Cannabinoid Signaling System Research Articles

A Critical Evaluation of Terpenoid Signaling at Cannabinoid CB1 Receptors in a Neuronal Model.

In addition to phytocannabinoids, cannabis contains terpenoids that are claimed to have a myriad of effects on the body. We tested a panel of five common cannabis terpenoids, myrcene, linalool, limonene, α-pinene and nerolidol, in two neuronal models, autaptic hippocampal neurons and dorsal root ganglion (DRG) neurons. Autaptic neurons express a form of cannabinoid CB1 receptor-dependent retrograde plasticity while DRGs express a variety of transient receptor potential (TRP) channels. Most terpenoids had little or no effect on neuronal cannabinoid signaling. The exception was nerolidol, which inhibited endocannabinoid signaling. Notably, this is not via inhibition of CB1 receptors but by inhibiting some aspect of 2-arachidonoylglycerol (2-AG) production/delivery; the mechanism does not involve reducing the activity of the 2-AG-synthesizing diacylglycerol lipases (DAGLs). Nerolidol was also the only terpenoid that activated a sustained calcium response in a small (7%) subpopulation of DRGs. In summary, we found that only one of five terpenoids tested had notable effects on cannabinoid signaling in two neuronal models. Our results suggest that a few terpenoids may indeed interact with some components of the cannabinoid signaling system and may therefore offer interesting insights upon further study.

Cannabinoid CB1 receptors regulate salivation

Saliva serves multiple important functions within the body that we typically take for granted, such as helping prepare food for swallowing and defense against oral pathogens. Dry mouth is a primary symptom of Sjӧgren’s syndrome and is a side effect of many drug treatments. Cannabis users frequently report dry mouth, but the basis for this is still unknown. If the effects occur via the endogenous cannabinoid signaling system, then this may represent a novel mechanism for the regulation of salivation. We examined expression of cannabinoid CB1 receptors in submandibular salivary gland using immunohistochemistry and tested regulation of salivation by THC and cannabinoid-related ligands. We now report that CB1 receptors are expressed in the axons of cholinergic neurons innervating the submandibular gland. No staining is seen in submandibular gland epithelial cells (acinar and ductal), or myoepithelial cells (MECs). Treatment with THC (4 mg/kg, IP) or the cannabinoid receptor agonist CP55940 (0.5 mg/kg) reduced salivation in both male and female mice 1 h after treatment. CBD had no effect on its own but reversed the effect of THC in a concentration-dependent manner. Neither the CB1 receptor antagonist SR141716 (4 mg/kg) nor the CB2-selective agonist JWH133 (4 mg/kg) had an effect on salivation. We also found that fatty acid amide hydrolase (FAAH), the enzyme that metabolizes the endocannabinoid anandamide and related lipids, regulates salivation. Salivation was reduced in FAAH knockout mice as well as mice treated with the FAAH blocker URB597 (4 mg/kg). URB597 had no effect in CB1 knockout mice. FAAH protein is detected intracellularly in acinar but not ductal epithelial cells. In lipidomics experiments, we found that FAAH knockout mice chiefly had elevated levels of acylethanolamines, including anandamide, and reduced levels of acyglycines. Our results are consistent with a model wherein endocannabinoids activate CB1 receptors on cholinergic axons innervating the submandibular gland. THC likely acts by plugging into this system, activating CB1 receptors to reduce salivation, thus offering a mechanism underlying the dry mouth reported by cannabis users.

Endocannabinoid regulation of behavior in response to negative affective states associated with alcohol abstinence

Alcohol use disorder (AUD) is characterized by elevated levels of alcohol intake followed by negative affective states when individuals halt alcohol consumption. In individuals suffering from AUD, alcohol taking and seeking is often hypothesized to be motivated by negative reinforcement, where individuals continue consuming alcohol in order to avoid the negative affective states that are triggered by abstinence. Females are particularly vulnerable to the effects of AUD and are more likely to drink to regulate this negative affect produced by abstinence. The endogenous cannabinoid (eCB) signaling system is heavily implicated in the mediation of both reward and reinforcement, and alcohol consumption has been shown to be positively correlated with release of the eCB ligand 2‐arachidonoylglycerol (2‐AG) within the reward neurocircuitry. Taking this into consideration, we took a multifaceted approach to investigate the mechanisms by which individuals avoid negative affective symptoms associated with prolonged alcohol abstinence, one of the leading causes of relapse. Using a mouse model of chronic alcohol consumption followed by prolonged forced abstinence (CDFA) and pharmacological enhancement of eCB with JZL184, we were able to examine how altered eCB signaling can affect how females respond to avoid negative outcomes. First, by using negative reinforcement operant conditioning, we show that increased eCB signaling via a single injection of JZL184 causes an increase in acquisition rate as compared to ethanol‐treated and control animals. After reaching acquisition, a difference in behavioral pattern is noted; when given the chance to either escape or avoid the foot shock, JZL‐treated animals consistently chose to escape the shock rather than fully avoid it, possibly indicating a reduced sensitivity to the aversive stimulus. Next, when comparing latency to first bite in the novelty‐suppressed feeding test (NSFT), a common protocol used to measure depression and anxiety, with acquisition rate, we found that, in ethanol‐treated animals, decreased latency to eat is negatively correlated with days to acquisition. Moreover, we are able to reverse this effect with a single dose of JZL184. Together, these data indicate that a single dose of JZL184 has the ability to cause long‐lasting effects on the eCB signaling system and, consequently, the reward circuitry. Moving forward it will be critical to directly link these effects between reinforcement learning and reward processing in order to develop a pharmacological intervention to reduce the negative affective states following alcohol abstinence.

Targeting diacylglycerol lipase reduces alcohol consumption in preclinical models.

Alcohol use disorder (AUD) is associated with substantial morbidity, mortality, and societal cost, and pharmacological treatment options for AUD are limited. The endogenous cannabinoid (eCB) signaling system is critically involved in reward processing and alcohol intake is positively correlated with release of the eCB ligand 2-Arachidonoylglycerol (2-AG) within reward neurocircuitry. Here we show that genetic and pharmacological inhibition of diacylglycerol lipase (DAGL), the rate limiting enzyme in the synthesis of 2-AG, reduces alcohol consumption in a variety of preclinical models ranging from a voluntary free-access model to aversion resistant-drinking and dependence-like drinking induced via chronic intermittent ethanol vapor exposure in mice. DAGL inhibition during either chronic alcohol consumption or protracted withdrawal was devoid of anxiogenic and depressive-like behavioral effects. Lastly, DAGL inhibition also prevented ethanol-induced suppression of GABAergic transmission onto midbrain dopamine neurons, providing mechanistic insight into how DAGL inhibition could affect alcohol reward. These data suggest reducing 2-AG signaling via inhibition of DAGL could represent an effective approach to reduce alcohol consumption across the spectrum of AUD severity.

Evidence that cannabinoid CB1 receptors regulate intraocular pressure via two opposing mechanisms

The cannabinoid signaling system regulates intraocular pressure (IOP) in the mouse via a complex system that includes three receptors: CB1, GPR18 and GPR119. In each case, activating the receptor lowers IOP, but CB1 receptors are found both at sites of aqueous humor inflow and outflow. As such, knockout mice for any of these receptors would be expected to have higher-than average, or at least unchanged, intraocular pressure. The current study investigates the unexpected observation that CB1 knockout mice have lower pressure than wild type counterparts by testing various regulators of cannabinoid signaling in murine models of IOP. We now report that a CB1 antagonist has differential effects on IOP: SR141716 raises IOP in standard light cycle (SLC) but lowers IOP in reverse light cycle (RLC). This is mimicked by ABD1085, a negative allosteric modulator of CB1. CB1 inhibitors lower IOP in both normotensive and hypertensive mouse eyes. The pressure-lowering effect is absent in CB1 knockout mice. IOP rebounds after the end of treatment but shows no sign of desensitization with daily treatment for a week. Unlike the positive cannabinoid effect, antagonist effects are not sex-dependent. We propose that there are two mechanisms of action for CB1, one that lowers IOP upon activation and a second with inverse sign that lowers IOP when CB1 is antagonized. The relatively lower pressure in CB1 knockout mouse eyes suggests that this second negative regulation of IOP is dominant.

THC Regulates Tearing via Cannabinoid CB1 Receptors.

PurposeAqueous deficiency dry eye (ADDE) is a chronic condition affecting millions, with symptoms ranging from a dry itchiness to blurred vision and accompanied by an increased risk of eye infections. ADDE typically arises from disorders of the lacrimal gland that produces tears necessary for eye lubrication. Cannabis users frequently report dry eye, but the basis for this is unknown. If the effects occur via the endogenous cannabinoid signaling system, then this may represent a novel mechanism for the regulation of tearing.MethodsWe examined expression of cannabinoid CB1 receptors in the lacrimal gland using immunohistochemistry, Western blotting, and PCR and tested tetrahydrocannabinol (THC) regulation of tearing in wild-type and CB1-null mice.ResultsWe now report that CB1 receptors are expressed in the axons of cholinergic neurons innervating the lacrimal gland. Little if any staining is seen in lacrimal gland epithelial cells (acinar and ductal) or myoepithelial cells (MECs). Activation of CB1 receptors by THC or the cannabinoid agonist CP55940 reduces tearing in male mice. In female mice, THC has no effect, but CP55940 increases tearing. In both sexes, the effect of CP55940 is absent in CB1 knockout mice. CB1 mRNA and protein levels are approximately four- to fivefold higher in males than females. In male knockouts, THC increases tearing, suggesting that THC also acts through different receptors.ConclusionsOur results suggest a novel, albeit sex-dependent, physiologic basis for the dry eye symptoms experienced by cannabis users: activation of neuronal CB1 receptors in the lacrimal gland reduces tearing.

New Approaches to Cancer Therapy: Combining Fatty Acid Amide Hydrolase (FAAH) Inhibition with Peroxisome Proliferator-Activated Receptors (PPARs) Activation.

Over the course of the past decade, peroxisome proliferator-activated receptors (PPARs) have been identified as part of the cannabinoid signaling system: both phytocannabinoids and endocannabinoids are capable of binding and activating these nuclear receptors. Fatty acid amide hydrolase (FAAH) hydrolyzes the endocannabinoid anandamide and other N-acylethanolamines. These substances have been shown to have numerous anticancer effects, and indeed the inhibition of FAAH has multiple beneficial effects that are mediated by PPARα subtype and by PPARγ subtype, especially antiproliferation and activation of apoptosis. The substrates of FAAH are also PPAR agonists, which explains the PPAR-mediated effects of FAAH inhibitors. Much like cannabinoid ligands and FAAH inhibitors, PPARγ agonists show antiproliferative effects on cancer cells, suggesting that additive or synergistic effects may be achieved through the positive modulation of both signaling systems. In this Miniperspective, we discuss the development of novel FAAH inhibitors able to directly act as PPAR agonists and their promising utilization as leads for the discovery of highly effective anticancer compounds.

Cannabinoids Stimulate the TRP Channel-Dependent Release of Both Serotonin and Dopamine to Modulate Behavior in C. elegans.

Cannabis sativa alters sensory perception and exhibits potential medicinal benefits. In mammals, cannabinoids activate two canonical receptors, CB1/CB2, as well additional receptors/ion channels whose overall contributions to cannabinoid signaling have yet to be fully assessed. In Caenorhabditis elegans, the endogenous cannabinoid receptor agonist, 2-arachidonoylglycerol (2-AG) activates a CB1 ortholog, NPR-19, to modulate behavior (Oakes et al., 2017). In addition, 2-AG stimulates the NPR-19 independent release of both serotonin (5-HT) and dopamine (DA) from subsets of monoaminergic neurons to modulate locomotory behaviors through a complex monoaminergic signaling pathway involving multiple serotonin and dopamine receptors. 2-AG also inhibits locomotion in remodeled monoamine receptor mutant animals designed to measure the acute release of either 5-HT or DA, confirming the direct effects of 2-AG on monoamine release. 2-AG-dependent locomotory inhibition requires the expression of transient receptor potential vanilloid 1 (TRPV1) and TRPN-like channels in the serotonergic or dopaminergic neurons, respectively, and the acute pharmacological inhibition of the TRPV1-like channel abolishes both 2-AG-dependent 5-HT release and locomotory inhibition, suggesting the 2-AG may activate the channel directly. This study highlights the advantages of identifying and assessing both CB1/CB2-dependent and independent cannabinoid signaling pathways in a genetically tractable, mammalian predictive model, where cannabinoid signaling at the molecular/neuronal levels can be correlated directly with changes in behavior.SIGNIFICANCE STATEMENT This study is focused on assessing CB1/CB2-independent cannabinoid signaling in a genetically tractable, whole-animal model where cannabinoid signaling at the molecular/neuronal levels can be correlated with behavioral change. Caenorhabditis elegans contains a cannabinoid signaling system mediated by a canonical cannabinoid receptor, NPR-19, with orthology to human CB1/CB2 (Oakes et al., 2017). The present study has characterized an NPR-19-independent signaling pathway that involves the cannabinoid-dependent release of both serotonin and dopamine and the expression of distinct TRP-like channels on the monoaminergic neurons. Our work should be of interest to those studying the complexities of CB1/CB2-independent cannabinoid signaling, the role of TRP channels in the modulation of monoaminergic signaling, and the cannabinoid-dependent modulation of behavior.

Changes in the Peripheral Endocannabinoid System as a Risk Factor for the Development of Eating Disorders

Eating Disorder (ED) is characterized by persistently and severely disturbed eating behaviours. They arise from a combination of long-standing behavioural, emotional, psychological, interpersonal, and social factors and result in insufficient nutrient ingestion and/or adsorption. The three main EDs are: anorexia nervosa, bulimia nervosa, and binge eating disorder. We review the role of peripheral endocannabinoids in eating behaviour. The neuronal pathways involved in feeding behaviours are closely related to catecholaminergic, serotoninergic and peptidergic systems. Accordingly, feeding is promoted by serotonin, dopamine, and prostaglandin and inhibited by neuropeptide Y, norepinephrine, GABA, and opioid peptides. The endocannabinoid system plays a role in EDs, and multiple lines of evidence indicate that the cannabinoid signalling system is a key modulatory factor of the activity in the brain areas involved in EDs as well as in reward processes. Besides their central role in controlling food behaviours, peripheral cannabinoids are also involved in regulating adipose tissue and insulin signalling as well as cell metabolism in peripheral tissues such as liver, pancreas, fatty tissue, and skeletal muscle. Altogether, these data indicate that peripheral cannabinoids can provide new therapeutic targets not only for EDs but also for metabolic disease.

Enantiomer-specific positive allosteric modulation of CB1 signaling in autaptic hippocampal neurons

The cannabinoid signaling system is found throughout the CNS and its involvement in several pathological processes makes it an attractive therapeutic target. Because orthosteric CB1 cannabinoid receptor ligands have undesirable adverse effects there has been great interest in the development of allosteric modulators – both negative (NAMs) and positive (PAMs) – of these receptors. NAMs of CB1 appeared first on the scene, followed more recently by PAMs. Because allosteric modulation can vary depending on the orthosteric ligand it is important to study their function in a system that employs endogenous cannabinoids. We have recently surveyed first generation NAMs using cultured autaptic hippocampal neurons. These neurons express depolarization induced suppression of excitation (DSE), a form of synaptic plasticity that is mediated by CB1 and 2-arachidonoyl glycerol (2-AG); they are therefore an excellent neuronal model of endogenous cannabinoid signaling in which to test CB1 modulators.In this study we find that while two related compounds, GAT211 and ZCZ011, each show PAM-like responses in autaptic hippocampal neurons, they also exhibit complex pharmacology. Notably we were able to separate the PAM- and agonist-like responses of GAT211 by examining the enantiomers of this racemic compound: GAT228 and GAT229. We find that GAT229 exhibits PAM-like behavior while GAT228 appears to directly activate the CB1 receptor.Both GAT229 and ZCZ011 represent the first PAMs that we have found to be effective in using this 2-AG utilizing neuronal model system. Because these compounds may exhibit both probe selectivity and biased signaling it will be important to test them with anandamide as well as other signaling pathways.

Neuroprotective Effects of β-Caryophyllene against Dopaminergic Neuron Injury in a Murine Model of Parkinson's Disease Induced by MPTP.

Parkinson’s disease (PD) is one of the most common neurodegenerative disorders and is characterized by the loss of dopaminergic neurons in the substantia nigra (SN). Although the causes of PD are not understood, evidence suggests that its pathogenesis is associated with oxidative stress and inflammation. Recent studies have suggested a protective role of the cannabinoid signalling system in PD. β-caryophyllene (BCP) is a natural bicyclic sesquiterpene that is an agonist of the cannabinoid type 2 receptor (CB2R). Previous studies have suggested that BCP exerts prophylactic and/or curative effects against inflammatory bowel disease through its antioxidative and/or anti-inflammatory action. The present study describes the neuroprotective effects of BCP in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced murine model of PD, and we report the results of our investigation of its neuroprotective mechanism in neurons and glial cells. In the murine model, BCP pretreatment ameliorated motor dysfunction, protected against dopaminergic neuronal losses in the SN and striatum, and alleviated MPTP-induced glia activation. Additionally, BCP inhibited the levels of inflammatory cytokines in the nigrostriatal system. The observed neuroprotection and inhibited glia activation were reversed upon treatment with the CB2R selective antagonist AM630, confirming the involvement of the CB2R. These results indicate that BCP acts via multiple neuroprotective mechanisms in our murine model and suggest that BCP may be viewed as a potential treatment and/or preventative agent for PD.

Cannabinoids Activate Monoaminergic Signaling to Modulate Key C. elegans Behaviors.

Cannabis sativa, or marijuana, a popular recreational drug, alters sensory perception and exerts a range of potential medicinal benefits. The present study demonstrates that the endogenous cannabinoid receptor agonists 2-arachidonoylglycerol (2-AG) and anandamide (AEA) activate a canonical cannabinoid receptor in Caenorhabditis elegans and also modulate monoaminergic signaling at multiple levels. 2-AG or AEA inhibit nociception and feeding through a pathway requiring the cannabinoid-like receptor NPR-19. 2-AG or AEA activate NPR-19 directly and cannabinoid-dependent inhibition can be rescued in npr-19-null animals by the expression of a human cannabinoid receptor, CB1, highlighting the orthology of the receptors. Cannabinoids also modulate nociception and locomotion through an NPR-19-independent pathway requiring an α2A-adrenergic-like octopamine (OA) receptor, OCTR-1, and a 5-HT1A-like serotonin (5-HT) receptor, SER-4, that involves a complex interaction among cannabinoid, octopaminergic, and serotonergic signaling. 2-AG activates OCTR-1 directly. In contrast, 2-AG does not activate SER-4 directly, but appears to enhance SER-4-dependent serotonergic signaling by increasing endogenous 5-HT. This study defines a conserved cannabinoid signaling system in C. elegans, demonstrates the cannabinoid-dependent activation of monoaminergic signaling, and highlights the advantages of studying cannabinoid signaling in a genetically tractable whole-animal model.SIGNIFICANCE STATEMENTCannabis sativa, or marijuana, causes euphoria and exerts a wide range of medicinal benefits. For years, cannabinoids have been studied at the cellular level using tissue explants with conflicting results. To better understand cannabinoid signaling, we have used the Caenorhabditis elegans model to examine the effects of cannabinoids on behavior. The present study demonstrates that mammalian cannabinoid receptor ligands activate a conserved cannabinoid signaling system in C. elegans and also modulate monoaminergic signaling, potentially affecting an array of disorders, including anxiety and depression. This study highlights the potential role of cannabinoids in modulating monoaminergic signaling and the advantages of studying cannabinoid signaling in a genetically tractable, whole-animal model.

Two Janus Cannabinoids That Are Both CB2 Agonists and CB1 Antagonists.

The cannabinoid signaling system includes two G protein-coupled receptors, CB1 and CB2 These receptors are widely distributed throughout the body and have each been implicated in many physiologically important processes. Although the cannabinoid signaling system has therapeutic potential, the development of receptor-selective ligands remains a persistent hurdle. Because CB1 and CB2 are involved in diverse processes, it would be advantageous to develop ligands that differentially engage CB1 and CB2 We now report that GW405833 [1-(2,3-dichlorobenzoyl)-5-methoxy-2-methyl-3-[2-(4-morpholinyl)ethyl]-1H-indole] and AM1710 [1-hydroxy-9-methoxy-3-(2-methyloctan-2-yl)benzo[c]chromen-6-one], described as selective CB2 agonists, can antagonize CB1 receptor signaling. In autaptic hippocampal neurons, GW405833 and AM1710 both interfered with CB1-mediated depolarization-induced suppression of excitation, with GW405833 being more potent. In addition, in CB1-expressing human embryonic kidney 293 cells, GW405833 noncompetitively antagonized adenylyl cyclase activity, extracellular signal-regulated kinase 1/2 phosphorylation, phosphatidylinositol 4,5-bisphosphate signaling, and CB1 internalization by CP55940 (2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctan-2-yl)phenol). In contrast, AM1710 behaved as a low-potency competitive antagonist/inverse agonist in these signaling pathways. GW405833 interactions with CB1/arrestin signaling were complex: GW405833 differentially modulated arrestin recruitment in a time-dependent fashion, with an initial modest potentiation at 20 minutes followed by antagonism starting at 1 hour. AM1710 acted as a low-efficacy agonist in arrestin signaling at the CB1 receptor, with no evident time dependence. In summary, we determined that GW405833 and AM1710 are not only CB2 agonists but also CB1 antagonists, with distinctive and complex signaling properties. Thus, experiments using these compounds must take into account their potential activity at CB1 receptors.

Promising cannabinoid-based therapies for Parkinson's disease: motor symptoms to neuroprotection.

Parkinson’s disease (PD) is a slow insidious neurological disorder characterized by a loss of dopaminergic neurons in the midbrain. Although several recent preclinical advances have proposed to treat PD, there is hardly any clinically proved new therapeutic for its cure. Increasing evidence suggests a prominent modulatory function of the cannabinoid signaling system in the basal ganglia. Hence, use of cannabinoids as a new therapeutic target has been recommended as a promising therapy for PD. The elements of the endocannabinoid system are highly expressed in the neural circuit of basal ganglia wherein they bidirectionally interact with dopaminergic, glutamatergic, and GABAergic signaling systems. As the cannabinoid signaling system undergoes a biphasic pattern of change during progression of PD, it explains the motor inhibition typically observed in patients with PD. Cannabinoid agonists such as WIN-55,212-2 have been demonstrated experimentally as neuroprotective agents in PD, with respect to their ability to suppress excitotoxicity, glial activation, and oxidative injury that causes degeneration of dopaminergic neurons. Additional benefits provided by cannabinoid related compounds including CE-178253, oleoylethanolamide, nabilone and HU-210 have been reported to possess efficacy against bradykinesia and levodopa-induced dyskinesia in PD. Despite promising preclinical studies for PD, use of cannabinoids has not been studied extensively at the clinical level. In this review, we reassess the existing evidence suggesting involvement of the endocannabinoid system in the cause, symptomatology, and treatment of PD. We will try to identify future threads of research that will help in the understanding of the potential therapeutic benefits of the cannabinoid system for treating PD.

Pharmacology: Cannabis in neurology--a potted review.

Discovery of the endogenous cannabinoid signalling system unleashed substantial new research into several neurological conditions. A recent systematic review suggests that medical marijuana can improve a number of symptoms—particularly spasticity—in multiple sclerosis, but cannabinoids can have adverse psychological effects and their comparative effectiveness is unknown.

Parsing the players: 2-arachidonoylglycerol synthesis and degradation in the CNS.

The endogenous cannabinoid signalling system, composed of endogenous cannabinoids, cannabinoid receptors and the enzymes that synthesize and degrade the endogenous cannabinoids, is much more complex than initially conceptualized. 2-Arachidonoylglycerol (2-AG) is the most abundant endocannabinoid and plays a major role in CNS development and synaptic plasticity. Over the past decade, many key players in 2-AG synthesis and degradation have been identified and characterized. Most 2-AG is synthesized from membrane phospholipids via sequential activation of a phospholipase Cβ and a diacylglycerol lipase, although other pathways may contribute in specialized settings. 2-AG breakdown is more complicated with at least eight different enzymes participating. These enzymes can either degrade 2-AG into its components, arachidonic acid and glycerol, or transform 2-AG into highly bioactive signal molecules. The implications of the precise temporal and spatial control of the expression and function of these pleiotropic metabolizing enzymes have only recently come to be appreciated. In this review, we will focus on the primary organization of the synthetic and degradative pathways of 2-AG and then discuss more recent findings and their implications, with an eye towards the biological and therapeutic implications of manipulating 2-AG synthesis and metabolism. This article is part of a themed section on Cannabinoids 2013. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-6.

Cannabinoid Neuromodulation in the Adult Early Visual Cortex

Sensory processing is an active process involving the interaction of ongoing cortical activity with incoming stimulus information. However, the modulators and circuits involved in this interaction are incompletely understood. One potential candidate is the cannabinoid-signaling system, which is known to modulate the dynamics of cortical networks. Here, we show that in the primate primary and secondary visual cortices, the cannabinoid CP55940 modulates not only population dynamics but also influences the dynamics of the stimulus-response relationship of individual neurons. At the population level, CP55940 decreases EEG power, LFP power, and LFP coherence. At the single-neuron level, intrinsic spike train dynamics appear relatively unchanged, but visual receptive fields are altered: CP55940 induced an overall delay and broadening of the temporal component of V1 and V2 spatiotemporal receptive fields. Our findings provide neurophysiologic evidence for a link between cannabinoid-signaling, network dynamics and the function of a canonical cortical circuit.

The CB2-preferring agonist JWH015 also potently and efficaciously activates CB1 in autaptic hippocampal neurons

The G protein coupled receptors CB1 and CB2 are targets for the psychoactive constituents of cannabis, chief among them Δ9-THC. They are also key components of the multifunctional endogenous cannabinoid signaling system. CB1 and CB2 receptors modulate a wide variety of physiological systems including analgesia, memory, mood, reward, appetite and immunity. Identification and characterization of selective CB1 and CB2 receptor agonists and antagonists will facilitate understanding the precise physiological and pathophysiological roles of cannabinoid receptors in these systems. This is particularly necessary in the case of CB2 because these receptors are sparsely expressed and problematic to detect using traditional immunocytochemical approaches.1-Propyl-2-methyl-3-(1-naphthoyl)indole (JWH015) is an aminoalkylindole that has been employed as a “CB2-selective” agonist in more than 40 published papers. However, we have found that JWH015 potently and efficaciously activates CB1 receptors in neurons. Using murine autaptic hippocampal neurons, which express CB1, but not CB2 receptors, we find that JWH015 inhibits excitatory postsynaptic currents with an EC50 of 216nM. JWH015 inhibition is absent in neurons from CB1−/− cultures and is reversed by the CB1 antagonist, SR141716 [200nM]. Furthermore, JWH015 partially occludes CB1-mediated DSE (∼35% remaining), an action reversed by the CB2 antagonist, AM630 [1 and 3μM], suggesting that high concentrations of AM630 also antagonize CB1 receptors.We conclude that while JWH015 is a CB2-preferring agonist, it also activates CB1 receptors at experimentally encountered concentrations. Thus, CB1 agonism of JWH015 needs to be considered in the design and interpretation of experiments that use JWH015 to probe CB2-signaling.

SiRNA knockdown of GPR18 receptors in BV-2 microglia attenuates <em>N</em>-arachidonoyl glycine-induced cell migration

BackgroundNeurons are known to employ the endogenous cannabinoid system to communicate with other cells of the CNS. Endocannabioid signaling recruits microglia toward neurons by engaging cannabinoid CB2 and abnormal cannabidiol (Abn-CBD) receptors. The Abn-CBD receptor is a prominent atypical cannabinoid receptor that had been discriminated by means of various pharmacological and genetic tools but remained to be identified at the molecular level. We recently introduced N-arachidonoyl glycine (NAGly) signaling via GPR18 receptors as an important novel signaling mechanism in microglial-neuronal communication. NAGly is an endogenous, enzymatically oxygenated metabolite of the endocannabinoid N-arachidonoyl ethanolamide (AEA). Our recent studies support strongly two hypotheses; first that NAGly initiates directed microglial migration in the CNS through activation of GPR18, and second that GPR18 is the Abn-CBD receptor. Here we present siRNA knockdown data in further support of these hypotheses.FindingsA GPR18-targetting siRNA pSUPER G418 GFP cDNA plasmid was created and transfected into BV-2 microglia. Successfully transfected GFP+ GPR18 siRNA BV-2 microglia displayed reduced GPR18 mRNA levels and immunocytochemical staining. Cell migration induced by 1 μM concentrations of NAGly, O-1602 and Abn-CBD were significantly attenuated in GFP+ cells.ConclusionsOur data provide definitive evidence that these compounds, characteristic of Abn-CBD receptor pharmacology, are acting via GPR18 in BV-2 microglia. A fuller understanding of the hitherto unidentified cannabinoid receptors such as GPR18; their molecular interactions with endogenous ligands; and how phytocannabinoids influence their signaling is vital if we are to comprehensively assess the function of the endogenous cannabinoid signaling system in human health and disease.

Δ9‐Tetrahydrocannabinol and N‐arachidonyl glycine are full agonists at GPR18 receptors and induce migration in human endometrial HEC‐1B cells

Endometriosis is a disorder in which the endometrium forms growths outside the uterus and is associated with chronic pain. Recent evidence suggests that endometrial motility plays a role in the aetiology of endometriosis. The endocannabinoid system regulates cellular migration. Given the growing involvement of the endocannabinoids in reproduction, we investigated the role of the endocannabinoid system in migration of endometrial cells. Migration of the human endometrial HEC-1B cells was assayed. Standard PCR techniques were used to determine the presence of the GPCR, GPR18, in HEC-1B cells, and p44/42 MAPK was assayed in stably transfected HEK293-GPR18 cells to determine receptor specificity for known cannabinoid agonists and antagonists. N-arachidonoyl ethanolamine (AEA) metabolism was measured, using HPLC/MS/MS for lipid analysis. AEA, Δ(9) -tetrahydrocannabinol (Δ(9) -THC) and N-arachidonoyl glycine (NAGly) induce migration of HEC-1B cells through cannabinoid CB(1) receptor-independent mechanisms. MAPK activation in HEK293-GPR18 cells revealed novel pharmacology for known CB(1) and CB(2) receptor ligands at GPR18 receptors, including Δ(9) -THC, which activates MAPK at nanomolar concentrations, whereas WIN 55212-2, CP55940, JWH-133 and JWH-015, and arachidonyl-1-hydroxy-2-propylamide (R1-methanandamide) had no effect. Moreover, HEC-1B migration and MAPK activation by NAGly and Δ(9) -THC were antagonized by Pertussis toxin, AM251 and cannabidiol. An understanding of the function and regulation of GPR18 and its molecular interactions with endogenous ligands, and how phytocannabinoids play a role with GPR18 signalling is vital if we are to comprehensively assess the function of the cannabinoid signalling system in human health and disease. This article is commented on by Alexander, pp. 2411-2413 of this issue and is part of a themed section on Cannabinoids in Biology and Medicine. To view Alexander visit http://dx.doi.org/10.1111/j.1476-5381.2011.01731.x. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.