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Published in final edited form as: Prog Neuropsychopharmacol Biol Psychiatry. 2023 Dec 20;130:110924. doi: 10.1016/j.pnpbp.2023.110924

Cannabinoid CB2 receptors and hypersensitivity to methamphetamine: vulnerability to schizophrenia

Ana Canseco-Alba a,b, Koichi Tabata c, Momoki Yukihiko c, Taharima Tabassum d, Yasue Hourichi d,e, Tadao Arinami f, Emmanuel S Onaivi b, Hiroki Ishiguro c,d,*
PMCID: PMC10872318  NIHMSID: NIHMS1957367  PMID: 38135096

Abstract

The human cannabinoid receptor 2 (CB2R) gene CNR2 has been associated with schizophrenia development. Inbred mice treated with the CB2R inverse agonist AM630 and challenged with methamphetamine (MAP) showed reduced prepulse inhibition (%PPI) response and locomotor hyperactivity, both behavioral measures in rodents that correlate with psychosis. Mice lacking CB2R on striatal dopaminergic neurons exhibit a hyperdopaminergic tone and a hyperactivity phenotype. Hyperdopaminergia plays a role in the etiology of schizophrenia. This study aimed to determine the direct role of CB2R, heterozygous Cnr2 gene knockout (Het) mice treated with MAP to induce behavioral sensitivity mimicking a schizophrenia-like human phenotype. Additionally, the study aims to explore the unique modulation of dopamine activity by neuronal CB2R. Conditional knockout DAT-Cnr2−/− mice were evaluated in response to MAP treatments for this purpose. Sensorimotor gating deficits in DAT-Cnr2−/− mice were also evaluated. Het mice developed reverse tolerance (RT) to MAP-enhanced locomotor activity, and RT reduced the %PPI compared to wild-type (WT) mice. DAT-Cnr2−/− mice showed an increased sensitivity to stereotypical behavior induced by MAP and developed RT to MAP. DAT-Cnr2−/− mice exhibit a reduction in %PPI and alter social interaction, another core symptom of schizophrenia. These results demonstrate that there is an interaction between neuronal CB2R and MAP treatment, which increases the risk of schizophrenia-like behavior in this mouse model. This finding provides evidence for further studies targeting CB2R as a potential schizophrenia therapy.

Keywords: cannabinoid CB2 receptor, schizophrenia, methamphetamine, dopaminergic neuron

1. Introduction

Several lines of evidence indicate a relationship between cannabis intake and a variety of psychiatric conditions (Hindley et al., 2020; Lowe et al., 2019); including the ones from psychotic spectrum disorders (D’Souza et al., 2004; Di Forti et al., 2019; Murray et al., 2017; Patel et al., 2020). Experimental studies in healthy humans showed that Δ9-THC (the main psychoactive component of Cannabis preparations, such as marijuana) produces a broad range of transient symptoms, behaviors, and cognitive deficits that resemble some aspects of endogenous psychoses (D’Souza et al., 2004). Neuroimage studies in humans, such as MRI or PET, have reported that the acute psychotomimetic effects of Δ9-THC have been correlated with functional activation of brain regions commonly implicated in psychotic symptoms, particularly striatal regions (Cupo et al., 2021). Moreover, a rare but severe side effect of synthetic ∆9-THC drugs, such as dronabinol (Marinol®) or Nabilone (Cesamet™), includes psychosis [FDA]. Early onset of use and daily use of high-potency cannabis have been associated with an increased subsequent risk of developing psychotic disorders/episodes from prospective epidemiological studies (Di Forti et al., 2019; Onaivi et al., 2008; Onaivi et al., 2012). The prevalence of schizophrenia is higher among patients with a history of cannabis use (Libuy et al., 2018). Cannabis use is considered to be a risk factor for the early onset of schizophrenia in people with a predisposition or exacerbation of the psychotic symptoms already present (Patel et al., 2020). All this evidence points to, first, the imperative to undertake pharmacological investigations to determine the effectiveness and safety of cannabis use, especially in individuals with, or prone to, mental health problems, psychotic disorders in particular; secondly, the implication of the endocannabinoid system in the pathophysiology of psychotic spectrum disorders.

The endocannabinoid system is composed mainly of the cannabinoid receptors type 1 and type 2 (CB1R and CB2R, respectively), the molecules derived from arachidonic acid which bind to the CBRs named endocannabinoids and the enzymes responsible for their synthesis and degradation (Cristino et al., 2020). While most studies have focused on CB1R, there is increasing recognition of the importance of understanding the central nervous system roles of CB2R in neuropsychiatric disorders (Ishiguro and Onaivi, 2017). Indeed, it is likely that CB1Rs and CB2Rs work independently, cooperatively, or both to regulate various biological processes (De Jesús et al., 2010; Delis et al., 2017; García-Cabrerizo and García-Fuster, 2016; Liu et al., 2017; Lopes et al., 2020; Pertwee, 2008).

Although the precise cellular localization of CB2Rs in the brain remains unclear, evidence regarding the localization of CB2Rs in glial cells, including microglia (Galán-Ganga et al., 2020; Maresz et al., 2005; Ni et al., 2019) and astrocytes (Eraso-Pichot et al., 2023) and also in neurons (Brusco et al., 2008; Gong et al., 2006; Li and Kim, 2015), is increasing. Its role in normal and pathological neurophysiology is a recent and interesting field.

Postmortem human brain and cell culture studies have shown that common polymorphisms of the CB2R gene (CNR2) are associated with psychiatric disorders and reduced CB2R functionality. However, although these hypofunctional genotypes have been implicated in schizophrenia, major depression, and substance abuse in humans (Ishiguro et al., 2010; Ishiguro et al., 2007; Onaivi et al., 2008; Onaivi et al., 2012; Ortega-Alvaro et al., 2011), the molecular mechanisms responsible for these CB2R alterations have not been well characterized or validated using behavioral experiments in animal models of psychiatric disorders. Environmental factors combined with genetic dysfunction drive the development of endophenotypes of psychiatric disorders, such that each environmental/acquired factor affects the neural networks in the brain in a unique manner (Ishiguro and Onaivi, 2017). It is well-established that striatal hyperdopaminergic is related to the symptomatology of schizophrenia (McCutcheon et al., 2020). It has also been recommended that neuronal CB2Rs modulate dopaminergic neuronal activity and dopamine-regulated behavior (Zhang et al., 2014). DAT-Cnr2−/− mice are conditional knockout (cKO) animals that do not express cannabinoid CB2Rs in midbrain dopamine neurons (Liu et al., 2017). In various tests, a hyperdopaminergic tone and a hyperactivity phenotype have been consistently observed in DAT-Cnr2−/− mice (Canseco-Alba et al., 2019).

Methamphetamine (MAP) has a causative role in cognitive deficits and Schneiderian first-rank symptoms, some of which may overlap with those of schizophrenia (Li et al., 2014; Shelly et al., 2016). MAP-induced disturbance of prepulse inhibition (PPI) has been used as an animal model of schizophrenia (Chao et al., 2012; Dai et al., 2004). In our previous study (Ishiguro et al., 2010), C57BL/6JJmsSLC mice were treated with CB2R ligands, and their behavior was assessed through PPI testing following treatment with either MK-801 (a noncompetitive N-methyl-D-aspartate receptor antagonist) or MAP. MK-801 and MAP administration reduced the enhanced PPI induced by combined treatment with the CB2R inverse agonist AM630 (6-iodopravadoline). Therefore, it was necessary to examine whether a reduction in prenatal CB2R function results in hypersensitivity to MAP and sensorimotor dysfunction, as shown in our previous study (Ishiguro et al., 2010).

We used Cnr2 Het mice treated with MAP to induce behavioral sensitivity mimicking the schizophrenia-like phenotype observed in humans to understand CB2R activity in schizophrenia better. A combination of congenital CB2R dysfunction and environmental factors may increase the susceptibility to schizophrenia. However, this could differ from the enhancement of acquired MAP toxicity by CB2R inhibition observed in the mouse model (Ishiguro et al., 2010). In addition, since hyperactivity and altered dopaminergic neurotransmission are crucial in models of schizophrenia, we decided to evaluate some of the MAP in DAT-Cnr2−/− mice. Another core symptom of schizophrenia, a reduction in social interaction, was also evaluated on Cnr2+/− ko mice and DAT-Cnr2−/− cko mice in basal conditions.

2. Materials and methods

2.1. Animals

This study used two strains of mice based on two different but complementary rationales: Cnr2 knockout (ko) mice and DAT-Cnr2−/− mice conditional knockout (cko). Cnr2 ko was provided by Prof. Buckley, California State Polytechnic University (Buckley, 2008). Heterozygous mating pairs obtained from the same litter were used to produce cohorts for this study. We used Cnr2 Het mice rather than homozygotes for the experiments to mimic human genetic CB2R hypofunction (Buckley, 2008). The generation of DAT-Cnr2−/− cko was described elsewhere (Liu et al., 2017). This strain was used to evaluate the hyperdopaminergic brain lacking midbrain-CB2R. Previous studies have shown that C57BL/6J mice as wild-type (C57BL/6J-WT) are the most suitable control for these animals.

Male mice were used in the experiments and were group-housed under a 12 h/12 h light/dark cycle with ad libitum access to food and water. The animal facility was maintained at a temperature and humidity of 25 °C and 40–55%, respectively. The mice were 7–9 weeks old and weighed 20–25 g during the experiments.

All animal procedures were performed according to protocols approved by the Institutional Animal Care and Use Committee of the University of Yamanashi (approval no: A29–45) and the William Paterson University Animal Care and Use Committee.

2.2. Drugs

Behavioral changes following the administration of a chemical stressor (MAP) were analyzed to determine the functional relationship between MAP treatment and CB2R-mediated changes. MAP (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 0.9% saline and administered intraperitoneally at a 0.01 ml/g body weight volume. The same volume of vehicle (saline) was administered to the control animals. Experiments were performed 15 minutes after MAP administration.

2.3. Induction of reverse tolerance (RT) protocol

Briefly, MAP administration was used to establish a model of schizophrenia-like behavior for assessment of either an acute response to a single dose or the development of reverse tolerance (RT) to the drug. RT, also referred to as sensitization, is a phenomenon in which, a specific behavioral, physiological, or cellular response increases over time rather than decreasing following repeated exposure to a particular drug of abuse. MAP-induced RT is considered to be a psychotic-like symptom. Mice were administered MAP (2 mg/kg, intraperitoneally) once daily for four consecutive days for the RT induction. One week after the final injection, mice were injected with MAP (0.5 mg/kg, intraperitoneally) as an acquired RT model, as previously described, with some modifications (Ago et al., 2006; Shuto and Nishi, 2011).

2.4. Behavioral testing

All behavioral tests were performed during the light phase (1:00 PM to 7:00 PM) under dimmed light in an experiment room under the same temperature and humidity conditions as those in the animal housing facility.

2.4.1. Spontaneous locomotor activity

Our previous study indicated that acute administration of MAP enhanced locomotor activity in Het mice compared to that in WT mice (Ishiguro et al., 2010). In this study, the locomotor activities in four groups of mice were monitored to determine the total distance traveled in individual cages. These cages were identical to the home cages where the mice had been housed before the tests. Data were collected every 10 minutes for 2 hours using ACTIMO-DATA software (National Instruments Co., Austin, TX, USA). The groups were as follows: Naïve mice received a single saline injection before the experiments. Groups 1 and 2 included Het and WT mice, respectively (n = 9 WT and 8 Het mice). Groups 3 and 4 included Het and WT mice subsequent to the development of RT to MAP (n = 17 WT and 11 Het mice).

The same protocol to evaluate RT to MAP was used for the DAT-Cnr2−/− and WT mice (n = 10 per group, 4 groups).

Since the acute effect of MAP on DAT-Cnr2−/− was not reported before, we evaluated the acute effects of MAP. Four independent groups of -WT and DAT-Cnr2−/− male mice (n = 10 per group) were evaluated for their locomotor activities (total distance traveled and stereotypic counts—e.g., sniffing, licking, gnawing, and grooming actions). The mice were observed in the open-field chamber for 20 min in response to the systemic administration of different doses of methamphetamine (1, 3, or 10 mg/kg) or vehicle (saline). Mice were individually placed at the center of an infrared photobeam-controlled open-field test chamber (43.2 × 43.2 × 30.5 cm; ENV-510; Med Associates Inc., St. Albans, VT, USA) connected to a computer and allowed to explore it freely.

2.4.2. Prepulse inhibition testing (PPI)

MAP was used as the chemical stressor. Our previous study indicated that acute administration of MAP in C57BL/6J mice reduced %PPI to a greater degree following treatment with a CB2R inverse agonist (AM630) (Ishiguro et al., 2010). In this study, one group (Group A) of mice was treated with a single intraperitoneal saline injection (n = 8 WT and 26 Het mice). The second group (Group B) of mice received four intraperitoneal saline injections. One week after the final injection, the mice were intraperitoneally injected with MAP (0.5 mg/kg, intraperitoneally; n = 14 WT and 13 Het mice). The third group (Group C) was treated with four intraperitoneal MAP injections (2 mg/kg). One week after the final injection, mice were intraperitoneally injected with saline (n = 10 WT and 13 Het mice). The fourth group (Group D) of mice was made to develop RT to MAP (n = 16 WT and 22 Het mice) as described in section 2.3.

The mice were placed in a cylindrical Plexiglas test chamber with an SR-Lab system (San Diego Instruments Inc., San Diego, CA). The experimental sessions consisted of a 5-minute acclimatization period with 70 dB broadband background white noise (continuous throughout the session), followed by three 120 dB acoustic pulses. The PPI sessions consisted of 10 blocks administered in five different trial types: a 40 ms startle pulse at 120 dB alone and four prepulse-plus-pulse trials (with a 20 ms prepulse at either 74 dB, 78 dB, 82 dB, or 86 dB, followed by a 40 ms startle stimulus at 120 dB after a 100 ms delay), presented in a pseudorandom order, with an average interval of 15 seconds. The maximum startle amplitude was recorded as the startle response. The PPI was calculated as a percentage score of each prepulse intensity using the following equation: %PPI=100 × [(mean startle amplitude in pulse-alone trials – mean startle amplitude in prepulse trials) /mean startle amplitude in pulse-alone trials]. %PPI was compared between the genotypes of each mouse group, as they were calculated based on the mouse startle amplitude, which differed among treatments, as shown in the locomotion analysis.

The PPI response was not evaluated before in DAT-Cnr2−/−, so the exploration of this response was evaluated similarly on DAT-Cnr2 in basal conditions.

2.4.3. Social preference task (SPT)

The SPT evaluates general sociability. Mice were habituated to the apparatus and the test room for 60 minutes on the day after the evaluation. The apparatus consisted of a three-chamber arena (30 cm length × 60 cm width × 22 cm height). Each chamber was connected to the other by an entrance (8.5 cm height and 7.5 width) and a retractable door. On the evaluation day, experimental mice were introduced into the middle chamber with both entrances closed. Once the door was opened, the animals were allowed to explore the arena freely. One of the chambers contains an empty cup, while the other contains a cup with a nonsexual “intruder” inside. The behavior of the mice was recorded without any researchers present. After 5 minutes, the mice were returned to their cages. The videos were double-blinded and analyzed. The time spent on the social investigation was recorded, and the preference ratio was obtained using the following formula: time spent socializing/time spent socializing + time spent exploring (not socially).

2.5. Statistical analyses

Locomotor activity among the treatment groups and genotypes was compared using a two-way analysis of variance (ANOVA). Post-hoc analysis was conducted using Tukey’s or Dunnett’s tests. Differences in PPI values were compared using one-way ANOVA with repeated measures at each acoustic level, followed by Tukey’s post-hoc test. The Mann–Whitney test was used for pairwise comparisons, like the SPR ratio U. All statistical analyses were performed using JMP software version 12 (SAS Institute, Tokyo, Japan). A P value of < 0.05 was considered statistically significant.

3. Results

3.1. MAP induce locomotor RT

Panel B. Black bar: WT mice with RT to MAP; dark gray bar: DAT-Cnr2−/− mice with RT to MAP; light gray bar: WT mice with saline treatment; white bar: DAT-Cnr2−/− mice with saline treatment. Following the development of RT to MAP, both genotypes exhibited an increased locomotion.

Data regarding locomotor RT induced by MAP are shown in Figure 1. In Cnr2 KO mice (Figure 1A), there were significant differences in locomotor activity between the treatment groups and genotypes [F(3,41) = 51.28, P < 0.0001; test of effects: Cnr2 genotype; F(1,1) = 13.11, P = 0.008; treatment, F(1,1) = 149.0, P < 0.0001; Figure 1A]. Post-hoc analyses showed significant differences between WT and Het mice following the development of RT to MAP, whereas no difference was observed in the saline treatment group. The analysis also revealed significant differences between the WT and Het mice treatment groups.

Figure 1. Locomotor changes in Cnr2 knockout and DAT-Cnr2−/− mice with reverse tolerance (RT) to methamphetamine (MAP).

Figure 1.

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Mean and standard error of the mean (SEM) of the total arbitrary locomotor counts of mice. Panel A. Black bar: WT mice with RT to MAP; dark gray bar: Het mice with RT to MAP; light gray bar: WT mice with saline treatment; white bar: Het mice with saline treatment. Following the development of RT to MAP, the mice exhibited greater locomotor activity than the saline-treated mice of each genotype. Only when the mice acquired RT to MAP increased locomotion by a greater amount in Het mice was seen than in WT mice. * P < 0.05.

In DAT-Cnr2−/− mice (Figure 1B), ANOVA revealed that there was a significant main effect of genotype [F(1, 18)=21.56, P = 0.001] and treatment [F(2,18)=48.86, P < 0.001]. Dunnett’s post-hoc comparison showed that MAP induces RT in both genotypes: C57BL/6J-WT and DAT-Cnr2−/− mice, although there is a trend to be higher in DAT-Cnr2−/− this comparison did not reach significance.

3.2. Acute effect of MAP on locomotor activity in DAT-Cnr2−/− mice

Data regarding the effects of different doses of MAP on distance traveled and stereotypic counts in DAT-Cnr2−/− are depicted in Figure 2. Two-way ANOVA revealed a significant effect of MAP treatment [F(3,72) = 129.55, P < 0.001] on distance traveled. The genotype factor did not reach significance, but there was a significant interaction effect of treatment × genotype [F(3,72) = 5.64, P = 0.002]. Post-hoc analysis using Dunnett’s test showed a significant increase in the distance traveled by C57BL/6J mice after MAP treatments at the 1 and 3 mg/kg doses, but not a higher dose (10 mg/kg). In DAT-Cnr2−/−cKO mice, the 3 mg/kg MAP dose significantly increased the distance traveled. In contrast, 10 mg/kg of MAP induced a significant decrease. Genotypes were also significantly different at the 3 mg/kg dose. In animals treated with a vehicle, the distance traveled by DAT-Cnr2−/−cKO mice was greater than that traveled by C57/6J-WT mice, a result that was consistent with the hyperactive phenotype of the former.

Figure 2. Effects of MAP in the total distance traveled and stereotypic counts in the open-field test of DAT-Cnr2−/−.

Figure 2.

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White bars represent the C57/6J-WT mice, and the black bars the DAT-Cnr2−/− cKO mice. Data are expressed as mean ± SEM (n = 10 mice per group). Two-way ANOVA followed by Dunnett’s post hoc test. *P < 0.05; MAP vs vehicle; + P < 0.05; C57/6J-WT vs DAT-Cnr2−/−.

Concerning stereotypic counts, two-way ANOVA revealed significant effects of genotype [F(1,72) = 89.60, P < 0.001], treatment [F(3,72) = 62.74, P < 0.001], and the treatment × genotype interaction [F(3,72) = 3.92, P = 0.012]. Post-hoc analysis showed that only the higher MAP dose (10 mg/kg) significantly increased stereotypic counts in C57BL/6J-WT animals. In DAT-Cnr2−/−cKO mice, all doses induced a significant increase in this parameter in comparison with the vehicle. Concerning the vehicle, there was a significant difference between the genotypes. However, an increase in this parameter was reported previously as part of the phenotype of the DAT-Cnr2−/−cKO. There was a significant difference between C57/6J-WT and DAT-Cnr2−/−cKO mice regarding the stereotypic counts induced by all MAP doses tested.

Remarkably, in C57/6J-WT mice, the 10 mg/kg MAP dose reduced the distance traveled because of increased stereotypical behaviors. MAP’s induction of stereotypic behaviors in DAT-Cnr2−/− was dose-dependent, reaching its peak at the same dose as with a decreased distance traveled. It is important to note that these animals were hyperactive and exhibited more stereotypical behaviors under basal conditions.

3.3. PPI testing in Cnr2 knockout mice

The results of PPI testing are shown in Figure 3. The letters on the left side of the graphs depict the experimental group. The naïve Het mice did not exhibit significant differences in %PPI between the genotypes (Het and WT mice) or between the Het and WT mice at each acoustic level, whereas a significant difference was observed between %PPI at each acoustic level [genotypes: F(1,1) = 0.008, P = 0.93; acoustic levels: F(3,3) = 24.7, P < 0.0001; genotypes and acoustic levels: F(3,3) = 0.72, P = 0.54, Figure 4A]. The second group of mice, analyzed as the control against the RT model mice (group C), also did not exhibit significant differences between the genotypes and between the Het and WT mice at each acoustic level, whereas a significant difference was observed between %PPI at each acoustic level [genotypes: F(1,1) = 0.02, P = 0.88; acoustic levels: F(3,3) = 5.8, P = 0.001; genotypes and acoustic levels: F(3,3) = 0.10, P = 0.96; Figure 4B]. Similarly, The third group of mice, analyzed as the control against the RT model mice (group D), did not exhibit significant differences between the genotypes and between the Het and WT mice at each acoustic level, whereas a significant difference was observed between %PPI at each acoustic level [genotypes: F(1,1) = 2.84, P = 0.10; acoustic levels: F(3,3) = 18.6, P = 0.56; genotypes and acoustic levels: F(3,3) = 0.68, P = 0.56, Figure 4C]. In the fourth group, after the development of RT to MAP, there was a significant difference in %PPI between the genotypes as well as between trials, whereas no significant differences were found between the Het and WT mice at each acoustic level [genotypes: F(1,1) = 15.7, P = 0.0001; acoustic levels: F(3,3) = 8.6, P < 0.0001; genotypes and acoustic levels: F(3,3) = 0.03, P = 0.99, Figure 4D]. Post-hoc analyses did not show significant differences between Het and WT mice at each acoustic level.

Figure 3. Prepulse inhibition (PPI) in WT (+/+) and Het (+/−) mice with/without reverse tolerance (RT) to methamphetamine (MAP).

Figure 3.

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The vertical axis shows the %PPI of the mice during four acoustic sessions of increasing loudness. The error bars show the standard error of the mean. Black bars: WT mice; gray bars: Het mice. (A) Mice treated with a single saline administration showed no difference in %PPI between the WT and Het groups. (B) Mice treated with a series of saline injections and MAP (0.5 mg/kg, ip) as the final injection showed no difference in %PPI between the WT and Het groups. (C) Mice treated with a series of MAP (2 mg/kg, ip) injections and saline as the final injection showed no difference in %PPI between the WT and Het groups. (D) Following the development of RT to MAP, a significant difference in %PPI between the WT and Het groups was observed.

Figure 4. Prepulse inhibition (PPI) in DAT-Cnr2−/−.

Figure 4.

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PPI (%) at two different prepulse intensities (+74 and +82 dB) in the PPI test. White bars represent WT mice, and grey bars represent the conditional ko mice DAT-Cnr2−/−. Data are presented as the mean ± SEM **p<0.01.

3.4. PPI testing in DAT-Cnr2−/− cko mice on basal conditions

The results of PPI testing in basal conditions of DAT-Cnr2−/− mice are shown in Figure 4. As can be seen, DAT-Cnr2−/− shows a significant reduction in %PPI compared to WT mice (P < 0.01).

3.5. Social preference task (SPT)

In Figure 5, the results of the SPT are depicted. Cnr2 ko (panel A) and DAT-Cnr2 (Panel B) mice spent less time socially interacting than their respective WT. A Mann-Whitney test indicated that the ratio was greater for WT mice (Mdn = 61) than for DAT-Cnr2 cko mice (Mdn =26) (P = 0.001).

Figure 5. Social preference test (SPT).

Figure 5.

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The vertical axis shows SFT ratio in basal conditions of Cnr2 HET mice (Panel A) and DAT-Cnr2 mice (Panel B). **P < 0.001.

4. Discussion

The findings of this study and those of our previous studies indicate that the combination of CNR2 gene dysfunction and MAP administration may replicate the genetic and environmental interaction involved in the occurrence of schizophrenia. In this study, Het mice appeared to exhibit hypersensitivity to MAP, which was particularly obvious regarding increased locomotor activity. Regarding MAP-induced PPI disturbances, previous studies have shown that combined treatment with MAP and the CB2R inverse agonist AM630 reduced %PPI in C57BL/6JJmsSLC mice (Ishiguro et al., 2010). In this study, Het mice that developed RT to MAP showed a significant reduction in %PPI compared to WT mice. Thus, prenatal or postnatal CB2R dysfunction seems to result in the development of sensorimotor dysfunction affecting behavioral responses to MAP administration. Our data derived from using this mouse model show that the genetically predetermined hypofunctionality of CB2Rs increases the susceptibility to develop schizophrenia-like behaviors when combined with stressors that are clinically known risk factors.

Also, our findings show that the deletion of the gene for CB2R in midbrain dopaminergic neurons increases the expression of psychosis-related behaviors. On basal conditions, DAT-Cnr2 cKO exhibits consistent hyperactivity (Canseco-Alba et al., 2019; Liu et al., 2020). In this study, we demonstrate that DAT-Cnr2 mice exhibit a reduced percentage of PPI and a reduced social preference under basal conditions. These three behavioral measurements are considered to be psychosis-like. Therefore, this is the first evidence that CB2R expressed in midbrain dopaminergic cells is relevant to psychosis models.

DAT-Cnr2 cKO mice have particularities related to psychostimulant effects (Canseco-Alba et al., 2019). This study is the first report on the effect of MAP, and it reveals an increase in the sensitivity to stereotypical behavior induced by MAP. This finding provides a possible explanation for the paradoxical effect on ambulatory behavior. However, stereotypy has been noted in patients with psychosis and also in several pharmacological animal models of psychosis. The hyperdopaminergic tone in DAT-Cnr2 cKO mice might contribute to this behavioral phenotype’s underlying neural mechanisms (Kibret et al., 2023).

The dopamine hypothesis of schizophrenia postulates, among other principles, that the positive symptoms of schizophrenia are a result, at least in part, of excessive dopamine neuronal firing originating in the midbrain and allowing excessive dopamine release in the limbic system, i.e., a hyperdopaminergic state (McCutcheon et al., 2020). MAP increases dopamine concentration in the synaptic cleft by reversing the transport direction of the DAT and also by decreasing the membrane-associated DAT in the presynaptic neurons, which can induce psychotic behaviors similar to the positive symptoms such as hallucination, seen in schizophrenia patients (Yui et al., 2000). DAT-Cnr2 cKO exhibits hypersensitivity to MAP-induced RT, which supports the results with Het Cnr2 ko mice.

CB2R was previously considered a strictly peripheral cannabinoid receptor with predominantly immune system-related functions. However, subsequent evidence indicated functional expression of CB2Rs in the brain (Onaivi et al., 2012). Several studies have linked a predisposition to psychosis and emotional disturbances in adolescence with increased vulnerability to certain types of stressors associated with the occurrence of schizophrenia and depression. For example, both maternal immune activation and adolescent cannabinoid exposure have been identified as major environmental risk factors for schizophrenia (Hollins et al., 2016). This study also examined risk factors affecting the mature mammalian mouse brain. Cannabis use during adolescence has been reported as a risk factor for schizophrenia (Chadwick et al., 2013), and recent studies using animal models have also shown that adolescence is a vulnerable period for the development of schizophrenia (Gomes et al., 2016). A recent meta-analysis did not establish a significant relationship between schizophrenia and maternal influenza infection (Selten and Termorshuizen, 2017), and prenatal exposure to maternal influenza has been reported to increase the risk of schizophrenia-spectrum disorders (Flinkkila et al., 2016). Although the influence of maternal immune activation on schizophrenia development is not apparent in the general population, future studies are required to clarify better the association between maternal immune activation and endocannabinoid system disorders. Newborn rats experiencing maternal deprivation exhibit anomalous behaviors even into adulthood, which has also been observed in patients with schizophrenia (Ratajczak et al., 2013). This is supported by animal studies that have highlighted a possible link between the endocannabinoid system and early maternal deprivation associated with an increase in the number of degenerated hippocampal neurons and astrocytes. Additionally, there are increased corticosterone and endocannabinoid 2-arachidonyl glycerol levels in the hippocampus (Llorente et al., 2008; Lopez-Gallardo et al., 2008). However, a genetic polymorphism study of diacylglycerol lipase alpha, which may directly regulate the levels of 2-arachidonyl glycerol, revealed a possible association with the development of alcoholism, but not with schizophrenia (Ishiguro et al., 2018). Thus, the functional involvement of CB2R in endocannabinoid metabolism is not entirely clear, and the gene-environment interactions underlying the development of schizophrenia via endocannabinoid system-mediated processes require further investigation.

Certain limitations of this study were noted. First, we employed a mouse model of schizophrenia. Therefore, further investigations with human participants are necessary. Second, we only examined adult brain stressors that could lead to the development of psychiatric symptoms. Although acute stressors may be associated with an increased risk of depression in adult humans, they do not necessarily lead to the development of schizophrenia. In future studies, we will investigate the possible interactions between CB2R hypofunction and the impact of stressors on adolescents to characterize the developmental roles of CB2R in schizophrenia. Schizophrenia is caused by a complex array of dysfunctions and alterations in several neural systems, including the dopamine and glutamate systems.

In conclusion, reduced CB2R functionality in the endocannabinoid system and CB2R-regulated hyperdopaminergic activity were observed to play a role in MAP-induced psychosis in the mouse model employed in this study and, possibly, the vulnerability to schizophrenia upon exposure to environmental stressors. These results demonstrate that targeting CB2R may be a potential therapeutic strategy for the treatment of schizophrenia.

Highlights.

  • The human cannabinoid receptor 2 (CB2R) gene CNR2 is associated with schizophrenia.

  • CB2R hypofunctionality increased schizophrenia-like behaviors in mice.

  • CB2R-regulated hyperdopaminergic activity was involved in MAP-induced psychosis.

Acknowledgments

The authors thank Professor Nancy Buckley, California State Polytechnic University, for donating the Cnr2 knockout mice used in this study.

Funding

HI was supported by the Public Interest Trust Research Aid Fund for Stress-Related Disease (with Commemoration of Imai Kimi) and JSPS KAKENHI (grant nos. 20390098, 23K07033). The Tokyo Metropolitan Institute of Medical Science supported YH. The William Paterson University, Dean’s Student Workers Program, and the NIH (grant nos. DA032890 and NIH-NIAAA AA027909) supported AC and ESO.

Role of the funding source

There was no involvement of the funding agencies in any procedures related to this research.

Abbreviations

CB1R

Cannabinoid receptor type 1

CB2R

Cannabinoid receptor type 2

MAP

Methamphetamine

PPI

Prepulse inhibition

SPT

Social preference task

WT

Wild type

Footnotes

Declaration of interest: None.

Conflicts of interest: None.

There was no human subjects analyzed in this research. All animal procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of Yamanashi (approval no: A29–45) and the William Paterson University Animal Care and Use Committee.

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