Decreased glial reactivity could be involved in the antipsychotic-like effect of cannabidiol

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Abstract

NMDA receptor hypofunction could be involved, in addition to the positive, also to the negative symptoms and cognitive deficits found in schizophrenia patients. An increasing number of data has linked schizophrenia with neuroinflammatory conditions and glial cells, such as microglia and astrocytes, have been related to the pathogenesis of schizophrenia. Cannabidiol (CBD), a major non-psychotomimetic constituent of Cannabis sativa with anti-inflammatory and neuroprotective properties induces antipsychotic-like effects. The present study evaluated if repeated treatment with CBD (30 and 60 mg/kg) would attenuate the behavioral and glial changes observed in an animal model of schizophrenia based on the NMDA receptor hypofunction (chronic administration of MK-801, an NMDA receptor antagonist, for 28 days). The behavioral alterations were evaluated in the social interaction and novel object recognition (NOR) tests. These tests have been widely used to study changes related to negative symptoms and cognitive deficits of schizophrenia, respectively. We also evaluated changes in NeuN (a neuronal marker), Iba-1 (a microglia marker) and GFAP (an astrocyte marker) expression in the medial prefrontal cortex (mPFC), dorsal striatum, nucleus accumbens core and shell, and dorsal hippocampus by immunohistochemistry. CBD effects were compared to those induced by the atypical antipsychotic clozapine. Repeated MK-801 administration impaired performance in the social interaction and NOR tests. It also increased the number of GFAP-positive astrocytes in the mPFC and the percentage of Iba-1-positive microglia cells with a reactive phenotype in the mPFC and dorsal hippocampus without changing the number of Iba-1-positive cells. No change in the number of NeuN-positive cells was observed. Both the behavioral disruptions and the changes in expression of glial markers induced by MK-801 treatment were attenuated by repeated treatment with CBD or clozapine. These data reinforces the proposal that CBD may induce antipsychotic-like effects. Although the possible mechanism of action of these effects is still unknown, it may involve CBD anti-inflammatory and neuroprotective properties. Furthermore, our data support the view that inhibition of microglial activation may improve schizophrenia symptoms.

Introduction

Individuals suffering from schizophrenia manifest a range of behavioral changes, including positive (delusions and hallucinations) and negative symptoms (social withdrawal, anhedonia), as well as cognitive impairment. Cognitive deficits and negative symptoms are present even before the onset of psychosis and are frequently associated with poor long-term outcome (Elvevag and Goldberg, 2000, Lesh et al., 2011). While the existing medications have proven effective in treating positive symptoms, their efficacy on negative symptoms and cognitive deficits is limited (Elvevag and Goldberg, 2000, Hanson et al., 2010), indicating a great need for new psychopharmacologic agents.

Although the etiology of schizophrenia is still unknown, evidence suggests that an impaired function of the prefrontal cortex mediated by a glutamate NMDA receptor hypofunction could be involved in the negative and cognitive symptoms of schizophrenia (Gonzalez-Burgos and Lewis, 2012, Nakazawa et al., 2012). This proposal is based essentially on studies showing that acute and chronic administration of NMDA receptor antagonists, such as phencyclidine, ketamine, and MK-801, in animals and healthy volunteers induces schizophrenia-like signs (Krystal et al., 1994, Jentsch and Roth, 1999). Moreover, when administered to schizophrenia patients these drugs can worse the psychotic symptoms (Krystal et al., 2005). Thus, animal models based on administration of these drugs have been widely used. However, even if most studies have employed acute administration of NMDA receptor antagonists, the effects induced by chronic treatment with these drugs are proposed to better represent the behavioral, neurochemical and neuroanatomical changes observed in schizophrenia patients (Jentsch and Roth, 1999).

An increasing number of clinical, epidemiological, and experimental data have linked schizophrenia with inflammatory conditions. In this context, glial cells, such as microglia and astrocytes, have been related to the pathogenesis of schizophrenia (Schnieder and Dwork, 2011, Monji et al., 2013). Microglia and astrocytes are the major immune cells in the central nervous system (CNS), regulating the induction as well as the limitation of inflammatory processes (Sofroniew and Vinters, 2010, Graeber et al., 2011).

Cannabidiol (CBD), a major non-psychotomimetic compound from Cannabis sativa, presents potential therapeutic effects in schizophrenia with several pre-clinical studies indicating that this drug induces antipsychotic-like effects (for review see Campos et al., 2012). These effects have also been described in open-label clinical studies (Zuardi et al., 1995, Zuardi et al., 2006) and in a recent controlled, randomized, double-blind clinical trial (Leweke et al., 2012). The mechanism of these effects is still unknown (Campos et al., 2012). However, besides its antipsychotic properties, CBD also induces anti-inflammatory and neuroprotective effects, which could contribute for its beneficial effects in schizophrenia. Indeed, a considerable number of preclinical studies have indicated that CBD attenuated increased glial reactivity associated to pathological conditions (Mecha et al., 2013, Perez et al., 2013, Schiavon et al., 2014). Yet, the involvement of these mechanisms in CBD antipsychotic effects has not been evaluated in animal models of schizophrenia.

Based on these pieces of evidence, we investigated whether repeated CBD treatment would attenuate the impairment in social interaction and novel object recognition (NOR) tests induced by chronic administration of the NMDA receptor antagonist MK-801. These tests have been widely used to study the negative symptoms and cognitive deficits, respectively, in animal models of schizophrenia (Ellenbroek and Cools, 2000, Rajagopal et al., 2014). Additionally, given that neuroinflammatory processes in schizophrenia may involve abnormal astrocyte and microglia functions (Rothermundt et al., 2009, Schnieder and Dwork, 2011, Monji et al., 2013, Catts et al., 2014) and that NMDA receptor antagonists induce neuronal damage and alter the expression of astrocyte and microglial markers (Nakki et al., 1995, Nakki et al., 1996), we also measured changes in the expression of neuronal (NeuN) and glial markers (GFAP, astrocytes; Iba-1, microglia) in brain structures related to the neurobiology of schizophrenia, such as the medial prefrontal cortex (mPFC), dorsal striatum (dSTR), nucleus accumbens (NAc) core and shell and dorsal hippocampus (dentate gyrus — DG, CA1 and CA3). CBD effects were compared to those induced by the atypical antipsychotic clozapine.

Section snippets

Animals

The experiments were performed using male C57BL/6J mice with 6 weeks of age at the beginning of treatment. Animals were housed in groups of four per cage (41 × 33 × 17 cm) in a temperature-controlled room (24 ± 1 °C) under standard laboratory conditions with free access to food and water and a 12 h light/dark cycle (lights on at 06:00 a.m.). Procedures were conducted in conformity with the Brazilian Society of Neuroscience and Behavior guidelines for the care and use of laboratory animals, which are in

CBD and clozapine effects on behavioral changes induced by repeated MK-801

Repeated MK-801 treatment impaired social interaction (F6,49 = 5.21, P < 0.0001, one-way ANOVA followed by S-N-K post-hoc test; P < 0.05 vs. vehicle + saline group, Fig. 3). CBD, at the dose of 60 mg/kg, and clozapine reversed the impaired social interaction induced by MK-801 treatment (S-N-K post-hoc test; P < 0.05 vs. vehicle + MK-801 group, Fig. 3). This change was also attenuated by CBD 30 mg/kg + MK-801 (S-N-K post-hoc test; P > 0.05 vs. vehicle + saline group).

In the NOR test, no significant differences in

Discussion

The present study shows that repeated administration of the NMDA receptor antagonist MK-801 for 28 days impaired social interaction and NOR tests performed 1 and 2 days after the end of the treatment, respectively. These results are consistent with those showing a PPI disruption induced by a similar treatment schedule with MK-801 (Gomes et al., 2015). Additionally, no change was observed in the EPM and open field tests. Lacks of changes in open field activity following repeated treatment with

Role of funding source

This research was supported by grants from FAPESP (FSG and EDB: 2012/17626-7; FVG: 2010/17343-0; 2012/14144-1), CNPq ( 470311/2012-6;Brazil), and Instituto de Salud Carlos III, Redes temáticas de Investigación Cooperativa en salud, RD2012/0028/0021 GRUPO UCM 951579 (Spain). The funding sources had no involvement in the study design, in the acquisition, analysis, or interpretation of the data, in the writing of the report, and in the decision to submit the paper for publication.

Contributors

FVG designed the study, performed the experiments, and wrote the first draft of the manuscript. RL and MLG also performed the immunohistochemistry experiments and analyzed the data. EDB, MPV and FSG were involved in conception and design of the study. Statistical analyses were conducted by FVG and FSG. All authors were involved in manuscript development, critically reviewed the manuscript for important intellectual content, and approved the final version.

Conflict of interest

The authors declare no conflicts of interest.

Acknowledgments

The authors thank José Carlos de Aguiar, Celia A. da Silva, Ángela A. Arrojo, and Lara Arnaldo for technical assistance. FVG has a FAPESP fellowship (2010/17343-0; 2012/14144-1). This research was supported by grants from FAPESP (FSG and EDB: 2012/17626-7), CNPq (470311/2012-6; Brazil), and Instituto de Salud Carlos III, Redes temáticas de Investigación Cooperativa en salud, RD2012/0028/0021 GRUPO UCM 951579 (Spain).

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