Endogenous Cannabinoid Signaling Research Articles

A Sex-Dependent Cannabinoid CB1 Receptor Role in Circadian Tearing of the Mouse.

We have shown that cannabinoid CB1 receptors regulate both salivation and tearing, but for tearing, this regulation is sex dependent with opposing effects by sex. We investigated a potential interplay of circadian and cannabinoid regulation of tearing. We measured cannabinoid and circadian regulation of tearing in CD1 strain mice as well as CB1 receptor protein expression using immunohistochemistry. We now report that CD1 strain mice have a circadian variation in basal tearing, differing by sex in terms of phase and amplitude. The amplitude of circadian variation in females is substantially dampened relative to males. Male CB1 receptor knockout mice do not differ from strain controls, but in female CB1 knockouts, the amplitude is enhanced and resembles that of WT males. This increased tearing is mimicked by the CB1 antagonist SR141716 (4 mg/kg, intraperitoneally [IP]), suggesting that tonic CB1 activation dampens female circadian tearing. Consistent with this, the cannabinoid receptor agonist CP55940 (0.5 mg/kg, IP) decreases tearing during the rest phase but increases tearing during the active phase in females. CB1 protein expression also differs by sex. While both males and females have CB1 receptors in parasympathetic inputs to the lacrimal gland, in female lacrimal glands, CB1 is also detected in myoepithethial cells. Mice have a sex-dependent circadian cycle of tearing. The endogenous cannabinoid signaling system appears to mediate some circadian effects, albeit in a sex-dependent manner and via distinct cellular targets.

Effects of in utero delta-9-tetrahydrocannabinol (THC) exposure on fetal and infant musculoskeletal development in a preclinical nonhuman primate model.

The endocannabinoid system (ECS) plays a major role in the maintenance of bodily homeostasis and adaptive response to external insults. It has been shown to regulate crucial physiological processes and behaviors, spanning nervous functions, anxiety, cognition, and pain sensation. Due to this broad activity, the ECS has been explored as a potential therapeutic target in the treatment of select diseases. However, until there is a more comprehensive understanding of how ECS activation by exogenous and endogenous ligands manifests across disparate tissues and cells, discretion should be exercised. Previous work has investigated how endogenous cannabinoid signaling impacts skeletal muscle development and differentiation. However, the effects of activation of the ECS by delta-9-tetrahydrocannabinol (THC, the most psychoactive component of cannabis) on skeletal muscle development, particularly in utero, remain unclear. To address this research gap, we used a highly translational non-human primate model to examine the potential impact of chronic prenatal THC exposure on fetal and infant musculoskeletal development. RNA was isolated from the skeletal muscle and analyzed for differential gene expression using a Nanostring nCounter neuroinflammatory panel comprised of 770 genes. Histomorphological evaluation of muscle morphology and composition was also performed. Our findings suggest that while prenatal THC exposure had narrow overall effects on fetal and infant muscle development, the greatest impacts were observed within pathways related to inflammation and cytokine signaling, which suggest the potential for tissue damage and atrophy. This pilot study establishes feasibility to evaluate neuroinflammation due to prenatal THC exposure and provides rationale for follow-on studies that explore the longer-term implications and functional consequences encountered by offspring as they continue to mature.

Endocannabinoid Hydrolase Inhibitors: Potential Novel Anxiolytic Drugs.

Over the past decade, the idea of targeting the endocannabinoid system to treat anxiety disorders has received increasing attention. Previous studies focused more on developing cannabinoid receptor agonists or supplementing exogenous cannabinoids, which are prone to various adverse effects due to their strong pharmacological activity and poor receptor selectivity, limiting their application in clinical research. Endocannabinoid hydrolase inhibitors are considered to be the most promising development strategies for the treatment of anxiety disorders. More recent effortshave emphasized that inhibition of two major endogenous cannabinoid hydrolases, monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), indirectly activates cannabinoid receptors by increasing endogenous cannabinoid levels in the synaptic gap, circumventing receptor desensitization resulting from direct enhancement of endogenous cannabinoid signaling. In this review, we comprehensively summarize the anxiolytic effects of MAGL and FAAH inhibitors and their potential pharmacological mechanisms, highlight reported novel inhibitors or natural products, and provide an outlook on future directions in this field.

CB1 Receptor Negative Allosteric Modulators as a Potential Tool to Reverse Cannabinoid Toxicity.

While the opioid crisis has justifiably occupied news headlines, emergency rooms are seeing many thousands of visits for another cause: cannabinoid toxicity. This is partly due to the spread of cheap and extremely potent synthetic cannabinoids that can cause serious neurological and cardiovascular complications-and deaths-every year. While an opioid overdose can be reversed by naloxone, there is no analogous treatment for cannabis toxicity. Without an antidote, doctors rely on sedatives, with their own risks, or 'waiting it out' to treat these patients. We have shown that the canonical synthetic 'designer' cannabinoids are highly potent CB1 receptor agonists and, as a result, competitive antagonists may struggle to rapidly reverse an overdose due to synthetic cannabinoids. Negative allosteric modulators (NAMs) have the potential to attenuate the effects of synthetic cannabinoids without having to directly compete for binding. We tested a group of CB1 NAMs for their ability to reverse the effects of the canonical synthetic designer cannabinoid JWH018 in vitro in a neuronal model of endogenous cannabinoid signaling and also in vivo. We tested ABD1085, RTICBM189, and PSNCBAM1 in autaptic hippocampal neurons that endogenously express a retrograde CB1-dependent circuit that inhibits neurotransmission. We found that all of these compounds blocked/reversed JWH018, though some proved more potent than others. We then tested whether these compounds could block the effects of JWH018 in vivo, using a test of nociception in mice. We found that only two of these compounds-RTICBM189 and PSNCBAM1-blocked JWH018 when applied in advance. The in vitro potency of a compound did not predict its in vivo potency. PSNCBAM1 proved to be the more potent of the compounds and also reversed the effects of JWH018 when applied afterward, a condition that more closely mimics an overdose situation. Lastly, we found that PSNCBAM1 did not elicit withdrawal after chronic JWH018 treatment. In summary, CB1 NAMs can, in principle, reverse the effects of the canonical synthetic designer cannabinoid JWH018 both in vitro and in vivo, without inducing withdrawal. These findings suggest a novel pharmacological approach to at last provide a tool to counter cannabinoid toxicity.

Pharmacological diacylglycerol lipase inhibition impairs contextual fear extinction in mice.

Acquisition and extinction of associative fear memories are critical for guiding adaptive behavioral responses to environmental threats, and dysregulation of these processes is thought to represent important neurobehavioral substrates of trauma and stress-related disorders including posttraumatic stress disorder (PTSD). Endogenous cannabinoid (eCB) signaling has been heavily implicated in the extinction of aversive fear memories and we have recently shown that pharmacological inhibition of 2-arachidonoylglycerol (2-AG) synthesis, a major eCB regulating synaptic suppression, impairs fear extinction in an auditory cue conditioning paradigm. Despite these data, the role of 2-AG signaling in contextual fear conditioning is not well understood. Here, we show that systemic pharmacological blockade of diacylglycerol lipase, the rate-limiting enzyme catalyzing in the synthesis of 2-AG, enhances contextual fear learning and impairs within-session extinction. In sham-conditioned mice, 2-AG synthesis inhibition causes a small increase in unconditioned freezing behavior. No effects of 2-AG synthesis inhibition were noted in the Elevated Plus Maze in micetested after fear extinction. These data provide support for 2-AG signaling in the suppression of contextual fear learning and the expression of within-session extinction of contextual fear memories.

Study on transcriptome characteristics of respiratory syncytial virus bronchiolitis in children by RNA sequencing

To explore the biological characteristics related to the pathogenesis and severity of respiratory syncytial virus (RSV) bronchiolitis by RNA sequencing of white blood cells in children with RSV bronchiolitis. This study is a case-control study. A total of 87 children diagnosed with bronchiolitis and RSV antigen positive and/or RSV nucleic acid positive in the pediatric respiratory department of the Second Affiliated Hospital of Wenzhou Medical University from October 2019 to April 2022 were selected as the case group. The case group was divided into three groups based on the condition: mild, moderate, and severe, and there were two groups according to the presence or absence of atopic symptoms: the atopic group and the non-atopic group, forty healthy children in the same period were selected as the control group. The whole blood leukocyte RNA of the children in the case group and the control group was extracted for RNA sequencing, and the data were analyzed to obtain differentially expressed genes (DEGs). Then, the immunobiological pathways and genes related to the pathogenesis, disease condition, and atopy were screened through Gene Ontology (GO) annotation, Kyoto Gene and Genome Encyclopedia (KEGG) annotation, and protein interaction network (PPI) construction methods. Construct the weighted gene co-expression network analysis (WGCNA) module to identify potential biological indicators related to disease severity.Compared with the control group, the case group had a total of 1 782 DEGs, including 1 586 upregulated genes and 196 downregulated genes. The GO pathway enrichment of DEGs is mainly enriched in molecular functions such as peroxidase activity and oxidoreductase activity. In the cytological components, it is mainly enriched in cytoplasmic vesicle lumen and secretory granule lumen. In biological processes, it is mainly enriched in processes such as neutrophil activation involved in immune responses, neutrophil degranulation, and neutrophil activation. KEGG analysis is mainly concentrated in the signal pathway of the viral protein interaction with cytokine and cytokine receptor. A PPI network was constructed to screen four genes at the core position, including CCL2, IL-10, MMP9 and JUN. The DEGs obtained by comparing different disease groups with the control group are mainly enriched in retrograde endocannabinoid signaling and cell apoptosis pathways. WGCNA analysis showed that the brown module related to oxygen saturation was most closely related to the disease, and its gene was mainly enriched in the RNA helicase retinoic acid inducible gene-I (RIG-I) like receptor signal pathway. There are 230 specific DEGs in the atopic group and 444 in the non-atopic group. KEGG enrichment analysis results show that both groups are enriched to NF-κB signaling pathway, the characteristic does not cause significant changes in immune response and transcriptome characteristics in children with RSV bronchiolitis. In conclusion, neutrophil activation, degranulation pathway and signal pathway of interaction between viral protein and cytokine and cytokine receptor are involved in the immune response of RSV bronchiolitis host. CCL2, IL-10, MMP9 and JUN genes may be associated with the pathogenesis. They might be potential biomarkers related to disease severity in RIG-I like receptors, cell apoptosis, and endogenous cannabinoid related signaling pathways.

Monoacylglycerol Lipase Inhibition Using ABX-1431 Attenuates Cerebral Ischaemia Early After Traumatic Brain Injury.

An early event in the pathology of traumatic brain injury (TBI) is a reduction in cerebral blood flow (CBF), which exacerbates secondary injury development and inhibits brain recovery. The endogenous cannabinoid system signalling (eCBs) might be critical in TBI recovery due to modulating synaptic activity and exerting neuroprotective and anti-inflammatory effects. In the brain, eCBs predominantly occur at cannabinoid receptor type 1 via the eCB 2-arachidonoylglycerol (2-AG). The aim of this work was to test the efficacy of potentiating 2-AG signalling by monoacylglycerol lipase (MAGL) inhibition using ABX-1431 immediately following TBI. Laser speckle contrast imaging (LSCI) was used to create a high-resolution map of regional cerebral blood flow (CBF) over the pericontusion cortical surface. In-vivo two-photon laser scanning microscopy (2PLSM) was used to monitor cerebral microcirculation (i.v. fluorescein isothiocyanate dextran, FITC) and mitochondrial respiration and brain tissue oxygen supply (nicotinamide adenine dinucleotide autofluorescence, NADH) during 4hours after CHI. After baseline imaging, male C57BL/6 J mice (10-12weeks, >28g) were subjected to a modified moderate Shohami weight-drop closed-head injury (CHI) followed by i.p. injection of ABX-1431 (5mg/kg) or vehicle 30 min after the insult (10 mice per group). Differences between groups and between time points were determined using two-way repeated measures (ANOVA) for multiple comparisons and post hoc testing with the statistical significance level set at p<0.05. Optical imaging revealed that CHI caused a decrease in regional CBF, arteriole diameters (vasospasm), and blood flow volume, leading to capillary microthrombosis and a reduction in capillary flow velocity. Compromised cerebral microcirculation led to the development of tissue hypoxia. ABX-1431 application, in a ~30-minute delay, mitigated the development of microvascular dysfunction, microthrombosis formation, and tissue hypoxia compared to the saline control group (p<0.05, starting 1hour after CHI). Therefore, MAGL inhibition by ABX-1431 attenuates cerebral ischaemia early after TBI. The observed 2-AG-mediated cerebrovascular relaxation might involve both a direct inhibition of smooth muscle contractility and a release of vasodilator mediator(s) from the endothelium.

Metabolomics study of APETx2 post-conditioning on myocardial ischemia-reperfusion injury.

Acid-sensing ion channels are activated during myocardial ischemia and are implicated in the mechanism of myocardial ischemia-reperfusion injury (MIRI). Acid-sensing ion channel 3 (ASIC3), the most pH-sensitive member of the ASIC family, is highly expressed in myocardial tissues. However, the role of ASIC3 in MIRI and its precise effects on the myocardial metabolome remain unclear. These unknowns might be related to the cardioprotective effects observed with APETx2 post-conditioning. Rat hearts subjected to Langendorff perfusion were randomly assigned to the normal (Nor) group, ischemia/reperfusion (I/R) group, ASIC3 blockade (AP) group. Rat hearts in group AP were treated with the ASIC3-specific inhibitor APETx2 (630nM). Molecular and morphological changes were observed to elucidate the role of ASIC3 in MIRI. Bioinformatics analyses identified differential metabolites and pathways associated with APETx2 post-conditioning. APETx2 post-conditioning stabilized hemodynamics in the isolated rat heart model of MIRI. It also reduced myocardial infarct size, mitigated mitochondrial damage at the ultrastructural level, and improved markers of myocardial injury and oxidative stress. Further more, we observed that phosphatidylcholine, phosphatidylethanolamine, citric acid, cyanidin 5-O-beta-D-glucoside, and L-aspartic acid decreased after MIRI. The levels of these metabolites were partially restored by APETx2 post-conditioning. These metabolites are primarily involved in autophagy and endogenous cannabinoid signaling pathways. ASIC3 is potentially a key player in MIRI. APETx2 post-conditioning may improve MIRI through specific metabolic changes. This study provides valuable data for future research on the metabolic mechanisms underlying the effects of APETx2 post-conditioning in MIRI.

Synaptic and cellular endocannabinoid signaling mechanisms regulate stress-induced plasticity of nucleus accumbens somatostatin neurons

Interneuron populations within the nucleus accumbens (NAc) orchestrate excitatory-inhibitory balance, undergo experience-dependent plasticity, and gate-motivated behavior, all biobehavioral processes heavily modulated by endogenous cannabinoid (eCB) signaling. While eCBs are well known to regulate synaptic plasticity onto NAc medium spiny neurons and modulate NAc function at the behavioral level, how eCBs regulate NAc interneuron function is less well understood. Here, we show that eCB signaling differentially regulates glutamatergic and feedforward GABAergic transmission onto NAc somatostatin-expressing interneurons (NAcSOM+) in an input-specific manner, while simultaneously increasing postsynaptic excitability of NAcSOM+ neurons, ultimately biasing toward vHPC (ventral hippocampal), and away from BLA (basolateral amygdalalar), activation of NAcSOM+ neurons. We further demonstrate that NAcSOM+ are activated by stress in vivo and undergo stress-dependent plasticity, evident as a global increase in intrinsic excitability and an increase in excitation-inhibition balance specifically at vHPC, but not BLA, inputs onto NAcSOM+ neurons. Importantly, both forms of stress-induced plasticity are dependent on eCB signaling at cannabinoid type 1 receptors. These findings reveal eCB-dependent mechanisms that sculpt afferent input and excitability of NAcSOM+ neurons and demonstrate a key role for eCB signaling in stress-induced plasticity of NAcSOM+-associated circuits.

Wwl70-induced ABHD6 inhibition attenuates memory deficits and pathological phenotypes in APPswe/PS1dE9 mice

Synaptic dysfunction plays a crucial role in the pathogenesis of Alzheimer's disease (AD). α/β-hydrolase domain-containing 6 (ABHD6) contributes to synaptic dysfunctions, and ABHD6 inhibition has shown potential therapeutic value in neurological disorders. However, the role of ABHD6 in AD has not been fully defined. In this study, we demonstrated that adeno-associated virus (AAV) mediated shRNA targeting ABHD6 in hippocampal neurons attenuated synaptic dysfunction and memory impairment of APPswe/PS1dE9 (APP/PS1) mice, while it didn't affect the amyloid-beta (Aβ) levels and neuroinflammation in the brains. In addition, intraperitoneal injection of wwl70, a specific inhibitor of ABHD6, improved synaptic plasticity and memory function in APP/PS1 mice, which might attribute to the activation of endogenous cannabinoid signaling. Furthermore, wwl70 significantly decreased the Aβ levels and neuroinflammation in the hippocampus of AD mice, and enhanced Aβ phagocytized by microglia. In conclusion, for the first time our data have shown that ABHD6 inhibition might be a promising strategy for AD treatment, and wwl70 is a potential candidate for AD drug development pipeline.

Cannabidiol and cannabis-inspired terpene blends have acute prosocial effects in the BTBR mouse model of autism spectrum disorder.

Cannabidiol (CBD) is a non-intoxicating phytocannabinoid with increasing popularity due to its purported therapeutic efficacy for numerous off-label conditions including anxiety and autism spectrum disorder (ASD). Those with ASD are commonly deficient in endogenous cannabinoid signaling and GABAergic tone. CBD has a complex pharmacodynamic profile that includes enhancing GABA and endocannabinoid signaling. Thus, there is mechanistic justification for investigating CBD's potential to improve social interaction and related symptoms in ASD. Recent clinical trials in children with ASD support CBD's beneficial effects in numerous comorbid symptoms, but its impact on social behavior is understudied. Here, we tested the prosocial and general anxiolytic efficacy of a commercially available CBD-rich broad spectrum hemp oil delivered by repeated puff vaporization and consumed via passive inhalation in the female cohort of the BTBR strain, a common inbred mouse line for preclinical assessment of ASD-like behaviors. We observed that CBD enhanced prosocial behaviors using the 3-Chamber Test with a different vapor dose-response relationship between prosocial behavior and anxiety-related behavior on the elevated plus maze. We also identified that inhalation of a vaporized terpene blend from the popular OG Kush cannabis strain increased prosocial behavior independently of CBD and acted together with CBD to promote a robust prosocial effect. We observed similar prosocial effects with two additional cannabis terpene blends from the Do-Si-Dos and Blue Dream strains, and further reveal that these prosocial benefits rely on the combination of multiple terpenes that comprise the blends. Our results illustrate the added benefit of cannabis terpene blends for CBD-based treatment of ASD.

Somatic and terminal CB1 receptors are differentially coupled to voltage-gated sodium channels in neocortical neurons

Endogenous cannabinoid signaling is vital for important brain functions, and the same pathways can be modified pharmacologically to treat pain, epilepsy, and posttraumatic stress disorder. Endocannabinoid-mediated changes to excitability are predominantly attributed to 2-arachidonoylglycerol (2-AG) acting presynaptically via the canonical cannabinoid receptor, CB1. Here, we identify a mechanism in the neocortex by which anandamide (AEA), another major endocannabinoid, but not 2-AG, powerfully inhibits somatically recorded voltage-gated sodium channel (VGSC) currents in the majority of neurons. This pathway involves intracellular CB1 that, when activated by anandamide, decreases the likelihood of recurrent action potential generation. WIN 55,212-2 similarly activates CB1 and inhibits VGSC currents, indicating that this pathway is also positioned to mediate the actions of exogenous cannabinoids on neuronal excitability. The coupling between CB1 and VGSCs is absent at nerve terminals, and 2-AG does not block somatic VGSC currents, indicating functional compartmentalization of the actions of two endocannabinoids.

Differential Enantiomer-Specific Signaling of Cannabidiol at CB1 Receptors

The two main constituents of cannabis are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). While Δ9-THC pharmacology has been studied extensively, CBD-long considered inactive-is now the subject of vigorous research related to epilepsy, pain, and inflammation and is popularly embraced as a virtual cure-all. However, our understanding of CBD pharmacology remains limited, although CBD inhibits cannabinoid CB1 receptor signaling, likely as a negative allosteric modulator. Cannabis synthesizes (-)-CBD, but CBD can also exist as an enantiomer, (+)-CBD. We enantioselectively synthesized both CBD enantiomers using established conditions and describe here a new, practical, and reliable, NMR-based method for confirming the enantiomeric purity of two CBD enantiomers. We also investigated the pharmacology of (+)-CBD in autaptic hippocampal neurons, a well-characterized neuronal model of endogenous cannabinoid signaling, and in CHO-K1 cells. We report the inhibition constant for displacing CP55,940 at CB1 by (+)-CBD, is 5-fold lower than (-)-CBD. We find that (+)-CBD is ∼10 times more potent at inhibiting depolarization-induced suppression of excitation (DSE), a form of endogenous cannabinoid-mediated retrograde synaptic plasticity. (+)-CBD also inhibits CB1 suppression of cAMP accumulation but with less potency, indicating that the signaling profiles of the enantiomers differ in a pathway-specific manner. In addition, we report that (+)-CBD stereoselectively and potently activates the sphingosine-1 phosphate (S1P) receptors, S1P1 and S1P3 These results provide an attractive method for synthesizing and distinguishing enantiomers of CBD and related phytocannabinoids and provide further evidence that these enantiomers have their own unique and interesting signaling properties. SIGNIFICANCE STATEMENT: Cannabidiol (CBD) is the subject of considerable scientific and popular interest, but we know little of the enantiomers of CBD. We find that the enantiomer (+)-CBD is substantially more potent inhibitor of cannabinoid CB1 receptors and that it activates sphingosine-1-phosphate receptors in an enantiomer-specific manner; we have additionally developed an improved method for the synthesis of enantiomers of CBD and related compounds.

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.

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.

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.

CAMP Signaling–Mediated Phosphorylation of Diacylglycerol Lipase α Regulates Interaction With Ankyrin-G and Dendritic Spine Morphology

BackgroundDiacylglycerol lipase α (DAGLα), a major biosynthetic enzyme for endogenous cannabinoid signaling, has emerged as a risk gene in multiple psychiatric disorders. However, its role in the regulation of dendritic spine plasticity is unclear. MethodsDAGLα wild-type or point mutants were overexpressed in primary cortical neurons or human embryonic kidney 293T cells. The effects of mutated variants on interaction, dendritic spine morphology, and dynamics were examined by proximity ligation assay or fluorescence recovery after photobleaching. Behavioral tests and immunohistochemistry were performed with ankyrin-G conditional knockout and wild-type male mice. ResultsDAGLα modulated dendritic spine size and density, but the effects of changes in its protein level versus enzymatic activity were different, implicating either a 2-arachidonoylglycerol (2-AG)–dependent or –independent mechanism. The 2-AG–independent effects were mediated by the interaction of DAGLα with ankyrin-G, a multifunctional scaffold protein implicated in psychiatric disorders. Using superresolution microscopy, we observed that they colocalized in distinct nanodomains, which correlated with spine size. In situ proximity ligation assay combined with structured illumination microscopy revealed that DAGLα phosphorylation upon forskolin treatment enhanced the interaction with ankyrin-G in spines, leading to increased spine size and decreased DAGLα surface diffusion. Ankyrin-G conditional knockout mice showed significantly decreased DAGLα-positive neurons in the forebrain. In mice, ankyrin-G was required for forskolin-dependent reversal of depression-related behavior. ConclusionsTaken together, ANK3 and DAGLA, both neuropsychiatric disorder genes, interact in a complex to regulate spine morphology. These data reveal novel synaptic signaling mechanisms and potential therapeutic avenues.

Preservation of spatial memory and neuroprotection by the fatty acid amide hydrolase inhibitor URB597 in a rat model of vascular dementia.

BackgroundChronic cerebral hypoperfusion (CCH) is a major risk factor for vascular dementia (VaD). There are currently no broadly effective prevention or treatment strategies for VaD, but recent studies have reported promising results following vascular bypass surgery and pharmacomodulation of the brain endocannabinoid system (ECS). In this study, early effects of encephalomyosynangiosis (EMS) bypass surgery and augmented endocannabinoid signaling on CCH-induced cognitive dysfunction and neuronal damage were investigated.MethodsAn animal model of VaD was established by bilateral common carotid artery occlusion (BCCAO). Cannabinoid signaling was upregulated by treatment with the fatty acid amide hydrolase inhibitor URB597 (URB). Spatial learning and memory, cerebral blood flow (CBF), revascularization, brain-derived neurotrophic factor (BDNF)-tropomyosin receptor kinase B (TrkB) signaling, and apoptosis were compared among Sham, BCCAO, BCCAO + EMS, BCCAO + URB, and BCCAO + URB + EMS groups. Spatial learning and memory were evaluated using the Morris water maze (MWM). The CBF in cortex and hippocampus was evaluated by 3-dimensional arterial spin labeling. The neovascularization was visualized by CD34 immunofluorescence staining, and BDNF-TrkB signaling protein expression levels were assessed by Western blotting.ResultsTreatment with URB597 but not EMS alone reversed the spatial learning and memory deficits induced by BCCAO. Neovascularization was enhanced after EMS surgery but not by URB597. Alternatively, there were no significant differences in CBF among treatment groups. Expression levels of BDNF and TrkB were significantly reduced by CCH compared to Sham treatment, and downregulation of both proteins was reversed by URB597 treatment but not EMS. BCCAO enhanced neuronal apoptosis, which was also reversed by URB597.ConclusionsAugmentation of endogenous cannabinoid signaling but not EMS protects against CCH-induced neurodegeneration and preserves spatial learning and memory, possibly by activating BDNF-TrkB signaling.

Cannabis use and posttraumatic stress disorder comorbidity: Epidemiology, biology and the potential for novel treatment approaches.

Cannabis use is increasing among some demographics in the United States and is tightly linked to anxiety, trauma, and stress reactivity at the epidemiological and biological level. Stress-coping motives are highly cited reasons for cannabis use. However, with increased cannabis use comes the increased susceptibility for cannabis use disorder (CUD). Indeed, CUD is highly comorbid with posttraumatic stress disorder (PTSD). Importantly, endogenous cannabinoid signaling systems play a key role in the regulation of stress reactivity and anxiety regulation, and preclinical data suggest deficiencies in this signaling system could contribute to the development of stress-related psychopathology. Furthermore, endocannabinoid deficiency states, either pre-existing or induced by trauma exposure, could provide explanatory insights into the high rates of comorbid cannabis use in patients with PTSD. Here we review clinical and preclinical literature related to the cannabis use-PTSD comorbidity, the role of endocannabinoids in the regulation of stress reactivity, and potential therapeutic implications of recent work in this area.