NAPE-PLD in the ventral tegmental area regulates reward events, feeding and energy homeostasis.

The N-acyl phosphatidylethanolamine-specific phospholipase D (NAPE-PLD) catalyzes the production of N-acylethanolamines (NAEs), a family of endogenous bioactive lipids, which are involved in various biological processes ranging from neuronal functions to energy homeostasis and feeding behaviors. Reward-dependent behaviors depend on dopamine (DA) transmission between the ventral tegmental area (VTA) and the nucleus accumbens (NAc), which conveys reward-values and scales reinforced behaviors. However, whether and how NAPE-PLD may contribute to the regulation of feeding and reward-dependent behaviors has not yet been investigated. This biological question is of paramount importance since NAEs are altered in obesity and metabolic disorders. Here, we show that transcriptomic meta-analysis highlights a potential role for NAPE-PLD within the VTA→NAc circuit. Using brain-specific invalidation approaches, we report that the integrity of NAPE-PLD is required for the proper homeostasis of NAEs within the midbrain VTA and it affects food-reward behaviors. Moreover, region-specific knock-down of NAPE-PLD in the VTA enhanced food-reward seeking and reinforced behaviors, which were associated with increased in vivo DA release dynamics in response to both food- and non-food-related rewards together with heightened tropism towards food consumption. Furthermore, midbrain knock-down of NAPE-PLD, which increased energy expenditure and adapted nutrient partitioning, elicited a relative protection against high-fat diet-mediated body fat gain and obesity-associated metabolic features. In conclusion, these findings reveal a new key role of VTA NAPE-PLD in shaping DA-dependent events, feeding behaviors and energy homeostasis, thus providing new insights on the regulation of body metabolism.

NAPE-PLD in the ventral tegmental area regulates reward events, feeding and energy homeostasis.

The N-acyl phosphatidylethanolamine-specific phospholipase D (NAPE-PLD) catalyzes the production of N-acylethanolamines (NAEs), a family of endogenous bioactive lipids, which are involved in various biological processes ranging from neuronal functions to energy homeostasis and feeding behaviors. Reward-dependent behaviors depend on dopamine (DA) transmission between the ventral tegmental area (VTA) and the nucleus accumbens (NAc), which conveys reward-values and scales reinforced behaviors. However, whether and how NAPE-PLD may contribute to the regulation of feeding and reward-dependent behaviors has not yet been investigated. This biological question is of paramount importance since NAEs are altered in obesity and metabolic disorders. Here, we show that transcriptomic meta-analysis highlights a potential role for NAPE-PLD within the VTA®NAc circuit. Using brain-specific invalidation approaches, we report that the integrity of NAPE-PLD is required for the proper homeostasis of NAEs within the midbrain VTA and it affects food-reward behaviors. Moreover, region-specific knock-down of NAPE-PLD in the VTA enhanced food-reward seeking and reinforced behaviors, which were associated with increased in vivo DA release dynamics in response to both food and non-food-related rewards together with heightened tropism towards food consumption. Furthermore, midbrain knock-down of NAPE-PLD, which increased energy expenditure and adapted nutrient partitioning, elicited a relative protection against high-fat diet-mediated body fat gain and obesity-associated metabolic features. In conclusion, these findings reveal a new key role of VTA NAPE-PLD in shaping DA-dependent events, feeding behaviors and energy homeostasis, thus providing new insights on the regulation of body metabolism.

Endocannabinoids produced in photoreceptor cells in response to light activate Drosophila TRP channels.

Drosophila phototransduction is a model for signaling cascades that culminate in the activation of transient receptor potential (TRP) cation channels. TRP and TRPL are the canonical TRP (TRPC) channels that are regulated by light stimulation of rhodopsin and engagement of Gαq and phospholipase Cß (PLC). Lipid metabolite(s) generated downstream of PLC are essential for the activation of the TRPC channels in photoreceptor cells. We sought to identify the key lipids produced subsequent to PLC stimulation that contribute to channel activation. Here, using genetics, lipid analysis, and Ca2+ imaging, we found that light increased the amount of an abundant endocannabinoid, 2-linoleoyl glycerol (2-LG), in vivo. The increase in 2-LG amounts depended on the PLC and diacylglycerol lipase encoded by norpA and inaE, respectively. This endocannabinoid facilitated TRPC-dependent Ca2+ influx in a heterologous expression system and in dissociated ommatidia from compound eyes. Moreover, 2-LG and mechanical stimulation cooperatively activated TRPC channels in ommatidia. We propose that 2-LG is a physiologically relevant endocannabinoid that activates TRPC channels in photoreceptor cells.

Activation of TRPV1 by Capsaicin or Heat Drives Changes in 2-Acyl Glycerols and N-Acyl Ethanolamines in a Time, Dose, and Temperature Dependent Manner.

Endocannabinoids (eCBs) and transient receptor potential (TRP) channels are associated with thermoregulation; however, there are many gaps in the understanding of how these signaling systems work together in responding to changes in temperature. TRPV1, a calcium-permeable ion channel, is activated by capsaicin, elevated temperature, the eCB Anandamide, and over 15 additional endogenous lipids. There is also evidence for signaling crosstalk between TRPV1 and the eCB receptor, CB1. We recently found that activation of TRPV1-HEK cells by capsaicin increases the production of the eCB, 2-arachidonoyl glycerol (2-AG), suggesting a molecular link between these receptors. Here, we tested the hypothesis that TRPV1 activation by capsaicin drives regulation of a wider-range of lipid signaling molecules and is time and dose-dependent. We also tested the hypothesis that changes in temperature that drive changes in calcium mobilization in TRPV1-HEK will likewise drive similar changes in lipid signaling molecule regulation. Lipid analysis was conducted by partial purification of methanolic extracts on C18 solid phase extraction columns followed by HPLC/MS/MS. Capsaicin increased the release of 2-acyl glycerols (2-AG, 2-linoleoyl glycerol, 2-oleoyl glycerol), in a concentration- and time-dependent manner, whereas levels of N-acyl ethanolamines (NAEs), including Anandamide, were significantly decreased. Analogous changes in 2-acyl glycerols and NAEs were measured upon ramping the temperature from 37 to 45°C. In contrast, opposite effects were measured when analyzing lipids after they were maintained at 27°C and then quickly ramped to 37°C, wherein 2-acyl glycerol levels decreased and NAEs increased. These results provide further evidence that the eCB system and TRPV1 have integrated signaling functions that are associated with the molecular response to temperature variation.

Development and implementation of SafeMedWaste, a chemical denaturant for non-hazardous disposal of controlled medications.

Substance use disorders are a significant public health issue. Options to dispose of controlled medications are limited, increasing the risk of diversion. Providing an alternative for disposal, a chemical denaturant, SafeMedWaste, was designed to destroy controlled substances irreversibly and safely be placed in non-hazardous landfills. Via HPLC-MS, four formulations of SafeMedWaste were tested with 34 different liquid controlled medications from DEA schedules I-V. Beta testing assessed the efficacy of SafeMedWaste in a clinical setting and on waste generated in a manufacturing setting. Furthermore, a formulation of SafeMedWaste was tested on solid controlled medications. All 34 of the liquid medications tested (e.g., amphetamine, diazepam, fentanyl, ketamine) were fully destroyed in SafeMedWaste within 2-24 h. Analysis of a beta test sample of SafeMedWaste containing fentanyl, midazolam, and morphine waste collected in a hospital showed full denaturation of these drugs in 24 h. Variants of SafeMedWaste were optimized to denature six different controlled substance waste samples from a manufacturing facility. In contrast to side-by-side studies with a charcoal disposal system using the same drugs, SafeMedWaste fully inactivated and destroyed the controlled substances in the waste streams. Another formulation of SafeMedWaste was tested on solid medications, which were fully denatured in 48-72 h. In conclusion, SafeMedWaste irreversibly denatures controlled medications that present a problem in our society.

Plasma endocannabinoids and cannabimimetic fatty acid derivatives are altered in gastroparesis: A sex- and subtype-dependent observation.

BACKGROUND: Gastroparesis (GP) is a motility disorder of the stomach presenting with upper gastrointestinal symptoms in the setting of delayed gastric emptying. Endocannabinoids are involved in the regulation of GI function including motility. However, their role in the pathophysiology of GP has not been sufficiently investigated. Our goal was to compare the circulating levels of endocannabinoids and cannabimimetic fatty acid derivatives in GP versus control subjects. METHODS: The study compared plasma concentrations of endocannabinoids and their lipoamine and 2-acyl glycerol congeners, measured by high-pressure liquid chromatography/tandem mass spectrometry (HPLC-MS-MS), in adult patients with diabetic gastroparesis (DM-GP; n = 24; n = 16 female), idiopathic gastroparesis (ID-GP; n = 19; n = 11 female), diabetic patients without GP (DM; n = 19; n = 10 female), and healthy controls (HC; n = 18; n = 10 female). Data, presented as mean ± SEM, were analyzed with ANOVA (Sidak post hoc). KEY RESULTS: Endocannabinoids anandamide (AEA: 0.5 ± 0.1 nMol/L) and 2-arachidonoyl glycerol (2-AG: 2.6 ± 0.7 nMol/L) were significantly lower in female DM-GP patients vs. DM females (AEA: 2.5 ± 0.7 nMol/L and 2-AG: 9.4 ± 3.3 nMol/L). Other monoacylglycerols including 2-palmitoyl glycerol and 2-oleoyl glycerol were also lower in female DM-GP patients compared to DM females. No changes were observed in men. CONCLUSIONS & INFERENCES: Endocannabinoids and other fatty acid derivatives with cannabimimetic properties are reduced in female DM-GP patients. Since GP, particularly with diabetic etiology, is more prevalent among women and since cannabinoids are antiemetic, this decrease in levels may contribute to symptom development in these subjects. Targeting the endocannabinoid system may be a future therapeutic option in DM-GP patients.

A new pedigree with thrombomodulin-associated coagulopathy in which delayed fibrinolysis is partially attenuated by co-inherited TAFI deficiency.

BACKGROUND: Thrombomodulin-associated coagulopathy (TM-AC) is a rare bleeding disorder in which a single reported p.Cys537* variant in the thrombomodulin gene THBD causes high plasma thrombomodulin (TM) levels. High TM levels attenuate thrombin generation and delay fibrinolysis. OBJECTIVES: To report the characteristics of pedigree with a novel THBD variant causing TM-AC, and co-inherited deficiency of thrombin-activatable fibrinolysis inhibitor (TAFI). PATIENTS/METHODS: Identification of pathogenic variants in hemostasis genes by next-generation sequencing and case recall for deep phenotyping. RESULTS: Pedigree members with a previously reported THBD variant predicting p.Pro496Argfs*10 and chain truncation in TM transmembrane domain had abnormal bleeding and greatly increased plasma TM levels. Affected cases had attenuated thrombin generation and delayed fibrinolysis similar to previous reported TM_AC cases with THBD p.Cys537*. Coincidentally, some pedigree members also harbored a stop-gain variant in CPB2 encoding TAFI. This reduced plasma TAFI levels but was asymptomatic. Pedigree members with TM-AC caused by the p.Pro496Argfs*10 THBD variant and also TAFI deficiency had a partially attenuated delay in fibrinolysis, but no change in the defective thrombin generation. CONCLUSIONS: These data extend the reported genetic repertoire of TM-AC and establish a common molecular pathogenesis arising from high plasma levels of TM extra-cellular domain. The data further confirm that the delay in fibrinolysis associated with TM-AC is directly linked to increased TAFI activation. The combination of the rare variants in the pedigree members provides a unique genetic model to develop understanding of the thrombin-TM system and its regulation of TAFI.

Long-Term Aberrations To Cerebellar Endocannabinoids Induced By Early-Life Stress.

Emerging evidence points to the role of the endocannabinoid system in long-term stress-induced neural remodeling with studies on stress-induced endocannabinoid dysregulation focusing on cerebral changes that are temporally proximal to stressors. Little is known about temporally distal and sex-specific effects, especially in cerebellum, which is vulnerable to early developmental stress and is dense with cannabinoid receptors. Following limited bedding at postnatal days 2-9, adult (postnatal day 70) cerebellar and hippocampal endocannabinoids, related lipids, and mRNA were assessed, and behavioral performance evaluated. Regional and sex-specific effects were present at baseline and following early-life stress. Limited bedding impaired peripherally-measured basal corticosterone in adult males only. In the CNS, early-life stress (1) decreased 2-arachidonoyl glycerol and arachidonic acid in the cerebellar interpositus nucleus in males only; (2) decreased 2-arachidonoyl glycerol in females only in cerebellar Crus I; and (3) increased dorsal hippocampus prostaglandins in males only. Cerebellar interpositus transcriptomics revealed substantial sex effects, with minimal stress effects. Stress did impair novel object recognition in both sexes and social preference in females. Accordingly, the cerebellar endocannabinoid system exhibits robust sex-specific differences, malleable through early-life stress, suggesting the role of endocannabinoids and stress to sexual differentiation of the brain and cerebellar-related dysfunctions.

Bioactive Lipids in Cancer, Inflammation and Related Diseases : Acute and Chronic Mild Traumatic Brain Injury Differentially Changes Levels of Bioactive Lipids in the CNS Associated with Headache.

Headache is a common complaint after mild traumatic brain injury (mTBI). Changes in the CNS lipidome were previously associated with acrolein-induced headache in rodents. mTBI caused similar headache-like symptoms in rats; therefore, we tested the hypothesis that mTBI might likewise alter the lipidome. Using a stereotaxic impactor, rats were given either a single mTBI or a series of 4 mTBIs 48 h apart. 72 h later for single mTBI and 7 days later for repeated mTBI, the trigeminal ganglia (TG), trigeminal nucleus (TNC), and cerebellum (CER) were isolated. Using HPLC/MS/MS, ~80 lipids were measured in each tissue and compared to sham controls. mTBI drove widespread alterations in lipid levels. Single mTBI increased arachidonic acid and repeated mTBI increased prostaglandins in all 3 tissue types. mTBI affected multiple TRPV agonists, including N-arachidonoyl ethanolamine (AEA), which increased in the TNC and CER after single mTBI. After repeated mTBI, AEA increased in the TG, but decreased in the TNC. Common to all tissue types in single and repeated mTBI was an increase the AEA metabolite, N-arachidonoyl glycine, a potent activator of microglial migration. Changes in the CNS lipidome associated with mTBI likely play a role in headache and in long-term neurodegenerative effects of repeated mTBI.

Elevated Levels of Arachidonic Acid-Derived Lipids Including Prostaglandins and Endocannabinoids Are Present Throughout ABHD12 Knockout Brains: Novel Insights Into the Neurodegenerative Phenotype.

Derived from arachidonic acid (AA), the endogenous cannabinoid (eCB) 2-arachidonoyl glycerol (2-AG) is a substrate for α/ß hydrolase domain-12 (ABHD12). Loss-of-function mutations of ABHD12 are associated with the neurodegenerative disorder polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract (PHARC). ABHD12 knockout (KO) mice show PHARC-like behaviors in older adulthood. Here, we test the hypothesis that ABHD12 deletion age-dependently regulates bioactive lipids in the CNS. Lipidomics analysis of the brainstem, cerebellum, cortex, hippocampus, hypothalamus, midbrain, striatum and thalamus from male young (3-4 months) and older (7 months) adult ABHD12 KO and age-matched wild-type (WT) mice was performed on over 80 lipids via HPLC/MS/MS, including eCBs, lipoamines, 2-acyl glycerols, free fatty acids, and prostaglandins (PGs). Aging and ABHD12 deletion drove widespread changes in the CNS lipidome; however, the effects of ABHD12 deletion were similar between old and young mice, meaning that many alterations in the lipidome precede PHARC-like symptoms. AA-derived lipids were particularly sensitive to ABHD12 deletion. 2-AG increased in the striatum, hippocampus, cerebellum, thalamus, midbrain, and brainstem, whereas the eCB N-arachidonoyl ethanolamine (AEA) increased in all 8 brain regions, along with at least 2-PGs. Aging also had a widespread effect on the lipidome and more age-related changes in bioactive lipids were found in ABHD12 KO mice than WT suggesting that ABHD12 deletion exacerbates the effects of age. The most robust effects of aging (independent of genotype) across the CNS were decreases in N-acyl GABAs and N-acyl glycines. In conclusion, levels of bioactive lipids are dynamic throughout adulthood and deleting ABHD12 disrupts the wider lipidome, modulating multiple AA-derived lipids with potential consequences for neuropathology.

Dissecting the Components of Sindbis Virus from Arthropod and Vertebrate Hosts: Implications for Infectivity Differences.

Sindbis virus (SINV) is an enveloped, single-stranded RNA virus, which is transmitted via mosquitos to a wide range of vertebrate hosts. SINV produced by vertebrate, baby hamster kidney (BHK) cells is more than an order of magnitude less infectious than SINV produced from mosquito (C6/36) cells. The cause of this difference is poorly understood. In this study, charge detection mass spectrometry was used to determine the masses of intact SINV particles isolated from BHK and C6/36 cells. The measured masses are substantially different: 52.88 MDa for BHK derived SINV and 50.69 MDa for C6/36 derived. Further analysis using several mass spectrometry-based methods and biophysical approaches indicates that BHK derived SINV has a substantially higher mass than C6/36 derived because in the lipid bilayer, there is a higher portion of lipids containing long chain fatty acids. The difference in lipid composition could influence the organization of the lipid bilayer. As a result, multiple stages of the viral lifecycle may be affected including assembly and budding, particle stability during transmission, and fusion events, all of which could contribute to the differences in infectivity.

Chemotherapy-induced cachexia dysregulates hypothalamic and systemic lipoamines and is attenuated by cannabigerol.

BACKGROUND: Muscle wasting, anorexia, and metabolic dysregulation are common side-effects of cytotoxic chemotherapy, having a dose-limiting effect on treatment efficacy, and compromising quality of life and mortality. Extracts of Cannabis sativa, and analogues of the major phytocannabinoid Δ9-tetrahydrocannabinol, have been used to ameliorate chemotherapy-induced appetite loss and nausea for decades. However, psychoactive side-effects limit their clinical utility, and they have little efficacy against weight loss. We recently established that the non-psychoactive phytocannabinoid cannabigerol (CBG) stimulates appetite in healthy rats, without neuromotor side-effects. The present study assessed whether CBG attenuates anorexia and/or other cachectic effects induced by the broad-spectrum chemotherapy agent cisplatin. METHODS: An acute cachectic phenotype was induced in adult male Lister-hooded rats by 6 mg/kg (i.p.) cisplatin. In total 66 rats were randomly allocated to groups receiving vehicle only, cisplatin only, or cisplatin and 60 or 120 mg/kg CBG (po, b.i.d.). Feeding behavior, bodyweight and locomotor activity were recorded for 72 hours, at which point rats were sacrificed for post-mortem analyses. Myofibre atrophy, protein synthesis and autophagy dysregulation were assessed in skeletal muscle, plasma metabolic profiles were obtained by untargeted 1H-NMR metabonomics, and levels of endocannabinoid-like lipoamines quantified in plasma and hypothalami by targeted HPLC-MS/MS lipidomics. RESULTS: CBG (120 mg/kg) modestly increased food intake, predominantly at 36-60hrs (p<0.05), and robustly attenuated cisplatin-induced weight loss from 6.3% to 2.6% at 72hrs (p<0.01). Cisplatin-induced skeletal muscle atrophy was associated with elevated plasma corticosterone (3.7 vs 13.1ng/ml, p<0.01), observed selectively in MHC type IIx (p<0.05) and IIb (p<0.0005) fibres, and was reversed by pharmacological rescue of dysregulated Akt/S6-mediated protein synthesis and autophagy processes. Plasma metabonomic analysis revealed cisplatin administration produced a wide-ranging aberrant metabolic phenotype (Q2Y=0.5380, p=0.001), involving alterations to glucose, amino acid, choline and lipid metabolism, citrate cycle, gut microbiome function, and nephrotoxicity, which were partially normalized by CBG treatment (Q2Y=0.2345, p=0.01). Lipidomic analysis of hypothalami and plasma revealed extensive cisplatin-induced dysregulation of central and peripheral lipoamines (29/79 and 11/26 screened, respectively), including reversible elevations in systemic N-acyl glycine concentrations which were negatively associated with the anti-cachectic effects of CBG treatment. CONCLUSIONS: Endocannabinoid-like lipoamines may have hitherto unrecognized roles in the metabolic side-effects associated with chemotherapy, with the N-acyl glycine subfamily in particular identified as a potential therapeutic target and/or biomarker of anabolic interventions. CBG-based treatments may represent a novel therapeutic option for chemotherapy-induced cachexia, warranting investigation in tumour-bearing cachexia models.

Cannabinoid CB2R receptors are upregulated with corneal injury and regulate the course of corneal wound healing.

CB2R receptors have demonstrated beneficial effects in wound healing in several models. We therefore investigated a potential role of CB2R receptors in corneal wound healing. We examined the functional contribution of CB2R receptors to the course of wound closure in an in vivo murine model. We additionally examined corneal expression of CB2R receptors in mouse and the consequences of their activation on cellular signaling, migration and proliferation in cultured bovine corneal epithelial cells (CECs). Using a novel mouse model, we provide evidence that corneal injury increases CB2R receptor expression in cornea. The CB2R agonist JWH133 induces chemorepulsion in cultured bovine CECs but does not alter CEC proliferation. The signaling profile of CB2R activation is activating MAPK and increasing cAMP accumulation, the latter perhaps due to Gs-coupling. Lipidomic analysis in bovine cornea shows a rise in acylethanolamines including the endocannabinoid anandamide 1 h after injury. In vivo, CB2R deletion and pharmacological block result in a delayed course of wound closure. In summary, we find evidence that CB2R receptor promoter activity is increased by corneal injury and that these receptors are required for the normal course of wound closure, possibly via chemorepulsion.

Broad and Region-Specific Impacts of the Synthetic Cannabinoid CP 55,940 in Adolescent and Adult Female Mouse Brains.

Relative to Δ9-tetrahydrocannabinol (THC), the synthetic cannabinoid CP 55,940 (CP) is significantly more potent and efficacious at cannabinoid receptors, the primary targets for endogenous cannabinoids (eCBs). eCBs belong to a large, interconnected lipidome of bioactive signaling molecules with a myriad of effects in optimal and pathological function. Recreational use of highly potent and efficacious synthetic cannabinoids is common amongst adolescents, potentially impacting brain development. Knowledge of the molecular outcomes of synthetic cannabinoid use will be important to develop more targeted therapies for synthetic cannabinoid intoxication and to prevent long-term disruption to the CNS. Here, we test the hypothesis that CP has age and region-dependent effects on the brain lipidome. Adolescent [post-natal day (PND) 35 and PND 50] and young adult female mice were given either an acute dose of CP or vehicle and brains were collected 2 h later. Eight brain regions were dissected and levels of ∼80 lipids were screened from each region using HPLC/MS/MS. CP had widespread effects on the brain lipidome in all age groups. Interestingly, more changes were observed in the PND 35 mice and more were reductions in a lipid's concentration, including region-dependent lowering of eCB levels. CP levels were highest in the cortex at PND 35, the hippocampus at PND 50, and in the cerebellum in the adult. These data provide novel insights into how high-potency, synthetic cannabinoids drive different, age-dependent, cellular signaling effects in the brain.

Cannabidiol's Upregulation of N-acyl Ethanolamines in the Central Nervous System Requires N-acyl Phosphatidyl Ethanolamine-Specific Phospholipase D.

Introduction: Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are bioactive cannabinoids. We recently showed that acute THC administration drives region-dependent changes in the mouse brain lipidome. This study tested the hypothesis that cell lines representing cell types present in the central nervous system (CNS), neurons (N18 cells), astrocytes (C6 glioma cells), and microglia (BV2 cells) would respond differently to THC, CBD, or their combination. This experimental strategy also allowed us to test the hypothesis that THC and CBD are metabolized differently if presented in combination and to test the hypothesis that responses to CBD are not like the fatty acid amide hydrolase (FAAH) inhibitor URB597. Finally, we tested the hypothesis that CBD's CNS effects would differ in the N-acyl phosphatidyl ethanolamine-specific phospholipase D (NAPE-PLD) knockout (KO) compared to wild-type (WT) mice. Methods: N18, C6, and BV2 cells were stimulated with 1 µM THC, 1 µM CBD, 1 µM THC:CBD, 1 µM URB597, or vehicle for 2 h and lipids extracted. Adult female WT and NAPE-PLD KO mice were injected with 3 mg/kg CBD or vehicle i.p., brains collected 2 h later, eight brain regions dissected, and lipids extracted. Extracted lipids were characterized and quantified using high-pressure liquid chromatography coupled with tandem mass spectrometry (HPLC/MS/MS). Results: Lipid levels in each cell type were differentially affected by THC, CBD, or THC:CBD with a few exceptions. In all cell lines, THC increased levels of arachidonic acid and CBD increased levels of N-acyl ethanolamines (NAEs), including N-arachidonoyl ethanolamine. More THC remained when cells were coincubated with CBD; however, levels of THC metabolites were cell-type dependent. CBD and URB597 caused very different lipid profiles in the cell-based assays with the primary similarity being increases in NAEs. CBD increased levels of NAEs in the WT hippocampus, cerebellum, thalamus, cortex, midbrain, and brainstem; however, NAEs did not increase in any brain region after CBD in NAPE-PLD KO mice. Conclusions: CBD and THC differentially modify the lipidome of the brain and CNS-type cell lines. Increases in NAEs observed after CBD treatment had previously been attributed to FAAH inhibition; however, data here suggest the alternative hypothesis that CBD is activating NAPE-PLD to increase NAE levels.

Δ9-Tetrahydrocannabinol and Cannabidiol Differentially Regulate Intraocular Pressure.

Purpose: It has been known for nearly 50 years that cannabis and the psychoactive constituent Δ9-tetrahydrocannabinol (THC) reduce intraocular pressure (IOP). Elevated IOP remains the chief hallmark and therapeutic target for glaucoma, a major cause of blindness. THC likely acts via one of the known cannabinoid-related receptors (CB1, CB2, GPR18, GPR119, GPR55) but this has never been determined explicitly. Cannabidiol (CBD) is a second major constituent of cannabis that has been found to be without effect on IOP in most studies. Methods: Effects of topically applied THC and CBD were tested in living mice by using tonometry and measurements of mRNA levels. In addition the lipidomic consequences of CBD treatment were tested by using lipid analysis. Results: We now report that a single topical application of THC lowered IOP substantially (∼28%) for 8 hours in male mice. This effect is due to combined activation of CB1 and GPR18 receptors each of which has been shown to lower ocular pressure when activated. We also found that the effect was sex-dependent, being stronger in male mice, and that mRNA levels of CB1 and GPR18 were higher in males. Far from inactive, CBD was found to have two opposing effects on ocular pressure, one of which involved antagonism of tonic signaling. CBD prevents THC from lowering ocular pressure. Conclusions: We conclude that THC lowers IOP by activating two receptors-CB1 and GPR18-but in a sex-dependent manner. CBD, contrary to expectation, has two opposing effects on IOP and can interfere with the effects of THC.

Revisiting cannabinoid receptor 2 expression and function in murine retina.

The cannabinoid receptor CB2 plays a significant role in the regulation of immune function whereas neuronal expression remains a subject of contention. Multiple studies have described CB2 in retina and a recent study showed that CB2 deletion altered retinal visual processing. We revisited CB2 expression using immunohistochemistry and a recently developed CB2-eGFP reporter mouse. We examined the consequence of acute vs. prolonged CB2 deactivation on the electroretinogram (ERG) responses. We also examined lipidomics in CB2 knockout mice and potential changes in microglia using Scholl analysis. Consistent with a published report, in CB2 receptor knockout mice see an increased ERG scotopic a-wave, as well as stronger responses in dark adapted cone-driven ON bipolar cells and, to a lesser extent cone-driven ON bipolar cells early in light adaptation. Significantly, however, acute block with CB2 antagonist, AM630, did not mimic the results observed in the CB2 knockout mice whereas chronic (7 days) block did. Immunohistochemical studies show no CB2 in retina under non-pathological conditions, even with published antibodies. Retinal CB2-eGFP reporter signal is minimal under baseline conditions but upregulated by intraocular injection of either LPS or carrageenan. CB2 knockout mice see modest declines in a broad spectrum of cannabinoid-related lipids. The numbers and morphology of microglia were unaltered. In summary minimal CB2 expression is seen in healthy retina. CB2 appears to be upregulated under pathological conditions. Previously reported functional consequences of CB2 deletion are an adaptive response to prolonged blockade of these receptors. CB2 therefore impacts retinal signaling but perhaps in an indirect, potentially extra-ocular fashion.

Δ9-Tetrahydrocannabinol changes the brain lipidome and transcriptome differentially in the adolescent and the adult.

Exposing the adolescent brain to drugs of abuse is associated with increased risk for adult onset psychopathologies. Cannabis use peaks during adolescence, with largely unknown effects on the developing brain. Cannabis' major psychoactive component, Δ9-tetrahydrocannabinol (THC) alters neuronal, astrocytic, and microglial signaling. Therefore, multiple cellular and signaling pathways are affected with a single dose of THC. The endogenous cannabinoids (eCBs), N-arachidonoyl ethanolamine (AEA) and 2-arachidonoyl glycerol (2-AG) are members of an interconnected lipidome that includes an emerging class of AEA structural analogs, the lipoamines, additional 2-acyl glycerols, free fatty acids, and prostaglandins (PGs). Lipids in this lipidome share many biosynthetic and metabolic pathways, yet have diverse signaling properties. Here, we show that acute THC drives age-dependent changes in this lipidome across 8 regions of the female mouse brain. Interestingly, most changes are observed in the adult, with eCBs and related lipids predominately decreasing. Analysis of THC and metabolites reveals an unequal distribution across these brain areas; however, the highest levels of THC were measured in the hippocampus (HIPP) in all age groups. Transcriptomic analysis of the HIPP after acute THC showed that like the lipidome, the adult transcriptome demonstrated significantly more changes than the adolescent. Importantly, the regulation of 31 genes overlapped between the adolescent and the adult, suggesting a conserved transcriptomic response in the HIPP to THC exposure independent of age. Taken together these data illustrate that the first exposure to a single dose of THC has profound effects on signaling in the CNS.

Metformin attenuates susceptibility to inflammation-induced preterm birth in mice with higher endocannabinoid levels.

Premature decidual senescence is a contributing factor to preterm birth. Fatty acid amide hydrolase mutant females (Faah-/-) with higher endocannabinoid levels are also more susceptible to preterm birth upon lipopolysaccharide (LPS) challenge due to enhanced decidual senescence; this is associated with mitogen-activated protein kinase p38 activation. Previous studies have shown that mechanistic target of rapamycin complex 1 (mTORC1) contributes to decidual senescence and promotes the incidence of preterm birth. In this study, we sought to attenuate premature decidual aging in Faah-/- females by targeting mTORC1 and p38 signaling pathways. Because metformin is known to inhibit mTOR and p38 signaling pathways, Faah-/- females were treated with metformin. These mice had a significantly lower preterm birth incidence with a higher rate of live birth after an LPS challenge on day 16 of pregnancy; metformin treatment did not affect placentation or neonatal birth weight. These results were associated with decreased levels of p38, as well as pS6, a downstream mediator of mTORC1 activity, in day 16 Faah-/-decidual tissues. Since metformin treatment attenuates premature decidual senescence with limited side effects during pregnancy, careful use of this drug may be effective in ameliorating specific adverse pregnancy events.

Chronic Adolescent Δ9-Tetrahydrocannabinol Treatment of Male Mice Leads to Long-Term Cognitive and Behavioral Dysfunction, Which Are Prevented by Concurrent Cannabidiol Treatment.

Introduction: The high prevalence of adolescent cannabis use, the association between this use and later psychiatric disease, and increased access to high-potency cannabis highlight the need for a better understanding of the long-term effects of adolescent cannabis use on cognitive and behavioral outcomes. Furthermore, increasing Δ9-tetrahydrocannabinol (THC) in high-potency cannabis is accompanied by a decrease in cannabidiol (CBD), thus an understanding of the interactions between CBD and THC in the neurodevelopmental effects of THC is also important. The current study examined the immediate and long-term behavioral consequences of THC, CBD, and their combination in a mouse model of adolescent cannabis use. Materials and Methods: Male CD1 mice received daily injections of THC (3 mg/kg), CBD (3 mg/kg), CBD+THC (3 mg/kg each), vehicle, or remained undisturbed in their home cage (no handling/injections), either during adolescence (postnatal day [PND] 28-48) or during early adulthood (PND 69-89). Animals were then evaluated with a battery of behavioral tests 1 day after drug treatment, and again after 42 drug-free days. The tests included the following: open field (day 1), novel object recognition (NOR; day 2), marble burying (day 3), elevated plus maze (EPM; day 4), and Nestlet shredding (day 5). Results: Chronic administration of THC during adolescence led to immediate and long-term impairments in object recognition/working memory, as measured by the NOR task. In contrast, adult administration of THC caused immediate, but not long term, impairment of object/working memory. Adolescent chronic exposure to THC increased repetitive and compulsive-like behaviors, as measured by the Nestlet shredding task. Chronic administration of THC, either during adolescence or during adulthood, led to a delayed increase in anxiety as measured by the EPM. All THC-induced behavioral abnormalities were prevented by the coadministration of CBD+THC, whereas CBD alone did not influence behavioral outcomes. Conclusion: These data suggest that chronic exposure to THC during adolescence leads to some of the behavioral abnormalities common in schizophrenia. Interestingly, CBD appeared to antagonize all THC-induced behavioral abnormalities. These findings support the hypothesis that adolescent THC use can impart long-term behavioral deficits; however, cotreatment with CBD prevents these deficits.