Effect of cannabidiol on endocannabinoid, glutamatergic and GABAergic signalling markers in male offspring of a maternal immune activation (poly I:C) model relevant to schizophrenia

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Abstract

The mainstay treatment for schizophrenia is antipsychotic drugs (APDs), which are mostly effective against the positive symptoms (e.g. hallucinations), but provide minimal benefits for the negative symptoms (e.g. social withdrawal) and cognitive deficits. We have recently shown that treatment with the non-intoxicating phytocannabinoid, cannabidiol (CBD), can improve cognition and social interaction deficits in a maternal immune activation (MIA) model relevant to the aetiology of schizophrenia, however, the mechanisms underlying this effect are unknown. An imbalance in the main excitatory (glutamate) and inhibitory (GABA) neurotransmitter systems in the brain plays a role in the pathophysiology of schizophrenia. Therefore, the endocannabinoid system could represent a therapeutic target for schizophrenia as a regulator of glutamate and GABA release via the CB1 receptor (CB1R). This study investigated the effects of chronic CBD treatment on markers of glutamatergic, GABAergic and endocannabinoid signalling in brain regions implicated in social behaviour and cognitive function, including the prefrontal cortex (PFC) and hippocampus (HPC). Time-mated pregnant Sprague-Dawley rats (n = 16) were administered poly I:C (4 mg/kg, i.v.) or saline (control) on gestational day 15. Male offspring were injected with CBD (10 mg/kg, i.p.) or vehicle twice daily from postnatal day 56 for 3 weeks. The prefrontal cortex (PFC) and hippocampus (HPC) were collected for post-mortem receptor binding and Western blot analyses (n = 8 per group). CBD treatment attenuated poly I:C-induced deficits in cannabinoid CB1 receptor binding in the PFC and glutamate decarboxylase 67, the enzyme that converts glutamate to GABA, in the HPC. CBD treatment increased parvalbumin levels in the HPC, regardless of whether offspring were exposed to poly I:C in utero. Conversely, CBD did not affect N-methyl-d-aspartate receptor and gamma-aminobutyric acid (GABA) A receptor binding or protein levels of fatty acid amide hydrolase, the enzyme that degrades the endocannabinoid, anandamide. Overall, these findings show that CBD can restore cannabinoid/GABAergic signalling deficits in regions of the brain implicated in schizophrenia pathophysiology following maternal poly I:C exposure. These findings provide novel evidence for the potential mechanisms underlying the therapeutic effects of CBD treatment in the poly I:C model.

Introduction

The prefrontal cortex (PFC) and hippocampus (HPC) are anatomically connected and synchrony between the two regions in important for normal brain function (Li et al., 2015). However, people with schizophrenia exhibit structural abnormalities in these brain regions, including reduced hippocampal volume and cortical thinning in the PFC (Dietsche et al., 2017; van Erp et al., 2016). In addition, patients show abnormal activity in the default mode network (i.e. the network of brain regions that are active at rest) and during memory tasks (e.g. evidenced by reduced gamma oscillations), suggesting that dysfunction in these regions may underlie the symptomatology of the disorder, particularly the negative and cognitive symptom domains (Gonzalez-Burgos and Lewis, 2012; Guo et al., 2017). Unfortunately, the negative and cognitive symptoms tend to precede the onset of psychosis, are persistent over the course of the disorder, and are associated with poor functional outcomes in patients (Lindenmayer et al., 2013; Barch and Ceaser, 2012). Although antipsychotic drugs (APDs) are generally effective at controlling the positive symptoms of schizophrenia (e.g. hallucinations and delusions), the drugs have poor efficacy against the negative symptoms (e.g. social withdrawal) (Lindenmayer et al., 2013) and cognitive deficits of schizophrenia (Goff et al., 2011), and in some cases can worsen cognition (Hill et al., 2010). Despite the introduction of newer ‘second generation’ APDs to the market, this drug class does not exhibit superior efficacy to ‘first generation’ APDs (reviewed in MacKenzie et al., 2018). Therefore, new therapeutic options that can address the negative and cognitive symptom domains of schizophrenia are required.

Cannabidiol (CBD), the main non-intoxicating phytocannabinoid found in the cannabis plant, has the potential to alleviate symptoms across a range of pathological conditions, including epilepsy, chronic pain, anxiety and movement disorders (e.g. Parkinson's disease) (Crippa et al., 2018; Osborne et al., 2017b). However, there are limited studies that have investigated the chronic effects of CBD treatment in schizophrenia. CBD (800 mg, 4 weeks) significantly improved symptoms (measured on the Positive and Negative Syndrome Scale (PANSS)) in acute paranoid schizophrenia patients in a manner comparable to amisulpride, but had a more favourable side effect profile (e.g. less body weight gain) (Leweke et al., 2012). Recent clinical trials have explored the therapeutic potential of CBD as an adjunct to existing APD medications of stable schizophrenia outpatients. After 6 weeks of treatment, PANSS scores of the CBD-treated group (1000 mg/day, 6 weeks) significantly improved compared to placebo, while cognition improved from baseline (assessed using Brief Assessment of Cognition in Schizophrenia), but fell short of statistical significance against placebo (p = 0.068) (McGuire et al., 2018). In contrast, a similar trial that used a lower dose of CBD (600 mg/day, 6 weeks) found no improvement on PANSS or the MATRICS Consensus Cognitive Battery compared to baseline (Boggs et al., 2018), suggesting that dosage may be a critical factor for the efficacy of CBD in schizophrenia, particularly in APD-treated patients. Our laboratory recently reported that CBD treatment improved cognition and social interaction in adult male rat offspring exposed to maternal immune activation (MIA) using polyinosinic-polycytidilic (poly I:C) acid (Osborne et al., 2017a), which mimics some of the positive and negative symptoms, and cognitive deficits observed in schizophrenia (Meyer and Feldon, 2012). However, the mechanisms by which CBD improves negative and cognitive phenotypes in poly I:C offspring is unknown.

The endogenous cannabinoid (eCB) system plays an important role in various physiological functions, including neuroprotection, synaptic plasticity, memory and reward processing. The cannabinoid CB1 receptor (CB1R) is the main receptor of the eCB system in the brain with high expression levels in regions involved in cognitive function, including the PFC and HPC (Lu and Mackie, 2016). Multiple studies have implicated eCB system dysregulation in the pathophysiology of schizophrenia, including alterations in CB1R expression (reviewed in Ferretjans et al., 2012), its endogenous ligand anandamide (AEA) (De Marchi et al., 2003; Giuffrida et al., 2004; Koethe et al., 2009; Leweke et al., 1999), and fatty acid amide hydrolase (FAAH), the enzyme primarily responsible for the degradation of AEA (Bioque et al., 2013; Takata et al., 2013). The eCB system is functionally linked to the major excitatory (glutamatergic) and inhibitory (GABAergic) neurotransmitter systems in the brain, as endogenous cannabinoids bind to the CB1R and dampen presynaptic glutamate and/or GABA release (Viveros et al., 2012). An imbalance in glutamatergic and GABAergic signalling in the brain has been implicated in the cognitive deficits of schizophrenia (Gonzalez-Burgos and Lewis, 2012). Post-mortem schizophrenia studies report alterations in the ionotropic glutamatergic N-methyl-d-aspartate receptor (NMDAR) and its obligatory GluN1 subunit (Catts et al., 2016), and gamma-aminobutyric acid alpha receptor (GABAAR) (Gonzalez-Burgos and Lewis, 2008), as well as a reduction in glutamate decarboxylase 67 (GAD67; the rate-limiting enzyme that converts glutamate to GABA) and the calcium binding protein parvalbumin (PV), expressed on GABAergic interneurons (Cohen et al., 2015). The interaction of CBD with the eCB, glutamatergic and GABAergic systems is not well understood. CBD blocks the effects of CB1R/CB2 combined receptor agonists (McPartland et al., 2015; Pertwee, 2008; Thomas et al., 2007), acting as a CB1R negative allosteric modulator (NAM) in vitro (Laprairie et al., 2015) and may increase AEA levels by inhibiting FAAH activity (Leweke et al., 2012). Studies show that CBD can prevent behavioural and neurochemical deficits induced by NMDAR antagonism (Gomes et al., 2015a; Gururajan et al., 2012), and can act as a positive allosteric modulator of the GABAAR to increase inhibitory tone (Bakas et al., 2017). Therefore, the aim of this study was to examine the effects of CBD treatment on eCB (CB1R binding density and FAAH protein levels), as well as glutamatergic (NMDAR binding and subunit levels) and GABAergic (GABAAR binding density, GAD67 and PV protein levels) markers in the brains of poly I:C offspring that exhibit cognitive deficits.

Section snippets

Ethics statement

Experimental procedures were approved by the Animal Ethics Committee of the University of Wollongong, NSW, Australia (AE15/05) and complied with the National Health and Medical Research Council (NHMRC), Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (NHMRC, 2013). All efforts were made to minimise the number and suffering of animals.

Animal experiments

The detailed methods used for the animal experiments have been reported previously (Osborne et al., 2017a) in accordance with

Results

To determine the potential mechanisms underlying the beneficial effects of CBD, eCB, NMDAR and GABAergic markers were analysed in the brains of control and poly I:C offspring. Representative autoradiographs of CB1R ([3H]SR141716A), NMDAR ([3H]MK-801) and GABAAR ([3H]Muscimol) total binding density (as well as non-specific binding for the respective ligands) in the PFC and HPC are shown (Fig. 1). There was no difference in receptor binding density along the dorsal-ventral axis of the HPC

Discussion

We have previously shown that CBD treatment restored working and recognition memory, as well as social interaction deficits in male poly I:C offspring (Osborne et al., 2017a), however, the effects of CBD treatment on brain neurochemistry had not been characterised. The present study examined the effect of CBD treatment on eCB, glutamatergic and GABAergic markers in the PFC and HPC of male poly I:C offspring. We have shown that CBD treatment restored poly I:C-induced deficits in CB1R binding

Conclusions

In the present study, CBD treatment reversed deficits in CB1R binding density in the PFC and hippocampal GAD67 protein levels in male poly I:C offspring. CBD also increased hippocampal PV levels regardless of in utero poly I:C exposure. CBD had no effect on FAAH protein levels, NMDAR or GABAAR binding density in either brain region examined, however, poly I:C offspring did not exhibit deficits in these markers. This is the first study to characterise the neurochemical changes that occur

Role of funding source

This work was supported by a University of Wollongong Faculty of Science, Medicine and Health Advancement Grant (2015/SPGA-S/02) awarded to KWG and XFH, Centre for Medical and Molecular Biosciences funding awarded to KWG, and a Daniel Beck Memorial Award from Neuroscience Research Australia and the Beck family awarded to ALO. ALO and JSL were supported by an Australian Government Research Training Program Scholarship from the University of Wollongong. IB is supported by a Postgraduate Research

Contributors

ALO, KWG, NS and XFH designed the study; ALO, JSL, IB and KWG performed the experiments; XFH and KAN provided some experimental reagents; ALO analysed the data and wrote the first draft of the manuscript; JSL, KWG, KAN, NS contributed to the interpretation of the data and final manuscript. All authors have approved the final manuscript.

Declaration of interest

The authors declare no conflict of interest.

Ethics statement

Experimental procedures were approved by the Animal Ethics Committee of the University of Wollongong, NSW, Australia (AE15/05) and complied with the National Health and Medical Research Council (NHMRC), Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (NHMRC, 2013). All efforts were made to minimise the number and suffering of animals.

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