Psilocin suppresses methamphetamine‐induced hyperlocomotion and acquisition of conditioned place preference via D2R‐mediated ERK signaling

Abstract Aim Psilocin is an active metabolite form of psilocybin and exerts psychoactive effects. Recent studies suggest that psilocin may have regulatory effects on abuse drugs, but the mechanisms remain unclear. In this study, we want to explore the effects of psilocin on methamphetamine (METH)‐induced alterations of behavior in mice and its molecular mechanisms. Methods Acute METH administration model and conditioned place preference (CPP) model were used to investigate the effects of psilocin on METH‐induced alterations of behavior. Western blot was used to detect the expression of proteins. Results In the acute 2 mg/kg METH administration model, 1 mg/kg psilocin counteracted METH‐induced elevation of activity. In the 1 mg/kg METH‐induced CPP model, 1 mg/kg psilocin inhibited CPP formation during the acquisition phase. However, psilocin did not impact METH extinction and relapse. Molecular results showed that the regulatory effect of psilocin on METH was underscored by altered expression of dopamine 2 receptor (D2R) and phosphorylated extra‐cellular signal‐regulated kinase (p‐ERK) in the prefrontal cortex (PFC), nucleus accumbens (NAc), and ventral tegmental area (VTA). Trifluoperazine (TFP)‐2HCl is a D2R inhibitor, and SCH772984 is a selective extra‐cellular signal‐regulated kinase (ERK) inhibitor that effectively inhibits ERK1/2 phosphorylation. The results indicated that 2 mg/kg TFP‐2HCl and 10 mg/kg SCH772984 blocked METH‐induced hyperactivity and acquisition of METH‐induced CPP. Conclusion Psilocin has regulatory effects on METH‐induced alterations of behavior in mice via D2R‐mediated signal regulation of ERK phosphorylation.


| INTRODUC TI ON
Psilocybin is an indole alkylamine extracted from hallucinogenic mushrooms. Psilocin (4-hydroxy-dimethyltryptamine) is a dephosphorylation production of psilocybin that easily crosses the bloodbrain barrier and exerts psychoactive effects. 1 A growing body of research suggests that psilocin influences cognition and emotion in both animals and humans, without affecting psychomotor stimulation, alertness, attentiveness, memory, or orientation. 2 Moreover, clinical research suggests that psilocin may be safe and effective for treating neuropsychiatric disorders, including depressive disorder, cancer-related anxiety, 3 and alcohol 4 and tobacco addiction. 5 Psilocin is a postsynaptic 5-HTR agonist that predominantly targets the 5-HT2AR subtype. 6 In this regard, 5-HTR agonists induceallosteric inhibition of dopamine receptor 2 (D2R) signaling, and hallucinogenic 5-HT2AR agonists increase the Bmax values of D2R antagonist binding sites. 7 Moreover, ic3 (the third intracellular loop of D2R containing adjacent arginine residues) and C-tail (the acidic glutamate residues of 5-HT2AR) play a key role in the dimerization of 5-HT2AR and D2R. 8 Vollenweider et al. 9 examined the binding of [11C] raclopride to D2R in healthy volunteers after placebo and psilocybin treatment and reported an increase in D2R occupancy by endogenous dopamine.

Methamphetamine (METH) is a neurotoxic psychostimulant.
More than 35 million people use amphetamine-type substances worldwide. 10 A recent study has indicated that the D2R plays a key regulatory role in the molecular and behavioral responses to drug abuse. 11 Acute METH induces psychostimulant effects including euphoria, alertness, and increased locomotor activity. Moreover, acute METH-induced psychotropic effects may contribute to the augmentation of subsequent drug-seeking and drug-taking behavior. 12 These effects are induced by the stimulation of neurotransmitter release via actions on the dopamine transporter (DAT), resulting in increased dopamine (DA) release. Indeed, acute METH increases D2R expression associated with activation of the mesolimbic dopaminergic pathway. 13 However, positron emission tomography and magnetic resonance imaging data in human indicate that persist beyond the period of METH consumption reduced the density of D2 receptor. The mesolimbic dopaminergic reward-pathway is the main neural pathway that regulates the reinforcing effects of addictive drugs and comprises DA neurons in the ventral tegmental area (VTA) that project to the nucleus accumbens (NAc) 14 and prefrontal cortex (PFC). 15 Research suggests that altered striatal DA-dependent behaviors are caused by elevated D2R expression and upregulated extracellular signal-regulated kinase (ERK) 1/2 activation, 16 and D2R over-activation leads to a decrease protein kinase B (AKT) phosphorylation in cortical neurons, eventually leading to neurite lesions. 17 Recent studies have suggested that ERK signaling may constitute a common pathway to different drugs of abuse, leading to the addictive state. 18 Chronic administration of METH in rats causes behavioral and molecular changes, including neurodegenerative effects that may be partially mediated by inhibition of phosphorylated protein kinase B (p-AKT). 19 In addition, D2R activation is modulated by ERK 20 and AKT phosphorylation levels. 21 Based on recent studies, we hypothesized that psilocin might have an effect on METH-induced alterations of behavior via the D2R-mediated signal regulation of ERK phosphorylation or AKT phosphorylation. Therefore, this study investigated the levels of D2R, p-AKT, AKT, phosphorylated ERK (p-ERK), and ERK expression in the PFC, NAc, and VTA in order to explore the molecular mechanisms of the effect of psilocin on METH-induced alterations of behavior in mice.

| Acute METH administration model
In the first part of the experiment, 24 mice were divided into four groups including control, 5, 1, and 0.25 mg/kg psilocin. In the second part of the experiment, 25 mice were divided into four groups including control, METH only, and, 1 or 0.25 mg/kg psilocin administered prior to i.p injection of METH. In the third part of the experiment, 36 mice were divided into six groups including control, 2 mg/ kg TFP-2HCl, 10 mg/kg SCH772984, METH, 2 mg/kg TFP-2HCl and 10 mg/kg SCH772984 administered prior to i.p. injection of METH.
In all three parts of the experiment, mouse activity was monitored in an open-field for 1 h. At the end of the experimentation, mice were sacrificed and the PFC, NAc and VTA were collected. Samples were stored at −80°C until subsequent use.

| CPP model
Four CPP apparatuses each consisting of two equal compartments (15 cm × 15 cm × 37 cm) were used in this study. One was a white compartment with a metal grid floor and the other was a black compartment with a metal mesh floor. A movable baffle was in the center of apparatus. During the test period, the mice was allowed to shuttle freely in the apparatus by opening the baffle, and during training period, the mice was restricted to one side of the apparatus by closing the baffle. In this study, pre-test results showed that mice spent more time in the black compartment than in the white compartment. Therefore, the black compartment was the "preferred-one" and the white compartment was the "non-preferred-one".
On day1, mice were pre-tested for 15 min. Mice with abnormal activity (low motor activity, <20 shuttles, or more than 600 s spent on either side) were excluded.
In the acquisition phase (days 2-9), on days 2, 4, 6, and 8, all groups were injected with saline and placed in the black compartment for 45 min. On days 3, 5, 7, and 9, the control group was injected with saline; the drug group was injected with 1 mg/kg psilocin, 2 mg/kg TFP-2HCl, or 10 mg/kg SCH772984; the METH group was injected with METH; and the METH with drug intervention group was injected with 1 mg/kg psilocin, 2 mg/kg TFP-2HCl, or 10 mg/kg SCH772984 30 min before METH administration. All groups were placed in the white compartment for 45 min. At post-test (day 10), all mice were allowed to explore the two compartments freely for 15 min.
In the extinction phase (days [11][12][13][14][15][16][17][18][19][20][21][22], four groups including control, PI, METH-Ext1, and METH-Ext2 were subjected to extinction training and testing. The METH group with CPP was divided into METH-Ext1 and METH-Ext2 groups. On day 11, four groups were injected with saline and placed in the black compartment for 45 min. On day 12, the control and METH-Ext1 groups were injected with saline; PI and METH-Ext2 groups were injected with psilocin, and all groups were placed in the white compartment for 45 min. On day 13, all mice were allowed to explore the two compartments freely for 15 min. This procedure was repeated three times over days 14-22. In the reinstatement phase, four groups including Control, PI, METH-Rein1 and METH-Rein2 were tested. The METH-Ext1 group was divided into METH-Rein1 and METH-Rein2 groups. The METH-Rein1 group was injected (i.p) with METH, and the METH-Rein2 group was injected (i.p) with 1 mg/kg psilocin prior to METH injections. The mice were then allowed to explore the two compartments freely for 15 min. At the end of the experimentation, the mice were sacrificed, and the PFC, NAc, and VTA were collected. Samples were stored at −80°C until subsequent use.

| Protein extraction
Samples were homogenized in RIPA buffer with protease inhibitor (PMSF). Proteins were extracted with a high-throughput tissue grinder (SCIENTZ-48). Subsequently, proteins were centrifuged at 12,000 g for 15 min at 4°C. Protein concentration was measured using a BCA assay. Loading buffer was added to the samples, and samples were boiled at 100°C for 5 min.

| Western blotting
The boiled protein samples were separated by 12% SDS-PAGE (15 μg per lane). The proteins were transferred to a polyvinylidene fluoride (PVDF) membrane, and 5% skim milk was used to block the membrane at room temperature for 4 h. Next, the PVDF membrane was incu-

| Statistical analysis
GraphPad Prism (version 8.0) was used for all statistical analyses.
All data are presented as means ± SEM. Bartlett's test for homogeneity of variance was used to ensure a normal distribution of data and all data were normally distributed. Acquisition and extinction of METH-induced CPP data were analyzed using two-way ANOVA.
The effects of different concentrations of psilocin, acute METH administration model, reinstatement of METH-induced CPP, and western blot data were analyzed using one-way ANOVA. All post hoc pairwise comparisons were performed using the Bonferroni test.

| Effects of different psilocin concentrations
Three different concentrations of psilocin 5, 1, and 0.25 mg/kg were injected into mice. Significant differences were observed between the control group and three concentrations of psilocin groups (F (3,21) = 5.300, p = 0.007), whereby the 5 mg/kg psilocin group exhibited increased activity compared to the control group (p < 0.05).
However, the activity of 1 and 0.25 mg/kg psilocin groups was not significantly different from that of the control group ( Figure 1).

| Regulatory effects of psilocin on acute METH administration model
We selected doses of 1 and 0.25 mg/kg psilocin for injection in the acute METH administration model. We observed significant differences between groups in the acute METH administration model (F (3,21) = 15.32, p < 0.001) (Figure 2), whereby the activity of the METH group was significantly increased compared to that of the control group (p < 0.001). In addition, 1 mg/kg psilocin significantly inhibited METH-induced hyperactivity in mice, which was statistically significant compared to the METH group (p < 0.01). However, 0.25 mg/kg psilocin did not significantly affect METH-induced hyperactivity.

| Regulatory effects of psilocin on the acquisition of METH-induced CPP
A dose of 1 mg/kg psilocin was selected for injection in the METHinduced CPP model. The procedure of the CPP acquisition phase is presented in Figure 3A. We observed a significant interaction on the acquisition phase. Post-test indicated that the CPP scores of the METH group were significantly increased compared to those of the control group (p < 0.001). Further, injection of the 1 mg/kg psilocin before METH significantly inhibited the increase in CPP scores of the METH group (p < 0.001). CPP scores were higher in the group that received 1 mg/kg psilocin prior to the METH group injection than in the control group (p < 0.05). No significant difference in CPP scores was observed between the 1 mg/kg psilocin group and the control group ( Figure 3B).

| Regulatory effects of psilocin on the extinction of METH-induced CPP
The procedure of the CPP extinction phase is presented in Figure 4A.
In the extinction phase, the METH group was divided into two groups: the METH-Ext1 group was injected with saline, and METH-Ext2 group was injected with 1 mg/kg psilocin. The psilocin and control groups underwent continuous training. We observed a significant interaction (F (9,75) = 5.177, p < 0.001) and significant main effects of time

| Regulatory effects of psilocin on the reinstatement of METH-induced CPP
The procedure of the CPP reinstatement phase is presented in Figure 5A. In the reinstatement phase, the METH-Ext1 group was divided into METH-Rein1 and METH-Rein2 groups. Mice in the METH-Rein1 group were injected with METH, while mice in the METH-Rein2 group were injected with 1 mg/kg psilocin 30 min before METH injection. We observed a significant difference in the CPP reinstatement phase (F (3,19)    intervention groups). We observed significant differences in intervention effects in the acute METH administration model (F (5,30) = 90.06, p < 0.001) (Figure 8), whereby the activity of the METH group was significantly increased compared to that of the control group (p < 0.001). In addition, 2 mg/kg TFP-2HCl and 10 mg/ kg SCH772984 significantly inhibited METH-induced hyperactivity in mice compared to that in the METH group (both p < 0.001). No significant differences were observed between the 2 mg/kg TFP-2HCl, 10 mg/kg SCH772984, and control groups.

| Regulatory effects of TFP-2HCl and SCH772984 on the acquisition of METH-induced CPP
With regard to the acquisition of METH-induced CPP, administration of 1 mg/kg psilocin before METH more strongly regulated p-ERK expression levels than p-AKT expression levels in the PFC, NAc, and VTA of the METH group. To further investigate the molecular mechanisms of action of psilocin, 2 mg/kg TFP-2HCl and 10 mg/kg SCH772984 were injected into mice in the acquisition of the METH-induced CPP model. The procedure of the CPP acquisition phase is presented in Figure 9A. We observed a significant interaction (F (5,41)  In previous studies, a higher dose (4 mg/kg) of psilocin affected locomotor, 22 however, a lower psilocin dose (1 mg/kg) did not significantly affect locomotor behavior relative to that of control. Indeed, the physical and psychological dependence potential of magic mushrooms has been reported to be low. 23 METH induces behaviors such as hyperlocomotion, CPP, and self-administration. 24 Neuroimaging studies have demonstrated that METH causes cortical and striatal abnormalities that induce addictionrelated phenotypes, thereby promoting compulsive drug use. 25 In this study, 1 mg/kg psilocin counteracted METH-induced elevation of activity. Moreover, in the acquisition phase, 1 mg/kg psilocin inhibited CPP formation. This study highlights the potential of psilocin for the treatment of acute and chronic METH-induced alterations of behavior. Psilocin is a postsynaptic 5-HTR2A agonist. 6 Previous research has demonstrated that direct infusion of 5-HT inhibited DA activity and reduced DA-mediated functions including spontaneous locomotor activity, amphetamine-induced locomotor activity and stereotypies, turning behavior, and extra-pyramidal functions. 26 Psilocin and psilocybin have recently received growing attention in addiction research. 27 For example, a recent study reported that psilocin significantly reduced the percentage of drinking and heavy drinking days in alcohol-dependent patients. 28 Crucially, after treatment with psilocybin, 80% of tobaccodependent patients had quit smoking after 6 months. 5 F I G U R E 4 Effects of 1 mg/kg psilocin on the extinction of METH-induced CPP. (A) Experimental protocol. (B) Psilocin on the extinction of CPP. Data are presented as mean ± SEM (n = 5-10). *p < 0.05, **p < 0.01, ***p < 0.001 vs the control group Recent studies have reported the existence of D2R-5-HT2AR heteromers in cellular models whereby the ic3 region plays a key role in the dimerization of 5-HT2AR and D2R. 8 Moreover, there is an opposing action of 5-HT on DA in the nigrostriatal system. 29 For instance, decreasing the density of 5-HT transporters results in a disinhibition of the mesolimbic DA system and inhibitory effects of 5-HT2 mediated by the local dendritic release of DA which promotes D2R activation. 30 In this study, we examined D2R expression given that previous have reported a decrease in AKT signaling in the VTA during chronic morphine. 37 In addition, both in METH acute administration model and in the acquisition phase of CPP 1 mg/kg psilocin injected before METH could reverse regulate the level of p-ERK expression more significant than p-AKT in the PFC, NAc and VTA of the METH group.
In addition, in both the METH acute administration model and acquisition phase of CPP, administration of 1 mg/kg psilocin before METH more strongly regulated the expression level of p-ERK than that of p-AKT in the PFC, NAc, and VTA in the METH group. Based on its pharmacological characteristics, the p-ERK assay provides a robust readout of D2R activation and has a medium throughput, which may effectively screen many compounds. 20 Moreover, Akt dephosphorylation is a late biochemical reaction stimulated by DA receptors, 21 and prolonged stimulation of D2-class receptors leads to specific dephosphorylation of AKT on its regulatory Thr308. 38 In the present study, we observed that the inhibitory effects of psilocin on METH-induced expression of D2R and p-ERK were potentially associated with the blocking effect of psilocin on METHinduced hyperactivity and acquisition of METH-induced CPP. To determine the role of D2R and p-ERK in METH-induced hyperactivity and acquisition of METH-induced CPP, we examined behavioral changes in mice after administering trifluoperazine 2HCl (TFP-2HCl) and SCH772984 to inhibit D2R and p-ERK, respectively. TFP-2HCl is a phenothiazine derivative that exerts specific actions in the brain by inhibiting postsynaptic D2Rs. Moreover, trifluoperazine blocks postsynaptic D2Rs in midbrain limbic and cortical projections. 39 Reports have demonstrated that 2-3 mg/kg TFP-2HCl affects the central nervous system in mice. [40][41][42] SCH772984 is a selective ERK1/2 inhibitor that binds to unphosphorylated and inactive ERK1/2 and effectively inhibits ERK1/2 phosphorylation. 43 Previous research has reported that the effective dose range of SCH772984 in mouse models is 10-20 mg/kg. [44][45][46] In conclusion, 1 mg/kg psilocin counteracted METH-induced hy-  (B) TFP-2HCl and SCH772984 inhibited CPP acquisition. Data are presented as mean ± SEM (n = 7-10). ***p < 0.001 vs the control group; # p < 0.05, ## p < 0.01 vs the METH group

CO N FLI C T O F I NTE R E S T
We have no conflicts of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data included in this study are available from the corresponding author upon reasonable request.