Cannabidiol is a behavioral modulator in BTBR mouse model of idiopathic autism.

Introduction: The prevalence of Autism Spectrum Disorder (ASD) has drastically risen over the last two decades and is currently estimated to affect 1 in 36 children in the U.S., according to the center for disease control (CDC). This heterogenous neurodevelopmental disorder is characterized by impaired social interactions, communication deficits, and repetitive behaviors plus restricted interest. Autistic individuals also commonly present with a myriad of comorbidities, such as attention deficit hyperactivity disorder, anxiety, and seizures. To date, a pharmacological intervention for the treatment of core autistic symptoms has not been identified. Cannabidiol (CBD), the major nonpsychoactive constituent of Cannabis sativa, is suggested to have multiple therapeutic applications, but its effect(s) on idiopathic autism is unknown. We hypothesized that CBD will effectively attenuate the autism-like behaviors and autism-associated comorbid behaviors in BTBR T+Itpr3tf/J (BTBR) mice, an established mouse model of idiopathic ASD. Methods: Male BTBR mice were injected intraperitoneally with either vehicle, 20 mg/kg CBD or 50 mg/kg CBD daily for two weeks beginning at postnatal day 21 ± 3. On the final treatment day, a battery of behavioral assays were used to evaluate the effects of CBD on the BTBR mice, as compared to age-matched, vehicle-treated C57BL/6 J mice. Results: High dose (50 mg/kg) CBD treatment attenuated the elevated repetitive self-grooming behavior and hyperlocomotion in BTBR mice. The social deficits exhibited by the control BTBR mice were rescued by the 20 mg/kg CBD treatment. Discussion: Our data indicate that different doses for CBD are needed for treating specific ASD-like behaviors. Together, our results suggest that CBD may be an effective drug to ameliorate repetitive/restricted behaviors, social deficits, and autism-associated hyperactivity.

Studies of involvement of G-protein coupled receptor-3 in cannabidiol effects on inflammatory responses of mouse primary astrocytes and microglia.

Cannabidiol (CBD) exhibits anti-inflammatory and neuroprotective properties and is suggested to be effective in the pre-clinical and clinical treatment of illnesses of the central nervous system (CNS). Two major types of CNS glial cells, astrocytes and microglia, play critical roles in the development and pathogenesis of CNS diseases. However, the mechanisms by which CBD plays an anti-inflammatory and neuroprotective role for these glial cells have not been fully elucidated. In this study, we examined the effects of CBD on the inflammatory response of mouse primary astrocytes and microglia. We also investigated whether the effect of CBD on cytokine release is mediated by the G protein coupled receptor 3 (GPR3), which was recently identified as a novel receptor for CBD. Our results showed that CBD inhibited inflammatory responses of astrocytes and microglia stimulated with lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) ligand in vitro and in vivo. In addition, CBD reduced the phosphorylation of STAT3 and NF-κB signaling pathways in LPS-stimulated astrocytes. However, the inhibitory effect of CBD on pro-inflammatory cytokine production was independent of GPR3 expression in both types of glial cells. Thus, although CBD is effective in ameliorating the activation of astrocytes and microglia, its mechanism of action still requires further study. Our data support the concept that CBD may have therapeutic potential for neurological disorders that involve neuroinflammation.

Involvement of dopamine receptor in the actions of non-psychoactive phytocannabinoids.

Nematode Caenorhabditis elegans (C. elegans) exhibited a vigorous swimming behavior in liquid medium. Addition of dopamine inhibited the swimming behavior, causing paralysis in 65% of wild-type nematodes. Interestingly, phytocannabinoids cannabidiol (CBD) or cannabidivarin (CBDV), caused paralysis in 40% of the animals. Knockout of DOP-3, the dopamine D2-like receptor critical for locomotor behavior, eliminated the paralysis induced by dopamine, CBD, and CBDV. In contrast, both CBD and CBDV caused paralysis in animals lacking CAT-2, an enzyme necessary for dopamine synthesis. Co-administration of dopamine with either CBD or CBDV caused paralysis similar to that of either phytocannabinoid treatment alone. These data support the notion that CBD and CBDV act as functional partial agonists on dopamine D2-like receptors in vivo. The discovery that dopamine receptor is involved in the actions of phytocannabinoids moves a significant step toward our understanding of the mechanisms for medical uses of cannabis in the treatment of neurological and psychiatric disorders.

Discovery of endogenous inverse agonists for G protein-coupled receptor 6.

The orphan G protein-coupled receptor 6 (GPR6) is highly expressed in the striatum and has been linked to multiple striatal pathologies. The identification of endogenous ligands and their mechanisms of action at GPR6 will help to elucidate the physiological and pathological roles of the receptor. In the current study, we tested the concentration-dependent effects of a variety of endocannabinoid-like N-acylamides on GPR6 signaling. Here, we demonstrate for the first time that N-arachidonoyl dopamine, N-docosahexaenoyl dopamine, N-oleoyl dopamine and N-palmitoyl dopamine exert inverse agonism at GPR6. This effect was concentration-dependent, with potencies in the micromolar range, and functionally selective for ß-arrestin2 recruitment. Structure-activity relationship studies demonstrate that both the N-acyl side chain and the dopamine head group are important for these ligands to act on GPR6. Our discovery of these N-acyl dopamines as endogenous inverse agonists for GPR6 moves us one step further in understanding the roles GPR6 play in neurodegenerative and neuropsychiatric disorders related to striatal dysfunction.

GPR3, GPR6, and GPR12 as novel molecular targets: their biological functions and interaction with cannabidiol.

The G protein-coupled receptors 3, 6, and 12 (GPR3, GPR6, and GPR12) comprise a family of closely related orphan receptors with no confirmed endogenous ligands. These receptors are constitutively active and capable of signaling through G protein-mediated and non-G protein-mediated mechanisms. These orphan receptors have previously been reported to play important roles in many normal physiological functions and to be involved in a variety of pathological conditions. Although they are orphans, GPR3, GPR6, and GPR12 are phylogenetically most closely related to the cannabinoid receptors. Using ß-arrestin2 recruitment and cAMP accumulation assays, we recently found that the nonpsychoactive phytocannabinoid cannabidiol (CBD) is an inverse agonist for GPR3, GPR6, and GPR12. This discovery highlights these orphan receptors as potential new molecular targets for CBD, provides novel mechanisms of action, and suggests new therapeutic uses of CBD for illnesses such as Alzheimer's disease, Parkinson's disease, cancer, and infertility. Furthermore, identification of CBD as a new inverse agonist for GPR3, GPR6, and GPR12 provides the initial chemical scaffolds upon which potent and efficacious agents acting on these receptors can be developed, with the goal of developing chemical tools for studying these orphan receptors and ultimately new therapeutic agents.

Novel inverse agonists for the orphan G protein-coupled receptor 6.

The orphan G protein-coupled receptor 6 (GPR6) displays unique promise as a therapeutic target for the treatment of neuropsychiatric disorders due to its high expression in the striatopallidal neurons of the basal ganglia. GPR6, along with closely related orphan receptors GPR3 and GPR12, are phylogenetically related to CB1 and CB2 cannabinoid receptors. In the current study, we performed concentration-response studies on the effects of three different classes of cannabinoids: endogenous, phyto-, and synthetic, on both GPR6-mediated cAMP accumulation and ß-arrestin2 recruitment. In addition, structure-activity relationship studies were conducted on cannabidiol (CBD), a recently discovered inverse agonist for GPR6. We have identified four additional cannabinoids, cannabidavarin (CBDV), WIN55212-2, SR141716A and SR144528, that exert inverse agonism on GPR6. Furthermore, we have discovered that these cannabinoids exhibit functional selectivity toward the ß-arrestin2 recruitment pathway. These novel, functionally selective inverse agonists for GPR6 can be used as research tools and potentially developed into therapeutic agents.