13.2 CANNABIDIOL AS AN ANTIPSYCHOTIC DRUG

Abstract Background The phytocannabinoid cannabidiol (CBD) attenuates the psychotomimetic effects produced by high doses of delta-9-tetrahydrocannabinol (THC), the main component of the Cannabis sativa plant. Corroborating this effect, several preclinical and clinical studies indicate that CBD has antipsychotic properties. The mechanisms responsible for these properties, however, remain unknown (Campos et al., Philos Trans R Soc Lond B Biol Sci 367:3364–782013). We have recently found that repeated CBD administration prevents the behavioral impairments, measured in the pre-pulse inhibition, social interaction and novel object recognition tests, induced in mice by repeated treatment (28 days) with the NMDA receptor antagonist MK-801. CBD also prevented the neural (measured by delta-FosB) and microglia activation, and the decrease in the number of parvalbumin-positive neurons, observed in the medial prefrontal cortex (Gomes et al., Int J Neuropsychopharmacol 18(5)2014, Schizophr Res 164:155–63, 2015). Currently, we are investigating if CBD could also reverse these changes once they have been established and the possible mechanisms of this effect. Methods Male C57BL/6J mice received intraperitoneal injections of MK-801 (0.25, 0.5 or 1 mg/kg, twice a day) for 7 or 14 days. To determine if these treatments regime would induce acute and long-term deficits, the social interaction (SI) test was performed 1 or 8 days after the end of the MK-801 treatment. Twenty-four hours after the SI, animals were submitted to the novel object recognition (NOR) test. Having established that 14 days of MK-801 induce both acute (24 h after) and long-term (8 days after) behavioral deficits in the SI and NOR tests, we investigated if repeated treatment with CBD (15, 30 or 60 mg/kg daily, i.p.) would reverse these changes. CBD treatment began 24h after the end of the MK-801 treatment and lasted for 7 days. Repeated clozapine (1 mg/kg) was used as a positive control. Forty-eight hours after the last injection, animals were submitted to SI and, 24-h later, to the NOR test. In a second experiment, independent groups of mice received, before each CBD injection, AM251 (a CB1 receptor inverse agonist, 0.1–0.3 mg/kg), AM630 (a CB2 receptor inverse agonist, 0.1–0.3 mg/kg), or the 5HT1A receptor antagonist WAY100635 (0.1–0.3 mg/kg). The data were analyzed by ANOVA followed by the Newman-Keuls test. Results MK-801 (0.5 mg/kg) administration for 14 days, but not for 7 days, impaired SI and NOR. Repeated CBD or clozapine treatment reversed these impairments. CB1 and CB2 antagonists (AM251 and AM630, respectively) failed to change CBD effect. However, its effect was blocked by pretreatment with the 5HT1A receptor antagonist WAY100635. Discussion Our findings show that a 2-week treatment with the NMDA receptor antagonist MK801 impairs social interaction and novel object recognition, which have been associated with negative and cognitive symptoms of schizophrenia, respectively. These behavioral deficits last for at least one week and are reversed by the atypical antipsychotic clozapine or CBD, reinforcing the proposal that this latter drug has antipsychotic-like properties. CBD effects seem to depend on facilitation of 5HT1A-mediated neurotransmission.


ENDOCANNABINOID MODULATION OF DOPAMINE NEUROTRANSMISSION Michael Bloomfield University College London
Overall Abstract: There are converging lines of evidence that the endocannabinoid system is involved in the pathophysiology of schizophrenia and that understanding these mechanisms may lead to novel treatment targets. In this symposium, we will present a series of experiments that link cannabinoid pharmacology to major fields of schizophrenia research including the dopaminergic, glutamatergic and serotonergic systems, glial cell function and the genetics of cognition. Dopamine is a major neurotransmitter implicated in the pathophysiology of schizophrenia. Thus, understanding processes that modulate dopaminergic signalling may lead to new insights into the biology and treatment of this disorder. The endocannabinoids anandamide and 2-arachidonoylglicerol (2-AG) modulate dopaminergic neural activity through interactions with CB1 and CB2 receptors. CB1 antagonists inhibit the effects of drugs that potentiate dopaminergic activity, such as cocaine. There is also evidence of interactions between CB1 and CB2 receptors in terms of cannabinoid-mediated changes in dopaminergic function. We will present new evidence that CB2 receptor antagonism opposes the inhibitory effects of rimonabant on cocaine-induced hyperlocomotion. Thus, highlighting the co-modulatory role of CB1 and CB2 receptors on dopaminergic function. We will then present a new study investigating the antipsychotic mode of action of cannabidiol (CBD). CBD attenuates the psychotomimetic effects of delta-9-tetrahydrocannabinol (THC) and there is evidence that CBD has antipsychotic effects in patients with psychosis. CBD prevents a range of behavioural impairments associated with the NMDA hypofunction model of psychosis measured in the pre-pulse inhibition, social interaction and novel object recognition tests following a two week exposure to the NMDA antagonist MK801. In addition, CBD, prevented neural (measured by delta-FosB) and microglia activation, and the decreased decrease in the number of medial prefrontal parvalbumin-positive neurons. The effects of CBD were blocked by pre-treatment with the 5-HT1A receptor antagonist WAY100635. This indicates that the antipsychotic effects of CBD may be mediated via 5HT1A-mediated mechanisms.
Next, we will describe the effects of CBD on glial cells. Glial cells, which express CB1 and CB2 receptors and synthesise endocannabinoid transmitters, have been implicated in schizophrenia whereby oligodendrocyte dysfunction has been associated with white matter deficits in the illness. In an investigation of the effects of CBD on a human oligodendrocyte culture (MO3.13), CBD administration resulted in diverse changes in the expression of proteins implicated in the pathophysiology of schizophrenia. Finally, we provide further evidence that polymorphisms in cannabinoid receptor genes are associated with cognitive impairments in humans. In particular, the rs12720071 polymorphism T/T allele is associated with impaired working memory in patients with psychosis.

ENDOCANNABINOID MODULATION OF DOPAMINE NEUROTRANSMISSION
Fabricio Moreira* ,1 1 Federal University of Minas Gerais Background: Dopamine is the major neurotransmitter implicated in schizophrenia pathology. Thus, understanding the processes modulating dopaminergic signalling may lead to new insights in the biology and treatment of this disorder. The endocannabinoids anandamide and 2-arachidonoylglicerol (2-AG) modulate neural activity through interactions CB1 and CB2 receptors. CB1 antagonists inhibit the effects of drugs that facilitate dopamine activity, such as cocaine. Similar to CB1 antagonists, CB2 agonists counteract the effects of cocaine in experimental animals. However, the functions of these receptors have been investigated separately. Here we test the hypothesis that CB1 and CB2 receptors interact to ameliorate the behavioural and molecular processes altered under hyperdopaminergic states. We also sought to identify the endocannabinoid involved in these effects. Methods: Male Swiss mice received cocaine injections to increase dopamine activity in the brain. The biological responses measured were hyperlocomotion, conditioned place preference, cFos expression and Erk protein phosphorylation in the nucleus accumbens. The animals received cannabinoid-related drugs before cocaine injections. The data were analysed with ANOVA followed by the Newman-Keuls test. Results: The CB1 receptor antagonist, rimonabant, and the CB2 receptor agonist, JWH133, inhibited cocaine-induced hyperlocomotion. Moreover, the CB2 antagonist, AM630, reversed the inhibitory effects of rimonabant in cocaine-induced hyperlocomotion, cFos expression, Erk phosphorylation and conditioned place preference. The inhibitors of anandamide and 2-AG hydrolysis, URB597 (FAAH inhibitor) and JZL184 (MGL inhibitor), respectively, were ineffective in inhibiting cocaine hyperlocomotion. However, when combined with a sub-effective dose of rimonabant, JZL184 (but not URB597), inhibited cocaine effects. Discussion: A CB2 antagonist reversed the effect of a CB1 antagonist, suggesting that these receptors modulate cocaine effects in opposite ways. Accordingly, increasing brain 2-AG levels inhibited cocaine effects only if CB1 is blocked and CB2 available. Thus, selective activation of CB2 receptors warrants further investigation as a new strategy for the treatment of psychiatric disorders resulting from hyperdopaminergic states.

CANNABIDIOL AS AN ANTIPSYCHOTIC DRUG
Francisco Guimaraes* ,1 , Naielly Rodrigues 1 , Nicole Silva 1 , Felipe Gomes 2 1 School of Medicine of Ribeirao Preto-University of Sao Paulo; 2 University of Pittsburgh Background: The phytocannabinoid cannabidiol (CBD) attenuates the psychotomimetic effects produced by high doses of delta-9-tetrahydrocannabinol (THC), the main component of the Cannabis sativa plant.
Corroborating this effect, several preclinical and clinical studies indicate that CBD has antipsychotic properties. The mechanisms responsible for these properties, however, remain unknown (Campos et al., Philos Trans R Soc Lond B Biol Sci 367:3364-782013). We have recently found that repeated CBD administration prevents the behavioral impairments, measured in the pre-pulse inhibition, social interaction and novel object recognition tests, induced in mice by repeated treatment (28 days) with the NMDA receptor antagonist MK-801. CBD also prevented the neural (measured by delta-FosB) and microglia activation, and the decrease in the number of parvalbumin-positive neurons, observed in the medial prefrontal cortex (Gomes et al., Int J Neuropsychopharmacol 18(5)2014, Schizophr Res 164:155-63, 2015. Currently, we are investigating if CBD could also reverse these changes once they have been established and the possible mechanisms of this effect. Methods: Male C57BL/6J mice received intraperitoneal injections of MK-801 (0.25, 0.5 or 1 mg/kg, twice a day) for 7 or 14 days. To determine if these treatments regime would induce acute and long-term deficits, the social interaction (SI) test was performed 1 or 8 days after the end of the MK-801 treatment. Twenty-four hours after the SI, animals were submitted to the novel object recognition (NOR) test. Having established that 14 days of MK-801 induce both acute (24 h after) and long-term (8 days after) behavioral deficits in the SI and NOR tests, we investigated if repeated treatment with CBD (15, 30 or 60 mg/kg daily, i.p.) would reverse these changes. CBD treatment began 24h after the end of the MK-801 treatment and lasted for 7 days. Repeated clozapine (1 mg/kg) was used as a positive control. Forty-eight hours after the last injection, animals were submitted to SI and, 24-h later, to the NOR test. In a second experiment, independent groups of mice received, before each CBD injection, AM251 (a CB1 receptor inverse agonist, 0.1-0.3 mg/kg), AM630 (a CB2 receptor inverse agonist, 0.1-0.3 mg/kg), or the 5HT1A receptor antagonist WAY100635 (0.1-0.3 mg/kg). The data were analyzed by ANOVA followed by the Newman-Keuls test. Results: MK-801 (0.5 mg/kg) administration for 14 days, but not for 7 days, impaired SI and NOR. Repeated CBD or clozapine treatment reversed these impairments. CB1 and CB2 antagonists (AM251 and AM630, respectively) failed to change CBD effect. However, its effect was blocked by pretreatment with the 5HT1A receptor antagonist WAY100635. Discussion: Our findings show that a 2-week treatment with the NMDA receptor antagonist MK801 impairs social interaction and novel object recognition, which have been associated with negative and cognitive symptoms of schizophrenia, respectively. These behavioral deficits last for at least one week and are reversed by the atypical antipsychotic clozapine or CBD, reinforcing the proposal that this latter drug has antipsychotic-like properties. CBD effects seem to depend on facilitation of 5HT1A-mediated neurotransmission.

EFFECTS OF CANNABINOIDS ON A HUMAN OLIGODENDROCYTE CULTURE: IMPLICATIONS FOR SCHIZOPHRENIA
Valéria Almeida* ,1 , Daniel Martins-De-Souza 1 1 University of Campinas (UNICAMP) Background: Preclinical studies have suggested the involvement of the endocannabinoid system in schizophrenia pathobiology. The effects of cannabinoid drugs in several animal models for schizophrenia have been used to understand the pathobiology of the disease, and to investigate potential treatments for schizophrenia symptoms. Alterations in endocannabinoid (eCB) signaling, such as cannabinoid receptor expression and anandamide levels, have also been investigated in animal models. In addition, in vitro studies have shown the molecular pathways and biological processes associated with cannabinoids' effects in some cell types, such as glial cell cultures. Glial cells, which express cannabinoid CB1 and CB2 receptors and synthesize eCBs, have been shown to be implicated in schizophrenia. Thus, the effects of cannabinoid drugs on these cells may contribute to our knowledge about the pathobiology of schizophrenia. Specifically, oligodendrocytes are associated with white matter deficits in schizophrenia. The modulation of their function, survival, and differentiation can result in new approaches to treat schizophrenia's white matter-associated deficits. Here we have investigated the effects of cannabidiol (CBD) on a human oligodendrocyte culture (MO3.13) in terms of protein expression. Methods: MO3.13 oligodendrocytes were treated with CBD (1µM) for 8h. Proteins were extracted from these cells, digested, and processed in a stateof-the-art LC-MS/MS system. Quantitative proteomics approaches were then employed in a label-free fashion. Differentially expressed proteins among the CBD treatment and controls were analyzed using systems biology in silico tools. Results: Analyses identified that several proteins were up-or down-regulated in response to CBD treatment. These proteins were analyzed in terms of biological processes, pathways, and functions. CBD affected the expression of 136 proteins. Some proteins such as the transient receptor potential channel (TRPM7), microtubule-associated proteins (MAP2 and MAP4), Rho GTPase activating proteins (21 and 23), and calcium channel voltagedependent T type alpha 1H (CACNA1H), among others possibly involved in schizophrenia pathobiology, were increased by CBD-treatment. Discussion: Studies have shown the effects of CBD on the treatment of schizophrenia; but the mechanisms involved in its antipsychotic properties are not fully understood. Herein, we observed that CBD modulated the expression of proteins that can be implicated in schizophrenia pathobiology. For instance, MAPs functions are related to cytoskeleton organization, differentiation, and migration of oligodendrocytes. Studies have shown a decrease of MAPs in schizophrenia patients; thus, increasing MAP2 and MAP4 by CBD may be an interesting mechanism to treat and prevent cytoskeleton impairments in oligodendrocytes and neurons in schizophrenia. Moreover, CBD increased the voltage gated channel (CACNA1H) that is involved in cannabinoid retrograde signaling and glutamate and GABAergic neurotransmission. CACNA1H modulates Ca2+ levels and the synaptic vesicle cycle. To note, we also found effects of CBD on pathways and biological processes involved with schizophrenia pathobiology, such as glucose metabolism, axon guidance, and inflammation mediated by cytokine signaling. In summary, these proteomic findings may provide an integrated picture of the role of endocannabinoid signaling in oligodendrocyte cells and possible implications for schizophrenia's pathobiology.

Federal University of Minas Gerais; 2 Federal University of Rio Grande do Sul
Background: Cognition is a major determinant of functioning in patients with schizophrenia. There is evidence that the endocannabinoid system influences cognition in human subjects, and participates in the pathophysiology of schizophrenia. In a previous study, we have shown that the expression of cannabinoid receptors (CB1R and CB2R) on peripheral lymphocytes is inversely correlated with performance in the Brief Assessment of Cognition in Schizophrenia (BACS), in patients with schizophrenia. Recently, CBRs polymorphisms have been associated with an increased risk for schizophrenia, structural changes in the central nervous systems and in cognitive performance of the patients. The aim of the present study was to investigate the association between CBRs polymorphisms and cognitive performance as assessed by the BACS. Methods: A sample of 85 stable medicated patients (61% men; age = 41.6 ± 12.2 years; illness duration = 12.8 ± 10.7 years) was enrolled in this study. Two CB1R polymorphisms (rs1049353; rs12720071) and one CB2R polymorphism (rs2229579) were tested.