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

Abstract

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 24 h post-treatment. 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. 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 anxiolytic effects.

Introduction

Approximately 30–40% of patients with major depressive disorder (MDD) have treatment-resistant depression (TRD), defined 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 and Hashimoto 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 al. 2018; Rong et al. 2018; Wilkinson et al. 2018). Therefore, safer and more effective therapeutic agents are required to treat patients with TRD. Carhart-Harris and colleagues have reported the rapid and lasting antidepressant effects of psilocybin, a hallucinogenic component of the psilocybe genus found in magic mushrooms (Hofmann et al. 1958), in patients with TRD (Carhart-Harris et al. 2016). Most patients with TRD had reduced depressive symptoms that lasted for at least three weeks (Carhart-Harris et al. 2018). These findings are supported by various clinical studies (Carhart-Harris et al. 2021; Davis et al. 2021; 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).

Psilocybin is known as one of the ‘‘serotonergic psychedelics’’ that activate the cortical serotonin 5-HT_2A receptor (5-HT2A), causing a hallucinatory effect (Gonzalez-Maeso et al. 2007; Nichols 2016); however, involvement of 5-HT2A in the therapeutic effects of psilocybin remains unclear. Although preclinical studies in mice have demonstrated the potential therapeutic effects of serotonergic psychedelics independent of 5-HT2A (Hesselgrave et al. 2021; Odland et al. 2021), other research groups have reported that 5-HT2A is required for the therapeutic effects of serotonergic psychedelics in rodents (Ripoll et al. 2006; Cameron et al. 2021; Cao et al. 2022). Therefore, it may be useful to investigate the antidepressant effects of various psychedelics with different affinities for 5-HT2A.

Serotonergic psychedelics are mainly classified into three structures: i.e., tryptamines such as psilocybin, ergolines such as lysergic acid diethylamide (LSD), and phenethylamines such as (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), 2,5-dimethoxy-4-methylamphetamine (DOM), 2,5-dimethoxy-4-bromoamphetamine (DOB), and (4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine (TCB-2) (McLean et al. 2006; Nichols 2018). Although tryptamine and ergoline derivatives are nonselective 5-HT receptor agonists, phenethylamine derivatives exhibit higher selectivity for 5-HT2A than other structures (Nichols 2012) (also see Table 1). In this study, we especially investigated the role of 5-HT2A in the post-acute effects of psychedelics on behavior models relevant to antidepressant, anxiety, and sociability using ethological (i.e., animal behavioral) analyses in mice. For instance, such emotional behaviors were tested in mice 24 h after (at the point at which the drug is no longer detectable in the mouse body, given its half-life (Hasler et al. 1997; McLean et al. 2006; de la Fuente Revenga et al. 2019)) administration of a psychedelic tryptamine such as psilocin, an active metabolite of psilocybin (psilocybin loses its phosphate portion and is completely converted to psilocin before entering the systemic circulation (Eivindvik et al. 1989)), and phenethylamines such as DOI and TCB-2. Additionally, spontaneous activity and hallucinatory-like behavior in mice 24 h after serotonergic psychedelic administrations were also examined. This study provides evidence for the use of 5-HT2A as a therapeutic target in MDD and related disorders.

Table 1.

Affinity values (Ki) of serotonergic psychedelics and antagonists towards 5-HT1A, 5-HT2A, and 5-HT2C

	5-HT receptor subtypes (Rats)
	5-HT1A	5-HT2A	5-HT2C	References
Psychedelics
Psilocin	49 nM	25 nM	10 nM	(Blair et al. 2000)
DOI	n.d	0.79 nM	3.01 nM	(Knight et al. 2004)
TCB-2	n.d	0.73 nM	n.d	(McLean et al. 2006)
Antagonists
Volinanserin	n.d	0.36 nM	88 nM	(Sorensen et al. 1993)
SB242048	n.d	158 nM	0.47 nM	(Majczyński et al. 2020)

n.d., not determined

Materials and methods

Animals

Male C57BL/6J mice (7–9 weeks old) were obtained from Japan SLC, Inc. (Hamamatsu, Japan). Unless otherwise stated, the mice were group-housed (4–5 mice/cage). The mice were kept in a well-regulated environment (24 ± 1°C, 55 ± 5% humidity) according to a 12-h light/dark cycle (lights on at 7:00 a.m.) and provided food and tap water ad libitum. All experiments followed the guidelines established by the Japanese Pharmacological Society and Institute for Experimental Animals at Meijo University. Protocols were approved by the Animal Ethics Board of Meijo University [permit no. (2017–2022)-11]. All efforts were made to minimize animal suffering and to reduce the number of animals used.

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 verified by nuclear magnetic resonance analysis (Avance III 600, Bruker, MA, USA). (±)-1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane (DOI), fluoxetine, and imipramine were obtained from Sigma-Aldrich (St. Louis, MO, USA). 4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide (TCB-2) was obtained from Tocris Biosciences (Minneapolis, MN, USA). 6-chloro-2,3-dihydro-5-methyl-N-[6-[(2-methyl-3-pyridinyl)oxy]-3-pyridinyl]-1H-indole-1-carboxamide dihydrochloride (SB242084) and (R)-(2,3-dimethoxyphenyl)(1-(2-(4-fluorophenyl)ethyl)piperidin-4-yl methanol (volinanserin) were obtained from Cayman Chemical (Ann Arbor, MI, USA). Psilocin was dissolved into final doses of 1.5, 2.0, and 4.0 mg/kg in 0.9% NaCl solution (saline). DOI concentration was adjusted to 10 mg/mL using dimethyl sulfoxide (DMSO; Sigma-Aldrich) and dissolved in saline to obtain the final doses of 0.05, 0.1, 0.25, 1.0, and 2.0 mg/kg. The concentration of TCB-2 was adjusted to 10 mg/mL using DMSO and dissolved to final doses of 0.5, 1.0, and 5.0 mg/kg in saline. The concentration of fluoxetine was adjusted to 50 mg/mL using DMSO and dissolved to a final dose of 10 mg/kg using saline. SB242084 concentration was adjusted to 10 mg/mL using DMSO and dissolved to final 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 final dose of 1.0 mg/kg using saline. The imipramine dose was adjusted to 10 mg/kg using saline. All drugs were administered intraperitoneally (i.p.) at a volume of 10 mL/kg.

Ethological analyses

Except for the head-twitch response (HTR) test, different mice were used for each respective test. In the HTR test, HTR in mice was measured twice, both immediately and 24 h after administration. In other tests, mice that had not experienced any tests were used for ethological analysis, respectively. To counteract the effect of hallucination-like behavior induced within 60 min of psychedelic administration, the forced-swimming, tail-suspension, novelty-suppressed feeding, and three-chamber sociability tests were performed 24 h after psychedelic administration.

Forced-swimming test

Forced-swimming test (FST) was performed to measure depression-like behavior as described previously, with minor modifications (Cryan et al. 2005; 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°C) 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. The effect of psilocin was analyzed not only 24 h after drug administration, but also every week for up to 3 weeks. All sessions were recorded using a video camera. The immobility time was measured visually by multiple experimenters in a double-blind manner.

Tail-suspension test

Tail-suspension test (TST) was carried out as described previously, with some modifications (Can et al. 2012). Mice were individually suspended in a tail suspension box (30 cm length × 30 cm width × 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 height) filled 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 HTR of mice was observed immediately after psychedelic administration, some of which were monitored 24 h later, again. The HTR consists of rapid side-to-side head movements and is used to test hallucination-related behaviors in rodents (Halberstadt and Geyer 2011). Mice were placed in an observation cage (29 cm long × 18 cm wide × 12 cm heigh) under moderate light conditions (15 lx) immediately and 24 h after drug administration. The HTR was recorded for 30 min using a video camera. Drug-induced HTR (defined as rapid rotations of the head that can be distinguished from species-appropriate grooming or scratching behaviors) was counted as described previously, with minor modifications (Gonzalez-Maeso et al. 2007; Ibi et al. 2017) in a double-blind manner.

Three-chamber sociability test

A three-chamber sociability test was used to assess sociability and interest in social novelty and discrimination. This test was carried out as described previously, with some modifications (Moy et al. 2004; De Gregorio et al. 2021). The three-chamber apparatus (62 cm long × 41 cm wide × 22.5 cm height; BrainScience Idea. Co., Ltd.) consisted of three chambers (left, center, and right) measuring 21 cm long × 41 cm wide × 22.5 cm height, 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 purified 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 following formula: [(interaction time with stranger1–interaction time with empty cage) / (interaction time with stranger1 + interaction time with empty cage) × 100]. The social novelty index was similarly calculated using the following formula: [(interaction time with stranger2– interaction time with stranger1) / (interaction time with stranger2 + interaction time with stranger1) × 100].

Locomotor activity

Locomotor activity was assessed as previously described (Ibi et al. 2020). Locomotor activity was performed 24 h after psychedelic administration to exclude the possibility that alternation of locomotor activity affects the performance in the ethological analyses, for instance, immobility time in the FST and TST. Each mouse was placed in a standard transparent rectangular rodent cage (40 cm long × 45 cm wide × 26 cm hight) under moderate light conditions (15 lx). The locomotor activity of each mouse was automatically measured for 15 min using digital counters with infrared sensors (Scanet SV-40; Melquest Co. Ltd., Toyama, Japan) 24 h after drug administration.

Statistical analysis

Prism 8 (GraphPad Software, Inc., San Diego, CA, USA) was used for statistical analyses and figure generation. Since it was impossible to assume that the behavioral data had a Gaussian distribution, the data were expressed as medians and interquartile ranges. Significance 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 significant.

Results

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, DOI, or TCB-2 (Fig. 1a). The doses 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), and TCB-2 (0.5, 1.0, and 5.0 mg/kg) were determined based on the previous reports (Fox et al. 2010; Halberstadt et al. 2011). 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. 1b; DOI: H(6) = 16.06, p = 0.0067, Fig. 1c; TCB-2: H(4) = 8.26, p = 0.035, Fig. 1d). In contrast, acute treatment with the antidepressants fluoxetine, a selective serotonin reuptake inhibitor, and imipramine, a tricyclic anti-depressant, 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. 1e). The doses of fluoxetine and imipramine were selected based on previous studies (Garcia et al. 2008; Zanos et al. 2015). These results suggest that psychedelics have antidepressant-like effects at the active dose in mice. As the active dose, 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. 1.

Effect of serotonergic psychedelic administrations on the immobility time in the FST in mice. Experimental schedule (a). The FST was performed 24 h after drug administration in mice, and the immobility time was measured for 15 min. The doses of psilocin (b), DOI (c), and TCB-2 (d) were examined in the FST at the doses indicated below the vertical line of each graph. The effects of fluoxetine at 10 mg/kg and imipramine at 5.0 mg/kg (e) were also examined. Values represent the median with interquartile range (n = 7–14). Significance levels: *p < 0.05, ^**p < 0.01, vs. vehicle (Kruskal–Wallis nonparametric one-way ANOVA followed by Bonferroni’s test). n.s.: not significant

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 (Fig. 2a). 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, consistent with a previous report that DOI decreases the immobility time in the FST in naïve mice (de la Fuente Revenga et al. 2021) (Table 2). Pretreatment with volinanserin prevented the reduction of immobility time in mice with serotonergic psychedelics in the FST (Kruskal–Wallis nonparametric ANOVA followed by Bonferroni’s test; psilocin: H(3) = 7.11, p = 0.029, Fig. 2b; DOI: H(4) = 11.22, p = 0.011, Fig. 2c; TCB-2: H(3) = 15.68, p = 0.00040, Fig. 2d) and TST (Kruskal–Wallis nonparametric ANOVA followed by Bonferroni’s test; psilocin: H(3) = 7.27, p = 0.026, Fig. 2e; DOI: H(4) = 9.95, p = 0.019, Fig. 2f; TCB-2: H(3) = 11.06, p = 0.0040, Fig. 2g).

Fig. 2.

Role of 5-HT2A in the antidepressant-like effect of serotonergic psychedelics in mice. Experimental schedule (a). FST (b-d) or TST (e–g) was carried out 24 h after administration of psilocin at 1.5 mg/kg (b, e), DOI at 0.1 mg/kg (c, f), or TCB-2 at 5.0 mg/kg (d, g) in mice, in which the immobility time was measured for 15 or 5 min, respectively. Volinanserin (Voli) at 1.0 mg/kg was injected 30 min before serotonergic psychedelic administration. Values represent the median with interquartile range (n = 6–22). Significance levels: *p < 0.05, ^**p < 0.01, ^***p < 0.001 vs. vehicle; ^#p < 0.05, ^##p < 0.01, vs. serotonergic psychedelic alone (Kruskal–Wallis nonparametric one-way ANOVA followed by Bonferroni’s test). n.s.: not significant

Table 2.

Summary of antidepressant- and anxiolytic-like behavioral changes in naïve mice treated with a single dose of serotonergic psychedelic

			The present study	Hibicke et al. 2020	de la Fuente Revenga et al. 2021	Odland et al. 2021	Singh et al. 2023
Age			7–9 weeks old	7–8 weeks old	10–20 weeks old	17–26 weeks old	Not reported
Species			Mouse	Rat	Mouse	Mouse	Mouse
Strain			C57BL/6J	WKY	C57BL/6NTac	NMRI	ICR
Sex			Male	Male	Male	Female	Male
Drug Dose (Administration route)			Psilocin 1.5 mg/kg (i.p.)	Psilocybin 1.0 mg/kg (i.p.)	DOI 2.0 mg/kg (i.p.)	Psilocybin 1.0 and 2.0 mg/kg (i.p.)	Psilocybin 4.4 mg/kg (i.p.)
			DOI 0.1 mg/kg (i.p.)	LSD 0.15 mg/kg (i.p.)		DOI 1.0 mg/kg (i.p.)
			TCB-2 5.0 mg/kg (i.p.)
Duration from administration to behavioral test
Antidepressant-like effect			24 h, 1, 2, and, 3 weeks	35 days	24 h and 7 days	n.d	n.d
Anxiolytic-like effect			24 h	40 or 41 days	24 h	30 min	60 min
Behavioral tests	Evaluated function	Measurements
Forced-swimming test	Antidepressant-like effect	Reduction of immobility time	Psilocin: ↑ (24 h, 1, 2, and 3 week)	Psilocybin: ↑	↑	n.d	n.d
			DOI: ↑ (24 h)	LSD: ↑
			TCB-2: ↑ (24 h)
Tail-suspension test	Antidepressant-like effect	Reduction of immobility time	Psilocin: ↑ (24 h)	n.d	n.d	n.d	n.d
			DOI: ↑ (24 h)
			TCB-2: ↑ (24 h)
Novelty-suppressed feeding test	Anxiolytic-like effect	Reduction of latency to eat	Psilocin: ↑	n.d	n.d	n.d	n.d
			DOI: =
			TCB-2: =
Elevated-plus maze test	Anxiolytic-like effect	Spent time in open arms	n.d	Psilocybin: ↑ (41 days)	n.d	n.d	n.d
				LSD: = (40 days)
Light–dark boxes preference test	Anxiolytic-like effect	Spent time in light box	n.d	n.d	=	n.d	n.d
Marble-burying test	Anxiolytic-like effect	Reduction of marble burying behavior	n.d	n.d	n.d	Psilocybin: ↑	↑
						DOI: ↑

^↑, Higher than vehicle-treated; =, no difference; n.d., not determined

Next, we investigated the contribution of 5-HT2C to the antidepressant-like effects of psilocin and DOI (Supplemental Fig. 1a) 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, where the doses of SB242084 were determined based on a previous study (Egashira et al. 2012). 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. 1b; DOI: H(3) = 7.87, p = 0.020, Supplemental Fig. 1c), 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. 2c) and TST (Fig. 2f) between vehicle-treated control and volinanserin-treated mice.

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 (Fig. 3a). Psilocin treatment significantly reduced the latency to feed (Fig. 3b). This is consistent with previous reports that psychedelics exhibit anxiolytic-like effects in naïve rodents (Hibicke et al. 2020; Odland et al. 2021; Singh et al. 2023) (Table 2). Anxiolytic-like effect of psilocin was not attenuated by pretreatment with volinanserin (Kruskal–Wallis nonparametric ANOVA followed by Bonferroni’s test; H(6) = 21.70, p = 0.00090; Fig. 3b). In contrast, DOI or TCB-2 administration did not affect the latency to feed in the NSFT (Fig. 3b). These results suggest that psilocin exerts an anxiolytic effect independent of 5-HT2A.

Fig. 3.

Role of 5-HT2A in the anxiolytic-like effect of serotonergic psychedelics in mice. Experimental schedule (a). Mice were administrated serotonergic psychedelic (psilocin at 1.5 mg/kg, DOI at 0.1 mg/kg, or TCB-2 at 5.0 mg/kg) 24 h before the NSFT, and food was restricted for 24 h until the test. During this test, latency to feed (b), feeding counts (c), and feeding time (d) were measured. Volinanserin (Voli) at 1.0 mg/kg was injected 30 min before psilocin administration. Values represent the median with interquartile range (n = 5–15). Significance levels: *p < 0.05, ^**p < 0.01, ^***p < 0.001 vs. vehicle (Kruskal–Wallis nonparametric one-way ANOVA followed by Bonferroni’s test). n.s.: not significant

To provide evidence that the decrease in the latency to eat was not due to a decrease in appetite, we measured the feeding counts and time. Psilocin and DOI did not affect feeding counts and time 24 h after psychedelic administration. However, mice treated with TCB-2 showed a significant increase in feeding counts and time compared to vehicle-treated control mice (p < 0.001, Fig. 3c; p < 0.0001, Fig. 3d) (Kruskal–Wallis nonparametric ANOVA followed by Bonferroni’s test; feeding counts: H(6) = 14.83, p = 0.011, Fig. 3c; feeding time: H(6) = 17.69, p = 0.0034, Fig. 3d). Further studies are needed to explain why TCB-2 affects feeding counts and time in this test.

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.

Fig. 4.

Effect of serotonergic psychedelic administrations on social behavior in the three-chamber sociability test in mice. The mice were first habituated to the experimental apparatus 24 h after psychedelic administration, and then sociability and social novelty sessions were performed sequentially. All sessions were conducted every 10 min for 15 min. Experimental schedule (top) and depiction (bottom) of the habituation (bottom left), sociability (bottom center), and social novelty (bottom right) sessions (a). In the sociability session, one cage contained an unfamiliar mouse (stranger1), while the other was empty. The subject mouse was allowed to freely explore the three chambers. In the social novelty session, another unfamiliar mouse (stranger2) was placed in the remaining empty cage opposite stranger1, and the subject mouse was allowed to explore freely within the three chambers. First, the time spent interacting with both empty cages was measured (habituation b). Next, the time spent interacting with either stranger1 or the empty cage was measured (sociability sessions; c and d). Finally, the time spent interacting with stranger1 or stranger2 was measured (social novelty sessions; e and f). To reduce variability among mice, we calculated the sociability (d) and social novelty (f) indices. The Wilcoxon matched-pairs signed rank test was used to compare the interaction times between cages in each group. Kruskal–Wallis nonparametric one-way ANOVA followed by Bonferroni’s test was used to compare sociability and social novelty indices and the interaction time with strangers1 or 2 among groups. Values represent the median with interquartile range (n = 6–15). Significance levels: *p < 0.05, ^**p < 0.01, ^****p < 0.0001, vs. empty (c), ^***p < 0.001 vs. stranger1 (e) (Wilcoxon matched-pairs signed rank test (b, c, e)). n.s.: not significant

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 (Fig. 5a), 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. 5b). Next, to assess the expression of hallucination-like behaviors in mice following the administration of serotonergic psychedelics, we investigated HTR for 30 min both immediately and 24 h after treatment with serotonergic psychedelics (Fig. 5a). The HTR was measured 24 h psychedelic treatment to remove the possibility that hallucination-like behavior develops 24 h after administration, i.e., when the antidepressant- and anxiolytic-like effects were observed in the ethological analyses in mice. 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. 5c–e). Interestingly, the mice showed no HTR induction even immediately after treatment with DOI (0.1 mg/kg) (Fig. 5d). 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. 5c; DOI: H(5) = 42.50, p < 0.0001, Fig. 5d; TCB-2: H(4) = 26.60, p < 0.0001, Fig. 5e).

Fig. 5.

Effect of serotonergic psychedelic administrations on the locomotor activities and the HTR in mice. Experimental schedule (a). Spontaneous locomotor activity was measured for 15 min 24 h after administration of psilocin at 1.5 mg/kg, DOI at 0.1 mg/kg, or TCB-2 at 5.0 mg/kg (b). HTR were counted for 30 min, both immediately and 24 h after administration of psilocin (c), DOI (d), and TCB-2 (e). Psychedelics dosages are indicated below the vertical line in each graph. Volinanserin (Voli) at 1.0 mg/kg was injected 30 min before psilocin administration. Values represent the median with interquartile range (n = 4–17). Significance levels: *p < 0.05, ^***p < 0.001, ^****p < 0.0001 vs. vehicle (Kruskal–Wallis nonparametric one-way ANOVA followed by Bonferroni’s test). n.s.: not significant

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 (Fig. 6a). 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. 6b). To further evaluate how long this effect of psilocin persisted, the FST was repeated every week for a month (Fig. 6c). As a result, psilocin significantly and persistently decreased immobility time for three weeks after administration (two-way ANOVA followed by Bonferroni’s test; F_{Treatment(1,35)} = 20.68, p < 0.0001; F_Time(2,35) = 2.13 p = 0.13; F_{Treatment x Time(2,38)} = 0.018, p = 0.98, Fig. 6d), but the effect was not observed four weeks after psilocin administration [p = 0.15 (Mann–Whitney U test, U = 15), median (interquartile range) immobility time (sec); vehicle: 603.7 (263.31–647.03), psilocin: 464 (175.87–482.85)]. These results suggested that psilocin, but not DOI or TCB-2, may have long-lasting antidepressant effects.

Fig. 6.

Long-lasting antidepressant-like effect of serotonergic psychedelics in the FST in mice. Experimental schedules (a). FST was carried out in mice a week after administration of psilocin at 1.5 mg/kg, DOI at 0.1 mg/kg, or TCB-2 at 5.0 mg/kg, in which the immobility time was measured for 15 min (b). Experimental schedules (c). The FST was repeatedly performed every week for 3 weeks in mice treated with psilocin, and immobility time was measured for 15 min (d). Values represent the median with interquartile range (n = 7–10). Significance levels: *p < 0.05, vs. vehicle (Kruskal–Wallis nonparametric one-way ANOVA followed by Bonferroni’s test), ^#p < 0.05 vs. vehicle group at the corresponding time (two-way ANOVA followed by Bonferroni’s test). n.s.: not significant

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 sniffing 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 non-hallucinogenic derivative of the psychedelic alkaloid ibogaine, in the FST of mice (Cameron et al. 2021). Based on these findings, the requirement of 5-HT2A for the therapeutic effects of serotonergic psychedelics is still controversial (Ripoll et al. 2006; Cameron et al. 2021; Cao et al. 2022). Considering our current study showing that volinanserin suppressed the antidepressant-like effects of serotonergic psychedelics, 5-HT2A activation by psychedelics may at least contribute to their antidepressant-like effects.

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. 3b), suggesting that psilocin induces anxiolytic effects independent of 5-HT2A receptors. In this context, DOI and TCB-2 may not have long-lasting anxiolytic effects because they have a higher selectivity for 5-HT2A than psilocin. Meanwhile, psilocin shows moderate to high affinity 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 affinity for serotonergic receptors, including 5-HT2A.

On the other hand, we should also discuss the possibility that the therapeutic effects of psychedelics cannot be solely explained by serotonergic receptors. For instance, pretreatment of antagonists for 5-HT1A (WAY100635), 5-HT2A (M100907), and 5-HT2C (SB242084) had no effect on the anxiolytic-like effects of psilocybin in the marble-burying test in mice (Odland et al. 2021; Singh et al. 2023), raising a possibility that non-serotonergic receptor may partially play a role in the therapeutic effect of psychedelics. The involvement of non-serotonergic receptors in antidepressant effect was supported by another study to demonstrate that psilocybin firmly binds to TrkB, the receptor for brain-derived neurotrophic factor (BDNF), a key player for antidepressants effect (Castren and Antila 2017), leading to antidepressant effects of psychedelics (Moliner et al. 2023). Taken together, the role of non-serotonergic receptors such as TrkB in the therapeutic effect of psychedelics also should be further investigated in the future.

The three main groups of psychedelics are tryptamines, ergolines, and phenethylamines (Nichols 2018). Previous studies have shown that ergolines such as LSD produced anxiolytic-like and prosocial effects in mice (De Gregorio et al. 2021, 2022), although we did not examine the therapeutic effects of only ergolines in the present study. The previous study has suggested that 5-HT2A may be required for the therapeutic effects of LSD (Holze et al. 2021). Accordingly, since phenethylamines such as DOI and TCB-2 have a higher affinity for 5-HT2A than other structures, they were employed in the present study, instead of ergolines. As shown in the Introduction, this supports the aim of the present study, i.e., investigation of the role of 5-HT2A in the therapeutic effects of psychedelics.

The selectivity of the antagonists used in this study should be discussed. Volinanserin is a potent and selective antagonist of 5-HT2A (Ki = 0.36 nM), and it slightly binds 5-HT2C (Ki = 88 nM, Table 1). In a previous study, pretreatment with volinanserin at 1.0 mg/kg blocked TCB-2-induced anxiolytic-like effects in the contextual fear conditioning test in rats (Hagsater et al. 2021), suggesting that volinanserin at 1.0 mg/kg may inhibit the therapeutic effect of psychedelics. Accordingly, we determined such dosage condition of volinanserin. SB-242084 is an antagonist for 5-HT2C (Ki = 0.47 nM, Table 1), with at least 100-fold more selectivity over other serotonergic receptors. In a previous study, SB242084 at the dose of 3.0 mg/kg significantly reversed the anxiolytic-like behavior caused by DOI in the marble-burying test in mice, whereas 1.0 mg/kg of SB242084 did not show such effect (Egashira et al. 2012). The doses of SB242084 used in this study should be sufficient to affect the effect of psychedelics. Taken together with the present results, the dosages of drugs used in the present study, not only psychedelics but also 5-HT2A/2C antagonists, could be pharmacologically reasonable and efficacious. Furthermore, from a clinical application perspective, considering that psychedelic reportedly exhibits similar pharmacological profile in both mouse and human brain tissue (Erkizia-Santamaría et al. 2022), the present report may help dose determination for clinical application/research of serotonergic psychedelics.

In the present study, psilocin, DOI, or TCB-2 did not affect the social behavior in naïve mice without stress in the three-chamber sociability test (Fig. 4). Conversely, administration with a sub-threshold dose of LSD several days reportedly increased prosocial effects in naïve mice (De Gregorio et al. 2021). Considering that this administration condition is entirely distinct from the current study, it is worthwhile to explore the prosocial effects of repeated administrations with sub-threshold doses of psilocin, DOI, and TCB-2. In addition, interestingly, rodents with either social isolation in early life or chronic social defeat stress in adulthood reportedly display social dysfunction (Garcia-Pardo et al. 2015; Jianhua et al. 2017; Rivera-Irizarry et al. 2020), which can be prevented by SSRI or ketamine treatment (Scarborough et al. 2021; Ortiz et al. 2022). Meanwhile, SSRI and ketamine 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. In addition, the vehicle-treated control mice exhibited social novelty/discrimination in the social novelty session, while mice treated with psychedelics did not (Fig. 4e), raising a possibility that psychedelics may impair social cognition; however, this likely contradicts a previous report that psilocybin improved cognitive dysfunction observed in a mouse model of autism (Buzzelli et al. 2023). Therefore, future studies should test the effects of psychedelics on social cognitive function in animal models of stress and/or disease.

Similar to previous reports (Halberstadt et al. 2011; 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. 5c, e). Additionally, DOI showed antidepressant-like effects in mice (Figs. 1c and 2c, f) but did not induce hallucination-like behavior immediately after administration, when given at the same concentration (Fig. 5d). 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. 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, 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 G_q-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 and Gao 2023). In the present study, DOI at 0.1 mg/kg showed an antidepressant-like effect without inducing HTR in mice, raising the possibility that DOI at the dose of 0.1 mg/kg may activate the β-arrestin pathway rather than the Gq-related pathway. This is also supported by a previous study demonstrating that the EC50 value of DOI for β-arrestin is even lower than that for Gq (Pottie et al. 2020). On the other hand, some studies showed that DOI may rapidly desensitize and/or downregulate 5-HT2A function/expression (Karaki et al. 2014; de la Fuente Revenga et al. 2022; Pędzich et al. 2022b), which may affect the results of subsequent behavioral tests. Future studies are needed to determine whether DOI at the dose of 0.1 mg/kg causes desensitization and/or downregulation of 5-HT2A, and whether it is biased toward β-arrestin-related pathway.

Second, the neuronal bases that isolate the antidepressant effects of psychedelics should be discussed. Previous studies have suggested that the activation of 5-HT2A in the cortex, such as the medial prefrontal and visual cortices, contributes to the hallucinatory-like effects of psychedelics in rodents (Willins and Meltzer 1997; Gonzalez-Maeso et al. 2007; Michaiel et al. 2019). Some reports have already suggested that the amygdala, the brain region that processes fear and anxiety, is partially involved in the antidepressant and anxiolytic effects of psilocybin (Carhart-Harris et al. 2017; Barrett et al. 2020; Effinger et al. 2023), however, few studies have examined the contribution of 5-HT2A in the amygdala to antidepressant- and anxiolytic-like effects of psychedelics (Pavlova et al. 2020; Pedzich et al. 2022a). If 5-HT2A activity in the cortex is not necessary for the antidepressant effect of psychedelics, it may be possible to separate the antidepressant effect from hallucinations by manipulating 5-HT2A activity in a brain region-specific manner. Taken together, the molecular and/or neuronal basis responsible for the anti-depressant effects of serotonergic psychedelics should be elucidated to isolate the antidepressant effects of the drugs from the hallucinatory and other effects of psychedelics.

Surprisingly, psilocybin administration has shown sustained antidepressant effects for at least 6 months in patients with TRD (Carhart-Harris et al. 2016). However, the effect of ketamine lasts for only 1–2 weeks at most (Rong et al. 2018; Wilkinson et al. 2018). Similarly, a previous preclinical study showed that the antidepressant-like effects of LSD and psilocybin persisted for more than five weeks. In contrast, the effect of ketamine lasts for only one week in naïve rats (Hibicke et al. 2020). The effects of psilocybin observed in rats are also supported by the present study (Table 2). Moreover, a multicriteria decision analysis of various abused drugs in the United Kingdom found that LSD and magic mushrooms (psilocybin) are less addictive and less dissociative than other drugs, such as ketamine, tobacco, and alcohol (Nutt et al. 2010). Given that the antidepressant effects of psilocybin (psilocin) last longer than those of ketamine (Rong et al. 2018; Wilkinson et al. 2018; Hibicke et al. 2020) and that psilocybin has fewer adverse effects including dependence and dissociation on users themselves than ketamine (Nutt et al. 2010), serotonergic psychedelics like psilocybin are considered superior to ketamine in terms of safety and efficacy.

Supplementary Material

The online version contains supplementary material available at https://doi.org/10.1007/s00210-023-02778-x.

Data availability

The datasets created during this investigation are available upon request from the corresponding author.