Cannabinoid Signaling Research Articles

Cannabinoid Cancer Biology and Prevention.

Plant-based, synthetic, and endogenous cannabinoids have been shown to control a diverse array of biological processes, including regulation of cell fate across cancers. Their promise as broad-based antitumor agents in preclinical models has led to the initiation of pilot clinical trials. Session 5 of the National Cancer Institute's Cannabis, Cannabinoids and Cancer Research Symposium provides an overview of this research topic. Overall, the presentations highlight cannabinoid signal transduction and specific molecular mechanisms underlying cannabinoid antitumor activity. They also demonstrate the broad-based antitumor activity of the plant-based, synthetic, and endogenous cannabinoid compounds. Importantly, evidence is presented demonstrating when cannabinoids may be contraindicated as a treatment for cancer, as in the case of human papilloma virus-meditated oropharynx cancer or potentially other p38 MAPK pathway-driven cancers. Finally, it is discussed that a key to advancing cannabinoids into the clinic is to conduct well-designed, large-scale clinical trials to determine whether cannabinoids are effective antitumor agents in cancer patients.

Systematic Review of Systemic and Neuraxial Effects of Acetaminophen in Preclinical Models of Nociceptive Processing.

Acetaminophen (APAP) in humans has robust effects with a high therapeutic index in altering postoperative and inflammatory pain states in clinical and experimental pain paradigms with no known abuse potential. This review considers the literature reflecting the preclinical actions of acetaminophen in a variety of pain models. Significant observations arising from this review are as follows: 1) acetaminophen has little effect upon acute nociceptive thresholds; 2) acetaminophen robustly reduces facilitated states as generated by mechanical and thermal hyperalgesic end points in mouse and rat models of carrageenan and complete Freund’s adjuvant evoked inflammation; 3) an antihyperalgesic effect is observed in models of facilitated processing with minimal inflammation (eg, phase II intraplantar formalin); and 4) potent anti-hyperpathic effects on the thermal hyperalgesia, mechanical and cold allodynia, allodynic thresholds in rat and mouse models of polyneuropathy and mononeuropathies and bone cancer pain. These results reflect a surprisingly robust drug effect upon a variety of facilitated states that clearly translate into a wide range of efficacy in preclinical models and to important end points in human therapy. The specific systems upon which acetaminophen may act based on targeted delivery suggest both a spinal and a supraspinal action. Review of current targets for this molecule excludes a role of cyclooxygenase inhibitor but includes effects that may be mediated through metabolites acting on the TRPV1 channel, or by effect upon cannabinoid and serotonin signaling. These findings suggest that the mode of action of acetaminophen, a drug with a long therapeutic history of utilization, has surprisingly robust effects on a variety of pain states in clinical patients and in preclinical models with a good therapeutic index, but in spite of its extensive use, its mechanisms of action are yet poorly understood.

Paracetamol (Acetaminophen) and the Developing Brain.

Paracetamol is commonly used to treat fever and pain in pregnant women, but there are growing concerns that this may cause attention deficit hyperactivity disorder and autism spectrum disorder in the offspring. A growing number of epidemiological studies suggests that relative risks for these disorders increase by an average of about 25% following intrauterine paracetamol exposure. The data analyzed point to a dose–effect relationship but cannot fully account for unmeasured confounders, notably indication and genetic transmission. Only few experimental investigations have addressed this issue. Altered behavior has been demonstrated in offspring of paracetamol-gavaged pregnant rats, and paracetamol given at or prior to day 10 of life to newborn mice resulted in altered locomotor activity in response to a novel home environment in adulthood and blunted the analgesic effect of paracetamol given to adult animals. The molecular mechanisms that might mediate these effects are unknown. Paracetamol has diverse pharmacologic actions. It reduces prostaglandin formation via competitive inhibition of the peroxidase moiety of prostaglandin H2 synthase, while its metabolite N-arachidonoyl-phenolamine activates transient vanilloid-subtype 1 receptors and interferes with cannabinoid receptor signaling. The metabolite N-acetyl-p-benzo-quinone-imine, which is pivotal for liver damage after overdosing, exerts oxidative stress and depletes glutathione in the brain already at dosages below the hepatic toxicity threshold. Given the widespread use of paracetamol during pregnancy and the lack of safe alternatives, its impact on the developing brain deserves further investigation.

RNA profiling of laser microdissected human trophoblast subtypes at mid-gestation reveals a role for cannabinoid signaling in invasion.

ABSTRACTHuman placental architecture is complex. Its surface epithelium, specialized for transport, forms by fusion of cytotrophoblast progenitors into multinucleated syncytiotrophoblasts. Near the uterine surface, these progenitors assume a different fate, becoming cancer-like cells that invade its lining and blood vessels. The latter process physically connects the placenta to the mother and shunts uterine blood to the syncytiotrophoblasts. Isolation of trophoblast subtypes is technically challenging. Upon removal, syncytiotrophoblasts disintegrate and invasive cytotrophoblasts are admixed with uterine cells. We used laser capture to circumvent these obstacles. This enabled isolation of syncytiotrophoblasts and two subpopulations of invasive cytotrophoblasts from cell columns and the endovascular compartment of spiral arteries. Transcriptional profiling revealed numerous genes, the placental or trophoblast expression of which was not known, including neurotensin and C4ORF36. Using mass spectrometry, discovery of differentially expressed mRNAs was extended to the protein level. We also found that invasive cytotrophoblasts expressed cannabinoid receptor 1. Unexpectedly, screening agonists and antagonists showed that signals from this receptor promote invasion. Together, these results revealed previously unseen gene expression patterns that translate to the protein level. Our data also suggested that endogenous and exogenous cannabinoids can affect human placental development.

In Case You Haven't Heard…

In “Hepatic cannabinoid signaling in the regulation of alcohol‐associated liver disease,” a study released by the National Institute on Alcohol Abuse and Alcoholism last month, researchers describe the role of the endocannabinoid (naturally occurring) system as a regulatory pathway in the pathophysiology of alcohol‐associated liver disease (ALD). Because of psychoactive side effects, pharmacological applications have not been successful. However, looking more closely at this system may help in the treatment of ALD, according to the study.

Alternative Splicing Mechanisms Underlying Opioid-Induced Hyperalgesia.

Prolonged use of opioids can cause opioid-induced hyperalgesia (OIH). The impact of alternative splicing on OIH remains partially characterized. A study of the absolute and relative modes of action of alternative splicing further the understanding of the molecular mechanisms underlying OIH. Differential absolute and relative isoform profiles were detected in the trigeminal ganglia and nucleus accumbens of mice presenting OIH behaviors elicited by chronic morphine administration relative to control mice. Genes that participate in glutamatergic synapse (e.g., Grip1, Grin1, Wnk3), myelin protein processes (e.g., Mbp, Mpz), and axon guidance presented absolute and relative splicing associated with OIH. Splicing of genes in the gonadotropin-releasing hormone receptor pathway was detected in the nucleus accumbens while splicing in the vascular endothelial growth factor, endogenous cannabinoid signaling, circadian clock system, and metabotropic glutamate receptor pathways was detected in the trigeminal ganglia. A notable finding was the prevalence of alternatively spliced transcription factors and regulators (e.g., Ciart, Ablim2, Pbx1, Arntl2) in the trigeminal ganglia. Insights into the nociceptive and antinociceptive modulatory action of Hnrnpk were gained. The results from our study highlight the impact of alternative splicing and transcriptional regulators on OIH and expose the need for isoform-level research to advance the understanding of morphine-associated hyperalgesia.

Cannabinoid and planar cell polarity signaling converges to direct placentation

Directed trophoblast migration toward the maternal mesometrial pole is critical for placentation and pregnancy success. Trophoblasts replace maternal arterial endothelial cells to increase blood supply to the placenta. Inferior trophoblast invasion results in pregnancy complications including preeclampsia, intrauterine growth restriction, miscarriage, and preterm delivery. The maternal chemotactic factors that direct trophoblast migration and the mechanism by which trophoblasts respond to these factors are not clearly understood. Here, we show that invasive trophoblasts deficient in Vangl2, a core planar cell polarity (PCP) component, fail to invade in maternal decidua, and this deficiency results in middle-gestational fetal demise. Previously, we have shown that tightly regulated endocannabinoids via G protein-coupled cannabinoid receptor CB1 are critical to the invasion of trophoblasts called spiral artery trophoblast giant cells (SpA-TGCs). We find that CB1 directly interacts with VANGL2. Trophoblast stem cells devoid of Cnr1 and/or Vangl2 show compromised cell migration. To study roles of VANGL2 and CB1 in trophoblast invasion in vivo, we conditionally deleted Cnr1 (coding CB1) and Vangl2 in progenitors of SpA-TGCs using trophoblast-specific protein alpha (Tpbpa)-Cre. We observed that signaling mediated by VANGL2 and CB1 restrains trophoblasts from random migration by keeping small GTPases quiescent. Our results show that organized PCP in trophoblasts is indispensable for their directed movement and that CB1 exerts its function by direct interaction with membrane proteins other than its canonical G protein-coupled receptor role.

Brief Report: Suspected Cannabis-Induced Mania and Psychosis in Young Adult Males with Autism Spectrum Disorder.

There is increasing interest in investigating cannabis for behavioral symptoms in individuals with autism spectrum disorder (ASD). The potential role of dysregulated cannabinoid signaling contributing to the pathophysiology of ASD is an area of active investigation. Results from retrospective and uncontrolled trials of cannabis in subjects with ASD have been published, reporting both potential benefits and adverse effects. Here, we describe the clinical course of three young adult males with ASD who developed mania or psychosis after the consistent use of cannabidiol and delta-9-tetrahydrocannabinol. Caution should be utilized with cannabis use in individuals with ASD until large-scale, replicated randomized controlled trials demonstrating efficacy, safety and tolerability have been published.

An Evaluation of Understudied Phytocannabinoids and Their Effects in Two Neuronal Models.

Cannabis contains more than 100 phytocannabinoids. Most of these remain poorly characterized, particularly in neurons. We tested a panel of five phytocannabinoids—cannabichromene (CBC), cannabidiolic acid (CBDA), cannabidivarin (CBDV), cannabidivarinic acid (CBDVA), and Δ9-tetrahydrocannabivarin (THCV) in two neuronal models, autaptic hippocampal neurons and dorsal root ganglion (DRG) neurons. Autaptic neurons expressed a form of CB1-dependent retrograde plasticity while DRGs expressed a variety of transient receptor potential (TRP) channels. CBC, CBDA, and CBDVA had little or no effect on neuronal cannabinoid signaling. CBDV and THCV differentially inhibited cannabinoid signaling. THCV inhibited CB1 receptors presynaptically while CBDV acted post-synaptically, perhaps by inhibiting 2-AG production. None of the compounds elicited a consistent DRG response. In summary, we find that two of five ‘minor’ phytocannabinoids tested antagonized CB1-based signaling in a neuronal model, but with very different mechanisms. Our findings highlight the diversity of potential actions of phytocannabinoids and the importance of fully evaluating these compounds in neuronal models.

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.

The Impact of Early Life Exposure to Cannabis: The Role of the Endocannabinoid System.

Cannabis use during pregnancy has continued to rise, particularly in developed countries, as a result of the trend towards legalization and lack of consistent, evidence-based knowledge on the matter. While there is conflicting data regarding whether cannabis use during pregnancy leads to adverse outcomes such as stillbirth, preterm birth, low birthweight, or increased admission to neonatal intensive care units, investigations into long-term effects on the offspring’s health are limited. Historically, studies have focused on the neurobehavioral effects of prenatal cannabis exposure on the offspring. The effects of cannabis on other physiological aspects of the developing fetus have received less attention. Importantly, our knowledge about cannabinoid signaling in the placenta is also limited. The endocannabinoid system (ECS) is present at early stages of development and represents a potential target for exogenous cannabinoids in utero. The ECS is expressed in a broad range of tissues and influences a spectrum of cellular functions. The aim of this review is to explore the current evidence surrounding the effects of prenatal exposure to cannabinoids and the role of the ECS in the placenta and the developing fetus.

Identification of BiP as a CB1 Receptor-Interacting Protein That Fine-Tunes Cannabinoid Signaling in the Mouse Brain.

Cannabinoids, the bioactive constituents of cannabis, exert a wide array of effects on the brain by engaging Type 1 cannabinoid receptor (CB1R). Accruing evidence supports that cannabinoid action relies on context-dependent factors, such as the biological characteristics of the target cell, suggesting that cell population-intrinsic molecular cues modulate CB1R-dependent signaling. Here, by using a yeast two-hybrid-based high-throughput screening, we identified BiP as a potential CB1R-interacting protein. We next found that CB1R and BiP interact specifically in vitro, and mapped the interaction site within the CB1R C-terminal (intracellular) domain and the BiP C-terminal (substrate-binding) domain-α. BiP selectively shaped agonist-evoked CB1R signaling by blocking an “alternative” Gq/11 protein-dependent signaling module while leaving the “classical” Gi/o protein-dependent inhibition of the cAMP pathway unaffected. In situ proximity ligation assays conducted on brain samples from various genetic mouse models of conditional loss or gain of CB1R expression allowed to map CB1R-BiP complexes selectively on terminals of GABAergic neurons. Behavioral studies using cannabinoid-treated male BiP+/− mice supported that CB1R-BiP complexes modulate cannabinoid-evoked anxiety, one of the most frequent undesired effects of cannabis. Together, by identifying BiP as a CB1R-interacting protein that controls receptor function in a signaling pathway- and neuron population-selective manner, our findings may help to understand the striking context-dependent actions of cannabis in the brain.SIGNIFICANCE STATEMENT Cannabis use is increasing worldwide, so innovative studies aimed to understand its complex mechanism of neurobiological action are warranted. Here, we found that cannabinoid CB1 receptor (CB1R), the primary molecular target of the bioactive constituents of cannabis, interacts specifically with an intracellular protein called BiP. The interaction between CB1R and BiP occurs selectively on terminals of GABAergic (inhibitory) neurons, and induces a remarkable shift in the CB1R-associated signaling profile. Behavioral studies conducted in mice support that CB1R-BiP complexes act as fine-tuners of anxiety, one of the most frequent undesired effects of cannabis use. Our findings open a new conceptual framework to understand the striking context-dependent pharmacological actions of cannabis in the brain.

Cannabinoid signaling promotes the de-differentiation and proliferation of Müller glia-derived progenitor cells.

Endocannabinoids (eCB) are lipid-based neurotransmitters that are known to influence synaptic function in the visual system. eCBs are also known to suppress neuroinflammation in different pathological states. However, nothing is known about the roles of the eCB system during the transition of Müller glia (MG) into proliferating progenitor-like cells in the retina. Accordingly, we used the chick and mouse model to characterize expression patterns of eCB-related genes and applied pharmacological agents to investigate how the eCB system impacts glial reactivity and the capacity of MG to become Müller glia-derived progenitor cells (MGPCs). We queried single cell RNA-seq libraries to identify eCB-related genes and identify cells with dynamic patterns of expression in damaged retinas. MG and inner retinal neurons expressed the eCB receptor CNR1, as well as enzymes involved in eCB metabolism. In the chick, intraocular injections of cannabinoids, 2-Arachidonoylglycerol (2-AG) and Anandamide (AEA), stimulated the formation of MGPCs. Cannabinoid Receptor 1 (CNR1)-agonists and Monoglyceride Lipase-inhibitor promoted the formation of MGPCs, whereas CNR1-antagonist and inhibitors of eCB synthesis suppressed this process. In damaged mouse retinas where MG activate NFkB-signaling, activation of CNR1 decreased and inhibition of CNR1 increased NFkB, whereas levels of neuronal cell death were unaffected. Surprisingly, retinal microglia were largely unaffected by increases or decreases in eCB-signaling in both chick and mouse retinas. We conclude that the eCB system in the retina influences the reactivity of MG and the formation of proliferating MGPCs, but does not influence the reactivity of immune cells in the retina.

Molecular signaling of synthetic cannabinoids: Comparison of CB1 receptor and TRPV1 channel activation

Recreational use of synthetic cannabinoids (SCs) is associated with desirable euphoric and relaxation effects as well as adverse effects including anxiety, agitation and psychosis. These SC-mediated actions represent a combination of potentiated cannabinoid receptor signaling and “off-target” receptor activity. The goal of this study was to compare the efficacy of various classes of SCs in stimulating CB1 receptors and activating “off-target” transient receptor potential (TRP) channels. Cannabinoid-type 1 (CB1) receptor activity was determined by measuring SC activation of G protein-gated inward rectifier K+ (GIRK) channels using a membrane potential-sensitive fluorescent dye assay. SC opening of vanilloid type-1 (TRPV1) channels was measured by recording intracellular Ca2+ transients. All of the SCs tested activated the GIRK channel with an efficacy of 4-fluoro MDMB-BUTINACA > 5-fluoro MDMB-PICA > MDMB-4en-PINACA ≈ WIN 55,212-2 > AB-FUBINACA > AM1220 ≈ JWH-122 N-(5-chloropentyl) > AM1248 > JWH-018 ≈ XLR-11 ≈ UR-144. The potency of the SCs at the CB1 receptor was 5-fluoro MDMB-PICA ≈ 4-fluoro MDMB-BUTINACA > AB-FUBINACA ≈ MDMB-4en-PINACA > JWH-018 > AM1220 > XLR-11 > JWH-122 N-(5-chloropentyl) > WIN 55,212-2 ≈ UR-144 > AM1248. In contrast, when tested at a SC concentration that produced a maximal effect on the Gi/GIRK channel, only XLR-11, UR-144 and AM1220 caused a significant activation of the TRPV1 channels. The TRPV1 channel/Ca2+ signal measured during application of 10 μM XLR-11 was similar to the signal induced by the endocannabinoid N-arachidonoylethanolamine (AEA). Thus, while various SCs share the ability to stimulate CB1 receptor/Gi signaling, they display limited efficacy in opening TRPV1 channels.

Glutamate Delta-1 Receptor Regulates Inhibitory Neurotransmission in the Nucleus Accumbens Core and Anxiety-Like Behaviors.

Glutamate delta-1 receptor (GluD1) is a member of the ionotropic glutamate receptor family expressed at excitatory synapses and functions as a synaptogenic protein by interacting with presynaptic neurexin. We have previously shown that GluD1 plays a role in the maintenance of excitatory synapses in a region-specific manner. Loss of GluD1 leads to reduced excitatory neurotransmission in medium spiny neurons (MSNs) in the dorsal striatum, but not in the ventral striatum (both core and shell of the nucleus accumbens (NAc)). Here, we found that GluD1 loss leads to reduced inhibitory neurotransmission in MSNs of the NAc core as evidenced by a reduction in the miniature inhibitory postsynaptic current frequency and amplitude. Presynaptic effect of GluD1 loss was further supported by an increase in paired pulse ratio of evoked inhibitory responses indicating reduced release probability. Furthermore, analysis of GAD67 puncta indicated a reduction in the number of putative inhibitory terminals. The changes in mIPSC were independent of cannabinoid or dopamine signaling. A role of feed-forward inhibition was tested by selective ablation of GluD1 from PV neurons which produced modest reduction in mIPSCs. Behaviorally, local ablation of GluD1 from NAc led to hypolocomotion and affected anxiety- and depression-like behaviors. When GluD1 was ablated from the dorsal striatum, several behavioral phenotypes were altered in opposite manner compared to GluD1 ablation from NAc. Our findings demonstrate that GluD1 regulates inhibitory neurotransmission in the NAc by a combination of pre- and postsynaptic mechanisms which is critical for motor control and behaviors relevant to neuropsychiatric disorders.

Cannabinoid Receptor 1 associates to different molecular complexes during GABAergic neuron maturation.

CB1 cannabinoid receptor is widely expressed in the central nervous system of animals from late prenatal development to adulthood. Appropriate activation and signaling of CB1 cannabinoid receptors in cortical interneurons are crucial during perinatal/postnatal ages and adolescence, when long-lasting changes in brain activity may elicit subsequent appearance of disorders in the adult brain. Here we used an optimized immunoprecipitation protocol based on specific antibodies followed by shot-gun proteomics to find CB1 interacting partners in postnatal rat GABAergic cortical neurons in vitro at two different stages of maturation. Besides describing new proteins associated with CB1 like dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex (DLAT), fatty acid synthase (FASN), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), voltage-dependent anion channel 1 (VDAC1), myosin phosphatase Rho-interacting protein (MPRIP) or usher syndrome type-1C protein-binding protein 1 (USHBP1), we show that the signaling complex of CB1 is different between maturational stages. Interestingly, the CB1 signaling complex is enriched at the more immature stage in mitochondrial associated proteins and metabolic molecular functions, whereas at more mature stage, CB1 complex is increased in maturation and synaptic-associated proteins. We describe also interacting partners specifically immunoprecipitated with either N-terminal or C-terminal CB1 directed antibodies. Our results highlight new players that may be affected by altered cannabinoid signaling at this critical window of postnatal cortical development.

Cannabinoid Modulation of Dopamine Release During Motivation, Periodic Reinforcement, Exploratory Behavior, Habit Formation, and Attention.

Motivational and attentional processes energize action sequences to facilitate evolutionary competition and promote behavioral fitness. Decades of neuropharmacology, electrophysiology and electrochemistry research indicate that the mesocorticolimbic DA pathway modulates both motivation and attention. More recently, it was realized that mesocorticolimbic DA function is tightly regulated by the brain’s endocannabinoid system and greatly influenced by exogenous cannabinoids—which have been harnessed by humanity for medicinal, ritualistic, and recreational uses for 12,000 years. Exogenous cannabinoids, like the primary psychoactive component of cannabis, delta-9-tetrahydrocannabinol, produce their effects by acting at binding sites for naturally occurring endocannabinoids. The brain’s endocannabinoid system consists of two G-protein coupled receptors, endogenous lipid ligands for these receptor targets, and several synthetic and metabolic enzymes involved in their production and degradation. Emerging evidence indicates that the endocannabinoid 2-arachidonoylglycerol is necessary to observe concurrent increases in DA release and motivated behavior. And the historical pharmacology literature indicates a role for cannabinoid signaling in both motivational and attentional processes. While both types of behaviors have been scrutinized under manipulation by either DA or cannabinoid agents, there is considerably less insight into prospective interactions between these two important signaling systems. This review attempts to summate the relevance of cannabinoid modulation of DA release during operant tasks designed to investigate either motivational or attentional control of behavior. We first describe how cannabinoids influence DA release and goal-directed action under a variety of reinforcement contingencies. Then we consider the role that endocannabinoids might play in switching an animal’s motivation from a goal-directed action to the search for an alternative outcome, in addition to the formation of long-term habits. Finally, dissociable features of attentional behavior using both the 5-choice serial reaction time task and the attentional set-shifting task are discussed along with their distinct influences by DA and cannabinoids. We end with discussing potential targets for further research regarding DA-cannabinoid interactions within key substrates involved in motivation and attention.

Cannabinoid Signaling in Auditory Function and Development.

Plants of the genus Cannabis have been used by humans for millennia for a variety of purposes. Perhaps most notable is the use of certain Cannabis strains for their psychoactive effects. More recently, several biologically active molecules within the plants of these Cannabis strains, called phytocannabinoids or simply cannabinoids, have been identified. Furthermore, within human cells, endogenous cannabinoids, or endocannabinoids, as well as the receptors and secondary messengers that give rise to their neuromodulatory effects, have also been characterized. This endocannabinoid system (ECS) is composed of two primary ligands—anandamide and 2-arachidonyl glycerol; two primary receptors—cannabinoid receptors 1 and 2; and several enzymes involved in biosynthesis and degradation of endocannabinoid ligands including diacylglycerol lipase (DAGL) and monoacylglycerol lipase (MAGL). Here we briefly summarize cannabinoid signaling and review what has been discerned to date with regard to cannabinoid signaling in the auditory system and its roles in normal physiological function as well as pathological conditions. While much has been uncovered regarding cannabinoid signaling in the central nervous system, less attention has been paid to the auditory system specifically. Still, evidence is emerging to suggest that cannabinoid signaling is critical for the development, maturation, function, and survival of cochlear hair cells (HCs) and spiral ganglion neurons (SGNs). Furthermore, cannabinoid signaling can have profound effects on synaptic connectivity in CNS structures related to auditory processing. While clinical cases demonstrate that endogenous and exogenous cannabinoids impact auditory function, this review highlights several areas, such as SGN development, where more research is warranted.

Expression of Cannabinoid Receptors in Mouse and Human Intrauterine Tissues: Insights into the Role of Endocannabinoid Signaling in Late Pregnancy

Abstract Introduction: Endocannabinoid signaling (ECS), mediated primarily by cannabinoid receptors (CNR) 1 and 2, is implicated in embryo implantation, decidualization, and placentation, but less is known about its role in late pregnancy or labour. Reports of elevated serum endocannabinoid concentrations during labour suggest that ECS may modulate uterine function leading up to and during parturition. Effects on uterine function likely vary depending on: the type of cannabinoid present, the intrauterine tissue, and CNR1/2 expression. Study of ECS gene expression in late pregnancy is therefore important to determine the contribution of this pathway to the normal and pathologic physiology of labour, for instance in preterm labour. Examining ECS gene expression in pregnancy may also contribute insight into the effects of exogenous cannabinoid consumption (reported in ~5% of pregnancies) on the late-pregnant uterus. To understand when and where cannabinoid signaling may impact uterine functions, we conducted an observational study on pregnant human and mouse intrauterine tissues. Methods: Human amnion, chorion, decidua, placenta, and upper/lower myometrium (n=6 participants) were biopsied at cesarean delivery (term, non-labour) to determine CNR expression by qPCR. Additional decidua (n=80) and upper/lower myometrium (n=82) samples were similarly obtained with and without labour, at term and preterm, for CNR expression analysis by qPCR. Mouse uteri for CNR expression analysis by qPCR were obtained from timed-mated C57BL6 mice at days 15-20 of pregnancy, in active labour, or post-partum. Human and mouse expression data were analyzed by Student’s t-test, one-way ANOVA (Bonferroni post-hoc), and Pearson’s correlation, as appropriate. Results: Term non-labour chorion, placenta, myometrium and decidua express CNRs, with 2-fold higher expression of CNR1 versus CNR2. In myometrium and decidua, neither CNR1 or CNR2 expression differed with labour status. CNR1 expression correlated positively with gestational age in decidua (r=0.346, p=0.0266) and lower segment myometrium (r=0.3667, p=0.0270) with labour. In mouse uteri, Cnr1 and Cnr2 expression significantly increased post-partum compared to mid/late pregnancy (vs day 19, p<0.0001 Cnr1; vs days 15-18, p<0.05 Cnr2). Conclusion: ECS may occur in the human decidua and myometrium throughout pregnancy and labour as CNR1 and CNR2 expression is maintained throughout. In our mouse model, higher Cnr1 and Cnr2 expression post-partum suggests that ECS may play a role in uterine resolution following delivery. Additional RNAseq analysis of the mouse tissues studied is underway to address whether expression of genes involved in production, transport or metabolism of cannabinoids are altered in late pregnancy or with the initiation of parturition.

The Impact of Cannabinoid Exposure on Glucocorticoid Receptor Signaling in Neural Stem Cells

Preterm birth-birth before 37 weeks of pregnancy-can cause many short- and long-term complications in newborns, including respiratory distress syndrome (RDS). RDS results from incomplete lung development and a surfactant deficiency, and it is a major factor of pre-term mortality. Synthetic glucocorticoids (sGCs) such as Betamethasone or Dexamethasone (Beta, Dex) are administered prenatally to women at risk of pre-term birth to prevent preterm complications. While sGCs are known to improve outcome, they also cause alterations in brain development and neural stem cell biology that are associated with long-term neurological defects.One common recreational drug used during pregnancy is cannabis. Some of the active components of cannabis include cannabinoids, which interact with the endocannabinoid receptor pathway in cells. Cannabinoids have been shown to induce proliferation and differentiation of embryonic neural stem cells (NSCs). We hypothesized that maternal cannabis use activates cannabinoid signaling pathways and leads to changes in glucocorticoid signaling in the developing brain. The purpose of this study was to determine whether cannabis use leads to a better or worse neurological outcome for children born pre-term and treated with sGCs for RDS.Neural stem cell neurospheres (NSCs) were isolated from the cerebral cortex of mice and treated with Vehicle (ethanol), Dex, cannabinoid receptor agonist WIN-55,212-2 (Win), or a combination WinDex. The transcriptional profile induced by exposure to Vehicle, Dex, and WinDex RNA were analyzed using microarray analyses examining the complete expressed genome. Gene Chip profiles indicated that both glucocorticoids and cannabinoids induce distinct transcriptional responses in E14.5 NSCs. The genes involved in proliferation-including S100a11, Jun, and Bex2-were repressed by Dex whereas WinDex rescued some of these expression profiles. Some genes encoding microRNA that inhibit our top target coding genes implicated in proliferation showed a greater induction by Dex compared to WinDex.Quantitative Polymerase Chain Reaction (qPCR) was performed to validate our genes of interest, including Adm, which has been shown to induce neural stem cell proliferation and differentiation. The biological impact of Winn on Dex-induced changes in NSC function were examined by in-vitro proliferation and differentiation studies using antibodies to Tuj1 (neurons), GFAP (glia), and CNPase (immature oligodendrocytes). The experiments indicate that Dex increased neuronal and oligodendrocyte differentiation, while WinDex appeared to reverse this phenotype in neurons.These studies suggest that cannabis use during pregnancy may limit the biological impact sGCs for preterm birth and lead to distinct cellular responses.