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Cannabidiol prevents methamphetamine-induced neurotoxicity by modulating dopamine receptor D1-mediated calcium-dependent phosphorylation of methyl-CpG-binding protein 2

In the past decade, methamphetamine (METH) abuse has sharply increased in the United States, East Asia, and Southeast Asia. METH abuse not only leads to serious drug dependence, but also produces irreversible neurotoxicity. Currently, there are no approved pharmacotherapies for the treatment of METH use disorders. Cannabidiol (CBD), a major non-psychoactive (and non-addictive) cannabinoid from the cannabis plant, shows neuroprotective, antioxidative, and anti-inflammatory properties under METH exposure. At present, however, the mechanisms underlying these properties remain unclear, which continues to hinder research on its therapeutic potential. In the current study, computational simulations showed that CBD and METH may directly bind to the dopamine receptor D1 (DRD1) via two overlapping binding sites. Moreover, CBD may compete with METH for the PHE-313 binding site. We also found that METH robustly induced apoptosis with activation of the caspase-8/caspase-3 cascade in-vitro and in-vivo, while CBD pretreatment prevented these changes. Furthermore, METH increased the expression of DRD1, phosphorylation of Methyl-CpG-binding protein 2 (MeCP2) at serine 421 (Ser421), and level of intracellular Ca2+ in-vitro and in-vivo, but these effects were blocked by CBD pretreatment. The DRD1 antagonist SCH23390 significantly prevented METH-induced apoptosis, MeCP2 phosphorylation, and Ca2+ overload in-vitro. In contrast, the DRD1 agonist SKF81297 markedly increased apoptosis, MeCP2 phosphorylation, and Ca2+ overload, which were blocked by CBD pretreatment in-vitro. These results indicate that CBD prevents METH-induced neurotoxicity by modulating DRD1-mediated phosphorylation of MeCP2 and Ca2+ signaling. This study suggests that CBD pretreatment may resist the effects of METH on DRD1 by competitive binding.

In the past decade, methamphetamine (METH) abuse has sharply increased in the United States, East Asia, and Southeast Asia. METH abuse not only leads to serious drug dependence, but also produces irreversible neurotoxicity. Currently, there are no approved pharmacotherapies for the treatment of METH use disorders. Cannabidiol (CBD), a major non-psychoactive (and non-addictive) cannabinoid from the cannabis plant, shows neuroprotective, antioxidative, and anti-inflammatory properties under METH exposure. At present, however, the mechanisms underlying these properties remain unclear, which continues to hinder research on its therapeutic potential. In the current study, computational simulations showed that CBD and METH may directly bind to the dopamine receptor D1 (DRD1) via two overlapping binding sites. Moreover, CBD may compete with METH for the PHE-313 binding site. We also found that METH robustly induced apoptosis with activation of the caspase-8/caspase-3 cascade in-vitro and in-vivo, while CBD pretreatment prevented these changes. Furthermore, METH increased the expression of DRD1, phosphorylation of Methyl-CpG-binding protein 2 (MeCP2) at serine 421 (Ser421), and level of intracellular Ca 2+ in-vitro and in-vivo, but these effects were blocked by CBD pretreatment. The DRD1 antagonist SCH23390 significantly prevented METH-induced apoptosis, MeCP2 phosphorylation, and Ca 2+ overload in-vitro. In contrast, the DRD1 agonist SKF81297 markedly increased apoptosis, MeCP2 phosphorylation, and Ca 2+ overload, which were blocked by CBD pretreatment in-vitro. These results indicate that CBD prevents METHinduced neurotoxicity by modulating DRD1-mediated phosphorylation of MeCP2 and Ca 2+ signaling. This study suggests that CBD pretreatment may resist the effects of METH on DRD1 by competitive binding.
Cannabidiol (CBD) is a primary non-psychoactive cannabinoid in the cannabis plant and exhibits considerable therapeutic potential in the treatment of neuropsychiatric disorders, including epilepsy, Parkinson's disease (PD), Alzheimer's disease (AD), depression, anxiety, psychosis, and drug dependence (Elsaid et al., 2019;Premoli et al., 2019;Vitale et al., 2021). Recent research has indicated that CBD exerts neuroprotective effects on METH-induced neurotoxicity in rats (Razavi et al., 2021). Furthermore, some evidence has suggested that CBD modulates several receptors in METH exposure, such as D1-like DA receptors (Nouri et al., 2021;Shen et al., 2022), D2-like DA receptors (Hassanlou et al., 2021), Sigma-1 receptors , and Toll-like type-4 receptors (TLR4) (Majdi et al., 2019). We previously showed that CBD may modulate DRD1 to attenuate METHinduced DA release (Shen et al., 2022). However, the underlying mechanism related to the neuroprotective effects of CBD on METH-induced neurotoxicity remains elusive. In the current study, we hypothesized that METH induces neurotoxicity via DRD1-mediated phosphorylation of MeCP2 at Ser421 with calcium influx, and CBD treatment prevents METH-induced neurotoxicity via modulation of DRD1.

Molecular docking
Molecular docking was performed using AutoDock v4.2.6 in accordance with previous research (Bian et al., 2019;Zhang et al., 2021). Structural information on rat DRD1 was obtained from the AlphaFold Protein Structure Database (https://alphafold.ebi. ac.uk/), and structural information on METH and CBD was obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/). First, preparation of DRD1 was performed using AutoDockTools v1.5.6, including the addition of hydrogens and calculation of atomic charges with the Kollman all-atom approach (Bian et al., 2019). The atomic charges of CBD and METH were calculated using the Gasteiger-Hückel approach with AutoDockTools v1.5. 6 (Bian et al., 2019). A three-dimensional (3D) search grid with 40 × 44 × 40 points was created using the AutoGrid algorithm, and the maximum number of poses per ligand was set to 200.
Next, all postures for CBD and METH were docked using AutoDock v4.2.6. The docking parameters were set to default, as per previous research (Zhang et al., 2021). The docking scores of all postures were extracted using AutoDockTools v1.5.6. The affinity score was greater than −5 kcal/mol, indicating a reliable docking process. The best docking posture was then selected. Finally, binding site analysis was performed using Discovery Studio Visualizer v4.5 and visualized using PyMol v2.5.2.

Cell counting kit-8 assay
Neurons were seeded at a density of 5 × 10 3 cells/well in a 96well plate. After the neurons were incubated with drugs following the above procedures, cell viability was detected using Cell counting kit-8 (CCK-8) reagent (Dalian Meilun Biotechnology Co. Ltd., #MA0218, Dalian, China). Absorbance was obtained on a microplate reader at 450 nm. Cell viability was calculated according to the formula provided by the kit.

Intracellular Ca 2+ detection
Intracellular Ca 2+ was detected as per previous study (Sun et al., 2015). For the in-vitro experiments, drug-treated neurons were rinsed three times with Hanks' balanced salt solution (HBSS, Sigma-Aldrich, #H6648, St. Louis, MO, United States) and incubated with HBSS containing 10 μM Fluo-3-AM solution (Sigma-Aldrich, #39294, St. Louis, MO, United States) for 60 min at 37°C. For the in-vivo experiments for Ca 2+ detection, tissue sections were first penetrated with HBSS containing 0.2% Triton X-100 and 0.1% sodium citrate (Solarbio Life Sciences, #C1032, Beijing, China) for 30 min, then incubated with HBSS containing 10 μM Fluo-3-AM solution for 60 min at 37°C. After incubation with Fluo-3-AM solution, the neurons and tissue sections were rinsed three times with HBSS. Observations were performed with a fluorescent microscope at a detection wavelength of 488 nm. Obtained images were analyzed with ImageJ.
Terminal deoxynucleotidyl transferase dUTP nick-end labeling staining Apoptosis was detected using a Terminal Deoxynucleotidyl Transferase dUTP Nick-end Labeling (TUNEL) assay kit (Sigma-Aldrich, #11684817 910 or #12156792910, St. Louis, MO, United States) according to the manufacturer's protocols. For the in-vitro experiments, neurons were fixed in 4% paraformaldehyde for 30 min and rinsed with PBS. For the invivo experiments, tissue sections were rinsed three times with PBS. After incubation with 3% H 2 O 2 for 10 min at room temperature, the neurons and tissue sections were rinsed with PBS, then penetrated with PBS containing 0.2% Triton X-100 and 0.1% sodium citrate for 2 min on ice. For TUNEL staining, the samples were incubated with TUNEL reaction mixture for 60 min at 37°C. After the samples were rinsed three times with PBS, DAPI was used to localize nuclei. A fluorescent microscope was used to obtain images with the detection wavelength at 488 nm or 580 nm. DAPI-and TUNEL-positive cells were counted using ImageJ (n = 5). TUNEL-positive cell ratio = TUNEL-positive cells/DAPI-positive cells.

Animals
Adult male Sprague-Dawley rats (8-9 weeks old, n = 40) were purchased from the Laboratory Animal Center, Kunming Medical University. The rats were housed in a humidity-(50% ± 10%) and temperature-controlled (22°C ± 1°C) room on a 12-h reverse light/ dark cycle. Food and water were available ad libitum. All animal procedures were conducted according to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of Kunming Medical University (permit number: kmmu2020403). The rats were randomly distributed into four groups: i.e., Saline (n = 10), CBD 40 mg/kg (n = 10), METH 15 mg/kg (n = 10), and CBD 40 mg/kg + METH 15 mg/kg (n = 10) groups.

Animal treatments
After 1 week of habituation, the rats received intraperitoneal injections of METH (eight injections, Frontiers in Pharmacology frontiersin.org 04 15 mg/kg/injection, 12-h intervals). Injections were performed at 08:00 and 20:00 for 4 days. This exposure paradigm was selected based on previous studies (Qiao et al., 2014;Huang et al., 2015;Xie et al., 2018;Xu et al., 2018;Huang et al., 2019;Chen et al., 2020). CBD was injected intraperitoneally at a dose of 40 mg/kg 1 h before METH Frontiers in Pharmacology frontiersin.org 05 administration, following our previous studies Shen et al., 2022). During treatment, body weights were recorded every day. In addition, METH-stereotyped behavior was observed as described previously (Sams-Dodd, 1998;Roberts et al., 2010;Qiao et al., 2014). Briefly, an observer blind to the experiment visually assessed several classified behaviors, including: (0) No repetitive head movements; 1) Weak repetitive side-to-side head movements; 2) Strong repetitive side-to-side head movements; and 3) Stationary stereotyped behavior with strong side-to-side or circular head movements. Rats were anesthetized 24 h after the last injection, and perfused transcardially with saline. After perfusion with saline, the brains of rats were removed, and the prefrontal cortex (PFC) and hippocampus (Hip) were dissected bilaterally on ice and stored at −80°C for detection of proteins. For tissue sections, the rats were perfused transcardially with 4% paraformaldehyde and brains were preserved in 4% paraformaldehyde at 4°C. After 2 weeks of fixation, the brains were sunk in increasing concentrations of sucrose (10%, 20%, and 30%). Brains were sectioned (15 μm) along the coronal plane incorporating the PFC and Hip on a freezing microtome (Leica Biosystems, #CM 1860, Nussloch, Germany) kept at −20°C.

Statistical analyses
All data were analyzed using SPSS v26.0 and presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to analyze data from parts A and B of the CCK-8 assay, with factors defined as METH treatments (corresponding to "METH dose" and "treatment time" levels). Three-way repeated-measures ANOVA was used to analyze data from the behavioral experiment, with factors defined as CBD treatment, METH treatment, and Time. Two-way ANOVA (no repeated measures) was used to analyze data from other in-vitro and in-vivo experiments. For the first part of the in-vitro experiments and other in-vivo experiments, factors were defined as CBD and METH treatments. For the second part of the in-vitro experiments, factors were defined as SCH23390 and METH treatments. For the third part of the in-vitro experiments, factors were defined as CBD and SKF81297 treatments. ANOVA was followed by Tukey HSD post-hoc tests. Before ANOVA, normality and homogeneity of equal variance were confirmed. The level of significance was set to 0.05.

Methamphetamine and cannabidiol bind with DA receptors D1
We first screened the METH-binding pocket in the DRD1 model from 7 reliable binding pockets. We chose the METH-binding pocket with the highest affinity (−5.8 kcal/mol). Specially, 12 amino acid residues of DRD1 formed the boundary of the pocket, including PHE-289, SER-198, ILE-103, ASN-292, PHE-288, PHE-313, ASP-102, TRP-321, THR-188, TRP-98, VAL-99, and LEU-190 ( Figure 1A). Among these amino acids, one hydrogen bond was observed between residue ASP-102 from transmembrane (TM) 3 and METH at 3.1 Å. In addition, residue PHE-288 from TM6 and METH formed a Pi-Pi T-shaped non-covalent interaction at 4.9 Å, while residue ILE-103 from TM3 and METH formed a Pi-Alkyl non-covalent interaction at 3.7 Å ( Figure 1A). We also performed docking simulation with the DRD1 model and CBD. In total, 15 reliable CBD-binding pockets were found, with the highest affinity of −7.3 kcal/mol. The best CBD-binding pocket in the DRD1 model consisted of 14 amino acid residues, including ALA-83, ASN-185, ARG-189, PHE-313, VAL-295,  Figure 1B). A hydrogen bond was observed between residue ASP-314 from TM7 and CBD at 1.9 Å, while residue ASP-187 from TM5 and CBD formed a Pi-Anion non-covalent interaction at 5.8 Å. In addition, several Alkyl-Alkyl interactions were found between CBD and TRP-89, CYS-186, ALA-83, ARG-189, and PHE-313. To determine whether CBD competes with METH for binding sites, we docked METH and CBD with DRD1 based on the above pockets simultaneously. Results showed that CBD was bound to DRD1 near the METHbinding pocket ( Figure 1C). Interestingly, for CBD and DRD1 interactions, the hydrogen bond between residue ASP-314 and CBD ( Figure 1B) was converted to van der Waals force ( Figure 1C), while the Alkyl-Alkyl interaction between residue PHE-313 ( Figure 1B) was transformed into a Pi-Sigma covalent bond ( Figure 1C). However, no differences were observed in the interactions between METH and DRD1 compared with METH docking alone ( Figure 1C). Overlaps between CBD-binding sites and METH-binding sites were found, e.g., PHE-313 and THR-188 ( Figure 1C). The bond strength between CBD and DRD1 was stronger than that of METH in PHE-313, but not that of METH in THR-188 ( Figure 1C). This is likely because the covalent Pi-Sigma bond is stronger than van der Waals force for drug loading (Li and Yang, 2015). Frontiers in Pharmacology frontiersin.org 07

Cannabidiol prevented methamphetamine-induced decrease in cell viability
Cultured neurons were treated with various concentrations of METH (50, 100, 200, 400, 800, 1,000 μM) for 24 h, with cell viability then detected using the CCK-8 assay. Cell viability decreased gradually with the increase in METH concentration [ Figure 2A; F (6, 14) = 30.603, p < 0.05, p < 0.01, p < 0.001]. Based on morphological changes in neurons and other previous studies (Sun et al., 2015;Chen et al., 2016), the 400 μM concentration of METH was chosen for subsequent experiments. The neurons were exposed to 400 μM METH for different times (3, 6, 12, 24, 48 h). As indicated in Figure 2B, cell viability showed a significant and timedependent decrease [ Figure 2B; F (5, 12) = 36.953, p < 0.01, p < 0.001]. These results suggest that METH induces neuronal death in a dose-dependent and time-dependent manner. As cellular damage was easy to detect, we also selected the 400 μM concentration of METH to incubate neurons for 24 h. Before METH exposure, the neurons were incubated with CBD (0.1, 1, and 10 μM) or vehicle (0.1% DMSO) for 1 h. Results showed that cell viability diminished significantly after 400 μM METH exposure [ Figure 2C; F (6, 14) = Cannabidiol blocked expression of DA receptors D1, phosphorylation of MeCP2, and apoptosis-related proteins induced by methamphetamine exposure in-vitro We next explored the mechanisms underlying the neuroprotective effects of CBD. To corroborate the cell viability findings, we detected the expression levels of apoptosis-related proteins (caspase-8, cleaved caspase-8, caspase-3, and cleaved caspase-3) in primary neurons. We found that METH significantly induced the expression levels of cleaved caspase-8 [ Figure

Cannabidiol prevented Ca 2+ overload and apoptosis induced by methamphetamine in neurons
As the phosphorylation of MeCP2 at Ser421 is calciumdependent (Chen et al., 2003;Buchthal et al., 2012), we further investigated the level of intracellular Ca 2+ in neurons. Results showed

Inhibition of DA receptors D1 activity prevented phosphorylation of MeCP2 and apoptosis-related protein expression induced by methamphetamine in-vitro
To clarify whether DRD1 signaling mediates apoptosis induced by METH, we applied the DRD1 antagonist SCH23390 to inhibit DRD1 activity in the neurons. Results showed that SCH23390 not only prevented METH-induced expression of cleaved caspase-8 [ Figure

Cannabidiol prevented growth inhibition and stereotyped behavior induced by methamphetamine in rats
To corroborate the findings that CBD resists neurotoxicity induced by METH in-vitro, we constructed a rat model using METH. We first investigated the effects of drug exposure on body weight in rats. Compared with the control group, repeated METH exposure inhibited normal weight gain in rats [ Figure 9A; F (1, 16) = 25 383.728, p < 0.001], while multiple CBD pretreatments prevented the inhibition induced by METH [ Figure 9A; F (1, 16) = 25 383.728, p < 0.05], although there was no significant effect when treated with CBD alone [ Figure 9A; F (1, 16) = 25 383.728, p =

FIGURE 10
Protein, intracellular Ca 2+ , and apoptosis levels in prefrontal cortex of rats repeatedly exposed to CBD and/or METH. (A) Levels of DRD1, pMeCP2, and apoptosis-related proteins in prefrontal cortex. (B) Intracellular Ca 2+ level in prefrontal cortex. (C) Apoptosis level in prefrontal cortex. ***p < 0.001 compared with control group, ### p < 0.001 compared with METH group; Tukey HSD post-hoc comparison after significant ANOVA.
Cannabidiol prevented DA receptors D1 expression, phosphorylation of MeCP2, Ca 2+ overload, and apoptosis induced by methamphetamine in prefrontal cortex of rats Repeated METH exposure affects cell proliferation and induces apoptosis in the PFC (Kim and Mandyam, 2014).
Thus, to further explore the neuroprotective effects of CBD, we investigated protein expression, intracellular Ca 2+ , and apoptosis levels in the PFC of rats. Results showed that repeated METH exposure markedly increased the expression levels of DRD1 [ Figure Figure 10C; F (3, 16) = 173.738, p < 0.001] induced by METH, but had no effect when administered alone. Thus, CBD shows neuroprotective effects in the PFC of rats.

FIGURE 11
Protein, intracellular Ca 2+ , and apoptosis levels in hippocampus of rats repeatedly exposed to CBD and/or METH. (A) Levels of DRD1, pMeCP2, and apoptosis-related proteins in hippocampus. (B) Intracellular Ca 2+ level in hippocampus. (C) Apoptosis level in hippocampus. ***p < 0.001 compared with control group, ### p < 0.001 compared with METH group; Tukey HSD post-hoc comparison after significant ANOVA.
Frontiers in Pharmacology frontiersin.org Cannabidiol blocked DA receptors D1 expression, phosphorylation of MeCP2, Ca 2+ overload, and apoptosis induced by methamphetamine in hip of rats As CBD promotes neurogenesis in the Hip of rats under chronic METH exposure (Razavi et al., 2021) and modulates the psychoactive properties of METH via DA receptors in the Hip of rats (Hassanlou et al., 2021;Nouri et al., 2021), we repeated the above experiments in the Hip. Results showed that METH exposure induced DRD1 [ Figure 11A;

Discussion
In the molecular docking simulations, we found that METH and CBD bound to residues of DRD1 with two overlapping binding sites. Moreover, CBD and METH competitively interacted for one of the sites. In the biological experiments, we found that METH significantly induced neurotoxicity with DRD1 activation, high MeCP2 phosphorylation at Ser421, and Ca 2+ overload in-vitro and in-vivo. However, CBD pretreatment effectively prevented METH-induced neurotoxicity by inhibiting DRD1, pMeCP2, and Ca 2+ influx in-vitro and in-vivo. In addition, the DRD1 antagonist SCH23390 significantly prevented METH-induced neurotoxicity, as well as pMeCP2 expression and Ca 2+ overload in-vitro. In contrast, the DRD1 agonist SKF81297 strongly induced neurotoxicity, MeCP2 phosphorylation, and Ca 2+ overload in-vitro, which were also blocked by CBD pretreatment.
METH is an amphetamine-type stimulant that primarily induces extracellular and cytoplasmic DA release by acting on DA transporters and vesicle monoamine transporter-2 (VMAT-2) (Kim et al., 2020). Abundant DA in the extracellular matrix and cytoplasm can produce DA quinones, superoxide anions, and hydrogen oxygen species via auto-oxidation, resulting in considerable oxidative damage (LaVoie and Hastings, 1999;Marshall and O'Dell, 2012). Thus, METH-induced DA release is closely related to the process of neurotoxicity (Kim et al., 2020). METH also induces the FasL-Fas death pathway and caspase-3 cleavage in rat neurons, which are mediated by DRD1 (Jayanthi et al., 2005). Here, we found that METH may bind directly with DRD1. Notably, METH exposure significantly induced apoptosis with activation of the caspase-8/caspase-3 cascade in-vitro and in-vivo, while the DRD1 antagonist SCH23390 markedly prevented the METH-induced caspase-8/caspase-3 cascade and neuronal apoptosis in-vitro. In contrast, the DRD1 agonist SKF81297 induced apoptosis via activation of the caspase-8/caspase-3 cascade in-vitro. These results indicate that DRD1 signaling plays a key role in METH-induced neurotoxicity, as reported in various studies (Ares-Santos et al., 2012;Ares-Santos et al., 2013;Friend and Keefe, 2013;Nguyen et al., 2018). However, some evidence suggests that other molecular pathways may also contribute to cell death under METH-induced neurotoxicity, including oxidative stress, mitochondrial stress, ER stress, UPS dysfunction, transcription factor changes, and autophagy (Jayanthi et al., 2021). Here, the DRD1 antagonist SCH23390 did not completely block the effects of METH on Ca 2+ influx, MeCP2 phosphorylation, caspase cascade, or neuronal apoptosis. Interestingly, DA induces the expression of DRD1 at low concentration (Sidhu et al., 1999) and functional activity of DRD1 also determines DA release (Shen et al., 2022). This positive feedback mechanism may partially explain why the DRD1 antagonist SCH23390 and DRD1 agonist SKF81297 affected DRD1 expression in our study.
On the other hand, growing evidence indicates that MeCP2 contributes to METH-induced behavioral disorders in rodents (Lewis et al., 2016;Wu et al., 2016;Fan et al., 2020). We observed serious stereotyped behavior in rats under repeated METH exposure, consistent with previous research (Roberts et al., 2010;Qiao et al., 2014). Notably, METH strongly induced stereotyped behavior in rats, with high phosphorylation of MeCP2 at Ser421 in the PFC and Hip. The DRD1 antagonist SCH23390 significantly blocked METH-induced phosphorylation of MeCP2 at Ser421 in the primary neurons of rats. In contrast, the DRD1 agonist SKF81297 strongly induced MeCP2 phosphorylation at Ser421 in-vitro. These results suggest that the phosphorylation of MeCP2 at Ser421 is mediated by DRD1, consistent with previous research (Deng et al., 2010). As the phosphorylation of MeCP2 at Ser421 is calcium-dependent (Chen et al., 2003;Buchthal et al., 2012), we further investigated the level of intracellular Ca 2+ , and found that METH exposure strongly increased Ca 2+ influx in-vitro and in-vivo. However, DRD1 inhibition significantly reduced the high level of intracellular Ca 2+ caused by METH, while DRD1 activation markedly increased intracellular Ca 2+ in the neurons. These results imply that DRD1-mediated phosphorylation of MeCP2 at Ser421 involves calcium signaling. Several studies suggest that changes in IEGs may also contribute to METHinduced neurotoxicity (Deng et al., 1999;Jayanthi et al., 2005; Frontiers in Pharmacology frontiersin.org Jayanthi et al., 2021). Interestingly, research has shown that phosphorylation of MeCP2 at Ser421 mediates the transcriptional activation of IEGs in psychostimulant-injected mice (Deng et al., 2010). Thus, these findings reveal the potential mechanism underlying METH-induced neurotoxicity involving DRD1-mediated calcium-dependent phosphorylation of MeCP2. CBD exhibits excellent therapeutic potential in neuropsychiatric disorders such as epilepsy, PD, AD, depression, anxiety, psychosis, and drug dependence (Elsaid et al., 2019;Premoli et al., 2019;Vitale et al., 2021). Under METH exposure, CBD shows neuroprotective (Razavi et al., 2021), antioxidative, and anti-inflammatory properties (Majdi et al., 2019;Karimi-Haghighi et al., 2020). In addition, CBD can restore the adaptation to threshold doses caused by repeated METH exposure (Khanegheini et al., 2021). Our results showed that CBD pretreatment prevented METH-induced caspase-8/ caspase-3 cascade and apoptosis in-vitro and in-vivo. During abstinence periods, CBD has been shown to promote neurogenesis in the Hip dentate gyrus in rats under chronic METH exposure (Razavi et al., 2021). In the current study, we found that CBD pretreatment markedly prevented the increase in caspase-8/caspase-3 cascade and apoptosis in the Hip of rats induced by repeated METH exposure. Given our results and those of the abovementioned studies, CBD shows substantial neuroprotective effects on METH-induced neurotoxicity. However, one major obstacle regarding CBD research is our relatively poor understanding of the mechanisms underlying its therapeutic potential. As CBD has a low-binding affinity toward traditional cannabinoid receptors (CB1-R and CB2-R) (Izzo et al., 2009;Bian et al., 2019;Vitale et al., 2021), determination of the main active target(s) of CBD is essential. Evidence indicates that CBD modulates DA receptors (Hassanlou et al., 2021;Nouri et al., 2021), Sigma-1 receptors , and TLR4 (Majdi et al., 2019). Here, we found that CBD may directly bind with DRD1 and compete with METH for the PHE-313 binding site. These two partially overlapping binding pockets likely affected ligand and receptor binding. This may explain why the type of interactions between CBD and DRD1 residues were variable when CBD and METH docked with the DRD1 model. Previous molecular docking evidence suggests that CBD is a potential binding ligand for DRD3 (Bian et al., 2019). Therefore, CBD may exert broader effects on DA receptors. In addition, our biological experiments showed that CBD also exhibited significant preventive effects on SKF81297-induced Ca 2+ influx, MeCP2 Ser421 phosphorylation, caspase-8/caspase-3 cascade, and apoptosis. These results suggest that the preventive effects of CBD on neurotoxicity may be mediated by DRD1.
However, METH-induced neurotoxicity not only involves direct damage to neurons, but also the participation of neuroinflammation (Shaerzadeh et al., 2018;Kim et al., 2020). Previous studies have shown that METH induces neuroinflammation via TLR4-or Sigma-1 receptor-related pathways (Kim et al., 2020). In addition, research suggests that METH-stimulative neuroinflammation may involve the participation of D1-like DA receptors , which are highly expressed in the microglia (Huck et al., 2015). Thus, the contribution of DRD1 to METH-induced neurotoxicity may not be limited to neurons. Furthermore, the phosphorylation of MeCP2 at Ser421 is also observed in microglia after psychostimulant exposure (Cotto et al., 2018). Therefore, the regulatory effects of DRD1-mediated phosphorylation of MeCP2 on METH-induced neurotoxicity may not be limited to neurons but could exert more global effects on multiple types of cells, providing a clear direction for future research.
In conclusion, our results suggest that METH induces neurotoxicity via DRD1-mediated calcium-dependent phosphorylation of MeCP2 at Ser421. Moreover, CBD significantly prevents METH-induced neurotoxicity via modulation of DRD1. Future research should focus on the contribution of neuroinflammation to neurotoxicity and explore the anti-inflammatory effects of CBD under METH exposure.

Data availability statement
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.

Ethics statement
The animal study was reviewed and approved by the Animal Care and Use Committee of Kunming Medical University.
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