketamine has serious adverse effects, such as sedation and dissociation (Sapkota et al., 2021). Although 60-80% of patients with TRD are responsive to ketamine, the effects typically last for only 1-2 weeks at most (Ballard et Goodwin et al., 2022, Gukasyan et al., 2022. In particular, a recent study revealed that psilocybin administration produces antidepressant effects in patients with MDD throughout a 12-month follow-up period (Gukasyan et al., 2022), indicating the incomparable long-term effects of psilocybin. Based on these reports, the FDA has approved psilocybin as a breakthrough medicine for clinical trials in MDD (Nutt et al., 2020).

Drugs
Psilocin was synthesized within the Japanese law according to the methods published by Shirota et al. (Shirota et al., 2003), and its purity was veri ed by nuclear magnetic resonance analysis (Avance III 600 mg/mL using DMSO and dissolved to nal doses of 0.5, 1.0, and 5.0 mg/kg in saline. The concentration of uoxetine was adjusted to 50 mg/mL using DMSO and dissolved to a nal dose of 10 mg/kg using saline. SB242084 concentration was adjusted to 10 mg/mL using DMSO and dissolved to nal doses of 1.0 and 3.0 mg/kg using saline. Volinanserin was adjusted to 10 mg/mL using DMSO and dissolved in saline to a nal dose of 1.0 mg/kg using saline. The imipramine dose was adjusted to 5.0 mg/kg using saline. All drugs were administered intraperitoneally (i.p.) at a dose of 10 mL/kg.

Forced-swimming test
Forced-swimming test (FST) was performed to measure depression-like behavior as described previously, with minor modi cations (Takaba et al., 2022). Mice were individually placed in a clear beaker (19.3 cm in height, 13.1 cm in diameter; IWAKI, Tokyo, Japan) at a depth of 15 cm under water (25 ± 1℃) to ensure that mice could not touch the bottom of the beaker with their hind paws and tails and; allowed to swim freely for 15 min. Mice are generally immobile when they make no further attempts to escape except for the necessary movements to maintain their balance. All sessions were recorded using a video camera. The immobility time was measured in a double-blind manner.

Tail-suspension test
Tail-suspension test (TST) was carried out as described previously, with some modi cations (Can et al., 2012). Mice were individually suspended in a tail suspension box (30 cm length x 30 cm width x 30 cm height; BrainScience Idea. Co., Ltd., Osaka, Japan) using a sellotape attached 1 cm from the end of the tail under moderate light conditions (20 lx) for 5 min. The immobility time was automatically recorded and analyzed using the Ethovision XT 12 automated tracking program (Noldus, Wageningen, Netherlands).

Novelty-suppressed feeding test
The novelty suppressed feeding test (NSFT) was performed to measure anxiety-like behavior by presenting regular chow to food-deprived mice (Elsayed et al., 2012). Mice were food-restricted for 24 h prior to the NSFT. A single food pellet was placed in the center of a bright light (100 lx) arena (31 cm long × 31 cm wide × 15 cm heigh) lled with wood chip bedding material. Each mouse was placed individually at the center of the apparatus and allowed to move freely in the cage for 10 min. The exploratory behaviors of all mice were recorded using a video camera. The latency to feed, feeding counts, and feeding time data of each mouse were collected in a double-blind manner.

Head-twitch response test
The head-twitch response (HTR) consists of rapid side-to-side head movements and is used to test hallucination-related behaviors in rodents  Co., Ltd.) consisted of three chambers (left, center, and right) measuring 21 cm long × 41 cm wide × 22.5 cm heigh, with walls separated by transparent acrylic. The walls to the center chamber had 4 cm width × 8 cm length cut-out doors, allowing movement between the chambers. Small wire cages (diameter, 10 cm) were placed in the right and left chambers, 4 cm from the walls. During habituation, the subject mouse was allowed to habituate to the apparatus and freely explore the three chambers for 15 min. An empty wire cage was placed in the right and left chambers under moderate light conditions (20 lx). During the sociability session, an unfamiliar male C57BL/6J mouse (stranger1) that had never met the subject mouse was placed in a wire cage in the left chamber, and an empty wire cage was placed in the right chamber. The subject mouse was introduced into the central chamber and allowed to freely explore the three chambers for 15 min. In the social novelty session, another unfamiliar male C57BL/6J mouse (stranger2) that had never met the subject mouse was placed in an empty wire cage in the right chamber, while the stranger1 remained in the cage in the left chamber. The subject mouse was introduced into the middle chamber and allowed to explore the three chambers freely for 15 min. After each session, the subject mouse was returned to its home cage for 10 min, and the three-chamber apparatus and wire cages were cleaned with puri ed water.
The time spent interacting with stranger1, stranger2, and the empty cage was automatically measured using the Ethovision XT 12 automated tracking program (Noldus). In order to reduce the variability among mice, the "sociability index" in each mouse was employed. This index was calculated using the

Statistical analysis
Prism 8 (GraphPad Software, Inc., San Diego, CA, USA) was used for statistical analyses, and gure generation. Since it was impossible to assume that the behavioral data had a Gaussian distribution, the data were expressed as medians and interquartile ranges. Signi cance was evaluated using the Kruskal-Wallis nonparametric one-way or two-way analysis of variance (ANOVA), followed by Bonferroni's test for multiple comparisons. For the three-chamber sociability test, the interaction times with the cages in the left and right chambers within each group were statistically compared using the Wilcoxon matched-pairs signed-rank test in all sessions. A value of p < 0.05 was considered statistically signi cant.  Fig. 1c). In contrast, acute treatment with the antidepressants uoxetine, 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.  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 a nity 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 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 signi cant 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 signi cantly 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.

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 signi cant 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 signi cant 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.

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 signi cantly and persistently decreased immobility time for three weeks after administration (two-way ANOVA followed by

Discussion
It is well known that 5-HT2A activation is necessary for psychedelic experiences (López-Giménez and González-Maeso, 2018), but the involvement of 5-HT2A in the therapeutic effects of psychedelics remains unknown. In a preclinical study, a single dose of psilocybin reportedly improved anhedonic behaviors in mice with chronic stress in the sucrose preference and female urine sni ng tests, which were not blocked by pretreatment with ketanserin, a 5-HT2A/2C antagonist (Hesselgrave et al., 2021). In contrast, pretreatment with ketanserin prevented the antidepressant-like effects of tabernanthalog, a nonhallucinogenic derivative of the psychedelic alkaloid ibogaine, in the FST of mice (Cameron et al., 2021). Based on these ndings, the requirement of 5-HT2A for the therapeutic effects of serotonergic psychedelics is still controversial (Ripoll et  Regarding effects other than antidepressant effects, the present study showed an anxiolytic-like effect of psilocin, but not of DOI or TCB-2, in the NSFT of mice; however, the observed effect of psilocin was not attenuated by pretreatment with volinanserin ( Fig. 3a), suggesting that psilocin induces anxiolytic effects independent of 5-HT2A receptors. In this context, DOI and TCB-2 may not have anxiolytic effects because they have a higher selectivity for 5-HT2A than psilocin. Meanwhile, psilocin shows moderate to high a nity for 5-HT2A, 5-HT1A, and 5-HT2C (Table 1) (Nichols, 2012). This raises the possibility that differences in the effects of psychedelics may be attributed to differences in their a nity for serotonergic receptors, including 5-HT2A.
In the present study, a single dose of psilocin, DOI, or TCB-2 did not affect the social behavior in naïve mice without stress in the three-chamber sociability test (Fig. 4). Interestingly, rodents with either social isolation in early life or chronic social defeat stress in adulthood reportedly display social dysfunction did not affect social function in naïve animals (Scarborough et al., 2021, Ortiz et al., 2022, which is consistent with the current study. This led us to consider whether the prosocial effects of acute psychedelic administration may become more evident in animal stress models. Therefore, future studies should test the prosocial effects of psychedelics in animal stress models. Similar to previous reports , Haberzettl et al., 2014, Klein et al., 2021, the current study demonstrated that hallucination-like behaviors occurred immediately after treatment with psilocin and TCB-2 in mice, although the effects were diminished 24 h after treatments (Fig. 5b, d). Additionally, DOI showed antidepressant-like effects in mice ( Fig. 1b and 2b, e) but did not induce hallucination-like behavior immediately after administration, even at the same concentration (Fig. 5c). These results suggest that the underlying mechanisms of the antidepressant effect may be different from those of hallucinations, even though both effects are evoked by 5-HT2A stimulation. Thus, if the antidepressant effect could be differentiated from the hallucinatory effects, this could potentially promote the development of highly effective antidepressants that do not induce hallucinations. To realize this, we should discuss two possibilities: the molecular and neuronal discrimination of the antidepressant effect.
First, the molecular aspects should be discussed. Since 5-HT2A is a Gα q protein-coupled receptor, it activates the Gq protein as a matter of course. On the other hand, signaling pathways of Gα i/o , Gα 12/13 , and β-arrestin, a scaffolding protein involved in receptor internalization and desensitization, are also activated by 5-HT2A stimulation (Pottie and Stove, 2022). A recent study with a newly created psychedelic analog suggested that the stimulation of the orthosteric binding pocket of 5-HT2A induces both hallucinatory and antidepressant effects via the Gq-mediated pathway, while stimulation of the allosteric pocket induces an antidepressant effect via the β-arrestin pathway without inducting hallucinations; that is, β-arrestin-biased 5-HT2A agonists may selectively lead to antidepressant effects (Cao et al., 2022, Yin andGao, 2023). In the present study, DOI at 0.1 mg/kg showed an antidepressantlike effect without inducing HTR in mice, raising the possibility that DOI may activate the β-arrestin pathway rather than the Gq-related pathway, acting as a β-arrestin-biased agonist.