Frequent low-impact exposure to THC during adolescence causes persistent sexually dimorphic alterations in the response to viral infection in mice.

Adolescent exposure to Δ9-tetrahydrocannabinol (THC) has enduring effects on energy metabolism and immune function. Prior work showed that daily administration of a low-impact dose of THC (5 mg/kg, intraperitoneal) during adolescence alters transcription in adult microglia and disrupts their response to bacterial endotoxin or social stress. To explore the lasting impact of adolescent THC exposure on the brain's reaction to viral infection, we administered THC (5 mg/kg, intraperitoneal) in male and female mice once daily on postnatal day (PND) 30-43. When the mice reached adulthood (PND 70), we challenged them with the viral mimic, polyinosinic acid:polycytidylic acid [Poly(I:C)], and assessed sickness behavior (motor activity, body temperature) and whole brain gene transcription. Poly(I:C) caused an elevation in body temperature which was lessened by prior THC exposure in female but not male mice. Adolescent THC exposure did not affect the locomotor response to Poly(I:C) in either sex. Transcriptomic analyses showed that Poly(I:C) produced a substantial upregulation of immune-related genes in the brain, which was decreased by THC in females. Additionally, the viral mimic caused a male-selective downregulation in transcription of genes involved in neurodevelopment and synaptic transmission, which was abrogated by adolescent THC treatment. The results indicate that Poly(I:C) produces complex transcriptional alterations in the mouse brain, which are sexually dimorphic and differentially affected by early-life THC exposure. In particular, adolescent THC dampens the brain's antiviral response to Poly(I:C) in female mice and prevents the transcriptional downregulation of neuron-related genes caused by the viral mimic in male mice.

Ceramide contributes to pathogenesis and may be targeted for therapy in VCP inclusion body myopathy.

Knock-in homozygote VCPR155H/R155H mutant mice are a lethal model of valosin-containing protein (VCP)-associated inclusion body myopathy associated with Paget disease of bone, frontotemporal dementia and amyotrophic lateral sclerosis. Ceramide (d18:1/16:0) levels are elevated in skeletal muscle of the mutant mice, compared to wild-type controls. Moreover, exposure to a lipid-enriched diet reverses lethality, improves myopathy and normalizes ceramide levels in these mutant mice, suggesting that dysfunctions in lipid-derived signaling are critical to disease pathogenesis. Here, we investigated the potential role of ceramide in VCP disease using pharmacological agents that manipulate the ceramide levels in myoblast cultures from VCP mutant mice and VCP patients. Myoblasts from wild-type, VCPR155H/+ and VCPR155H/R155H mice, as well as patient-induced pluripotent stem cells (iPSCs), were treated with an inhibitor of ceramide degradation to increase ceramide via acid ceramidase (ARN082) for proof of principle. Three chemically distinct inhibitors of ceramide biosynthesis via serine palmitoyl-CoA transferase (L-cycloserine, myriocin or ARN14494) were used as a therapeutic strategy to reduce ceramide in myoblasts. Acid ceramidase inhibitor, ARN082, elevated cellular ceramide levels and concomitantly enhanced pathology. Conversely, inhibitors of ceramide biosynthesis L-cycloserine, myriocin and ARN14494 reduced ceramide production. The results point to ceramide-mediated signaling as a key contributor to pathogenesis in VCP disease and suggest that manipulating this pathway by blocking ceramide biosynthesis might exert beneficial effects in patients with this condition. The ceramide pathway appears to be critical in VCP pathogenesis, and small-molecule inhibitors of ceramide biosynthesis might provide therapeutic benefits in VCP and related neurodegenerative diseases.

The endocannabinoid system in the adipose organ.

The endocannabinoid system is found in most, if not all, mammalian organs and is involved in a variety of physiological functions, ranging from the control of synaptic plasticity in the brain to the modulation of smooth muscle motility in the gastrointestinal tract. This signaling complex consists of G protein-coupled cannabinoid receptors, endogenous ligands for those receptors (endocannabinoids) and enzymes/transporters responsible for the formation and deactivation of these ligands. There are two subtypes of cannabinoid receptors, CB1 and CB2, and two major endocannabinoids, arachidonoylethanolamide (anandamide) and 2-arachidonoyl-sn-glycerol (2-AG), which are produced upon demand through cleavage of distinct phospholipid precursors. All molecular components of the endocannabinoid system are represented in the adipose organ, where endocannabinoid signals are thought to regulate critical homeostatic processes, including adipogenesis, lipogenesis and thermogenesis. Importantly, obesity was found to be associated with excess endocannabinoid activity in visceral fat depots, and the therapeutic potential of normalizing such activity by blocking CB1 receptors has been the focus of substantial preclinical and clinical research. Results have been mixed thus far, mostly owing to the emergence of psychiatric side effects rooted in the protective functions served by brain endocannabinoids in mood and affect regulation. Further studies about the roles played by the endocannabinoid system in the adipose organ will offer new insights into the pathogenesis of obesity and might help identify new ways to leverage this signaling complex for therapeutic benefit.

Atypical Endocannabinoid Signaling Initiates a New Form of Memory-Related Plasticity at a Cortical Input to Hippocampus.

Endocannabinoids (ECBs) depress transmitter release at sites throughout the brain. Here, we describe another form of ECB signaling that triggers a novel form of long-term potentiation (LTP) localized to the lateral perforant path (LPP) which conveys semantic information from cortex to hippocampus. Two cannabinoid CB1 receptor (CB1R) signaling cascades were identified in hippocampus. The first is pregnenolone sensitive, targets vesicular protein Munc18-1 and depresses transmitter release; this cascade is engaged by CB1Rs in Schaffer-Commissural afferents to CA1 but not in the LPP, and it does not contribute to LTP. The second cascade is pregnenolone insensitive and LPP specific; it entails co-operative CB1R/ß1-integrin signaling to effect synaptic potentiation via stable enhancement of transmitter release. The latter cascade is engaged during LPP-dependent learning. These results link atypical ECB signaling to the encoding of a fundamental component of episodic memory and suggest a novel route whereby endogenous and exogenous cannabinoids affect cognition.

Dysfunctional oleoylethanolamide signaling in a mouse model of Prader-Willi syndrome.

Prader-Willi syndrome (PWS), the leading genetic cause of obesity, is characterized by a striking hyperphagic behavior that can lead to obesity, type-2 diabetes, cardiovascular disease and death. The molecular mechanism underlying impaired satiety in PWS is unknown. Oleoylethanolamide (OEA) is a lipid mediator involved in the control of feeding, body weight and energy metabolism. OEA produced by small-intestinal enterocytes during dietary fat digestion activates type-α peroxisome proliferator-activated receptors (PPAR-α) to trigger an afferent signal that causes satiety. Emerging evidence from genetic and human laboratory studies suggests that deficits in OEA-mediated signaling might be implicated in human obesity. In the present study, we investigated whether OEA contributes to feeding dysregulation in Magel2m+/p- (Magel2 KO) mice, an animal model of PWS. Fasted/refed male Magel2 KO mice eat more than do their wild-type littermates and become overweight with age. Meal pattern analyses show that hyperphagia in Magel2 KO is due to increased meal size and meal duration rather than to lengthening of the intermeal interval, which is suggestive of a defect in mechanisms underlying satiation. Food-dependent OEA accumulation in jejunum and fasting OEA levels in plasma are significantly greater in Magel2 KO mice than in wild-type controls. Together, these findings indicate that deletion of the Magel2 gene is accompanied by marked changes in OEA signaling. Importantly, intraperitoneal administration of OEA (10mg/kg) significantly reduces food intake in fasted/refed Magel2 KO mice, pointing to a possible use of this natural compound to control hunger in PWS.

Lipid-enriched diet rescues lethality and slows down progression in a murine model of VCP-associated disease.

Valosin-containing protein (VCP)-associated disease caused by mutations in the VCP gene includes combinations of a phenotypically heterogeneous group of disorders such as hereditary inclusion body myopathy, Paget's disease of bone, frontotemporal dementia and amyotrophic lateral sclerosis. Currently, there are no effective treatments for VCP myopathy or dementia. VCP mouse models carrying the common R155H mutation include several of the features typical of the human disease. In our previous investigation, VCP(R155H/R155H) homozygous mice exhibited progressive weakness and accelerated pathology prior to their early demise. Herein, we report that feeding pregnant VCP(R155H/+) heterozygous dams with a lipid-enriched diet (LED) results in the reversal of the lethal phenotype in VCP(R155H/R155H) homozygous offspring. We examined the effects of this diet on homozygous and wild-type mice from birth until 9 months of age. The LED regimen improved survival, motor activity, muscle pathology and the autophagy cascade. A targeted lipidomic analysis of skeletal muscle and liver revealed elevations in tissue levels of non-esterified palmitic acid and ceramide (d18:1/16:0), two lipotoxic substances, in the homozygous mice. The ability to reverse lethality, increase survival, and ameliorate myopathy and lipids deficits in the VCP(R155H/R155H) homozygous animals suggests that lipid supplementation may be a promising therapeutic strategy for patients with VCP-associated neurodegenerative diseases.

Second-Generation Non-Covalent NAAA Inhibitors are Protective in a Model of Multiple Sclerosis.

Palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) are endogenous lipid mediators that suppress inflammation. Their actions are terminated by the intracellular cysteine amidase, N-acylethanolamine acid amidase (NAAA). Even though NAAA may offer a new target for anti-inflammatory therapy, the lipid-like structures and reactive warheads of current NAAA inhibitors limit the use of these agents as oral drugs. A series of novel benzothiazole-piperazine derivatives that inhibit NAAA in a potent and selective manner by a non-covalent mechanism are described. A prototype member of this class (8) displays high oral bioavailability, access to the central nervous system (CNS), and strong activity in a mouse model of multiple sclerosis (MS). This compound exemplifies a second generation of non-covalent NAAA inhibitors that may be useful in the treatment of MS and other chronic CNS disorders.

Fasting stimulates 2-AG biosynthesis in the small intestine: role of cholinergic pathways.

The endocannabinoids are lipid-derived signaling molecules that control feeding and energy balance by activating CB1-type cannabinoid receptors in the brain and peripheral tissues. Previous studies have shown that oral exposure to dietary fat stimulates endocannabinoid signaling in the rat small intestine, which provides positive feedback that drives further food intake and preference for fat-rich foods. We now describe an unexpectedly broader role for cholinergic signaling of the vagus nerve in the production of the endocannabinoid, 2-arachidonoyl-sn-glycerol (2-AG), in the small intestine. We show that food deprivation increases levels of 2-AG and its lipid precursor, 1,2-diacylglycerol, in rat jejunum mucosa in a time-dependent manner. This response is abrogated by surgical resection of the vagus nerve or pharmacological blockade of small intestinal subtype-3 muscarinic acetylcholine (m3 mAch) receptors, but not inhibition of subtype-1 muscarinic acetylcholine (m1 mAch). We further show that blockade of peripheral CB1 receptors or intestinal m3 mAch receptors inhibits refeeding in fasted rats. The results suggest that food deprivation stimulates 2-AG-dependent CB1 receptor activation through a mechanism that requires efferent vagal activation of m3 mAch receptors in the jejunum, which, in turn, may promote feeding after a fast.

Endocannabinoid signaling in the gut mediates preference for dietary unsaturated fats.

Dietary fat exerts a potent stimulatory effect on feeding. This effect is mediated, at least in part, by a cephalic mechanism that involves recruitment of the vagus nerve and subsequent activation of endocannabinoid signaling in the gut. Here, we used a sham-feeding protocol in rats to identify fatty-acid constituents of dietary fat that might be responsible for triggering small-intestinal endocannabinoid signaling. Sham feeding rats with a corn oil emulsion increased endocannabinoid levels in jejunum, relative to animals that received either mineral oil (which contains no fatty acids) or no oil. Sham-feeding emulsions containing oleic acid (18:1) or linoleic acid (18:2) caused, on average, a nearly 2-fold accumulation of jejunal endocannabinoids, whereas emulsions containing stearic acid (18:0) or linolenic acid (18:3) had no such effect. In a 2-bottle-choice sham-feeding test, rats displayed strong preference for emulsions containing 18:2, which was blocked by pretreatment with the peripherally restricted CB1 cannabinoid receptor antagonists, AM6546 and URB447. Our results suggest that oral exposure to the monoenoic and dienoic fatty acid component of dietary fat selectively initiates endocannabinoid mobilization in the gut, and that this local signaling event is essential for fat preference.

Overactivation of the Endocannabinoid System in Adolescence Disrupts Adult Adipose Organ Function in Mice.

Cannabis use stimulates calorie intake, but epidemiological studies show that people who regularly use it are leaner than those who don't. Two explanations have been proposed for this paradoxical finding. One posits that Δ9-tetrahydrocannabinol (THC) in cannabis desensitizes adipose CB1 cannabinoid receptors, stopping their stimulating effects on lipogenesis and adipogenesis. Another explanation is that THC exposure in adolescence, when habitual cannabis use typically starts, produces lasting changes in the developing adipose organ, which impacts adult systemic energy use. Here, we consider these possibilities in the light of a study which showed that daily THC administration in adolescent mice produces an adult metabolic phenotype characterized by reduced fat mass, partial resistance to obesity and dyslipidemia, and impaired thermogenesis and lipolysis. The phenotype, whose development requires activation of CB1 receptors in differentiated adipocytes, is associated with overexpression of myocyte proteins in the adipose organ with unchanged CB1 expression. We propose that adolescent exposure to THC causes lasting adipocyte dysfunction and the consequent emergence of a metabolic state that only superficially resembles healthy leanness. A corollary of this hypothesis, which should be addressed in future studies, is that CB1 receptors and their endocannabinoid ligands may contribute to the maintenance of adipocyte differentiation during adolescence.

Development of Potent and Selective Monoacylglycerol Lipase Inhibitors. SARs, Structural Analysis, and Biological Characterization.

New potent, selective monoacylglycerol lipase (MAGL) inhibitors based on the azetidin-2-one scaffold ((±)-5a-v, (±)-6a-j, and (±)-7a-d) were developed as irreversible ligands, as demonstrated by enzymatic and crystallographic studies for (±)-5d, (±)-5l, and (±)-5r. X-ray analyses combined with extensive computational studies allowed us to clarify the binding mode of the compounds. 5v was identified as selective for MAGL when compared with other serine hydrolases. Solubility, in vitro metabolic stability, cytotoxicity, and absence of mutagenicity were determined for selected analogues. The most promising compounds ((±)-5c, (±)-5d, and (±)-5v) were used for in vivo studies in mice, showing a decrease in MAGL activity and increased 2-arachidonoyl-sn-glycerol levels in forebrain tissue. In particular, 5v is characterized by a high eudysmic ratio and (3R,4S)-5v is one of the most potent irreversible inhibitors of h/mMAGL identified thus far. These results suggest that the new MAGL inhibitors have therapeutic potential for different central and peripheral pathologies.

Activation of type 5 metabotropic glutamate receptors and diacylglycerol lipase-α initiates 2-arachidonoylglycerol formation and endocannabinoid-mediated analgesia.

Acute stress reduces pain sensitivity by engaging an endocannabinoid signaling circuit in the midbrain. The neural mechanisms governing this process and molecular identity of the endocannabinoid substance(s) involved are unknown. We combined behavior, pharmacology, immunohistochemistry, RNA interference, quantitative RT-PCR, enzyme assays, and lipidomic analyses of endocannabinoid content to uncover the role of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG) in controlling pain sensitivity in vivo. Here, we show that footshock stress produces antinociception in rats by activating type 5 metabotropic glutamate receptors (mGlu(5)) in the dorsolateral periaqueductal gray (dlPAG) and mobilizing 2-AG. Stimulation of mGlu(5) in the dlPAG with DHPG [(S)-3,5-dihydroxyphenylglycine] triggered 2-AG formation and enhanced stress-dependent antinociception through a mechanism dependent upon both postsynaptic diacylglycerol lipase (DGL) activity, which releases 2-AG, and presynaptic CB(1) cannabinoid receptors. Pharmacological blockade of DGL activity in the dlPAG with RHC80267 [1,6-bis(cyclohexyloximinocarbonylamino)hexane] and (-)-tetrahydrolipstatin (THL), which inhibit activity of DGL-α and DGL-ß isoforms, suppressed stress-induced antinociception. Inhibition of DGL activity in the dlPAG with THL selectively decreased accumulation of 2-AG without altering levels of anandamide. The putative 2-AG-synthesizing enzyme DGL-α colocalized with mGlu(5) at postsynaptic sites of the dlPAG, whereas CB(1) was confined to presynaptic terminals, consistent with a role for 2-AG as a retrograde signaling messenger. Finally, virally mediated silencing of DGL-α, but not DGL-ß, transcription in the dlPAG mimicked effects of DGL inhibition in suppressing both endocannabinoid-mediated stress antinociception and 2-AG formation. The results indicate that activation of the postsynaptic mGlu(5)-DGL-α cascade triggers retrograde 2-AG signaling in vivo. This pathway is required for endocannabinoid-mediated stress-induced analgesia.

Assay of Monoacylglycerol Lipase Activity.

Monoacylglycerol lipase (MGL/MAGL/MGLL) is a serine hydrolase involved in the biological deactivation of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). 2-AG is the most abundant endogenous lipid agonists for cannabinoid receptors in the brain and elsewhere in the body. In the central nervous system (CNS), MGL is localized to presynaptic nerve terminals of both excitatory and inhibitory synapses, where it controls the regulatory actions of 2-AG on synaptic transmission and plasticity. In this chapter, we describe an in vitro method to assess MGL activity by liquid chromatography/mass spectrometry (LC/MS)-based quantitation of its reaction product. The method may be used to determine basal or altered MGL activity in cells or tissues after pharmacological, genetic, or biological interventions. In addition, the assay can be used for MGL inhibitor screening using purified recombinant enzyme or MGL-overexpressing cells.

Phytocannabinoids, the Endocannabinoid System and Male Reproduction.

The endocannabinoid system (ECS) is comprised of a set of lipid-derived messengers (the endocannabinoids, ECBs), proteins that control their production and degradation, and cell-surface cannabinoid (CB) receptors that transduce their actions. ECB molecules such as 2-arachidonoyl-sn-glycerol (2-AG) and anandamide (arachidonoyl ethanolamide) are produced on demand and deactivated through enzymatic actions tightly regulated both temporally and spatially, serving homeostatic roles in order to respond to various challenges to the body. Key components of the ECS are present in the hypothalamus-pituitary-gonadal (HPG) axis, which plays critical roles in the development and regulation of the reproductive system in both males and females. ECB signaling controls the action at each stage of the HPG axis through CB receptors expressed in the hypothalamus, pituitary, and reproductive organs such as the testis and ovary. It regulates the secretion of hypothalamic gonadotropin-releasing hormone (GnRH), pituitary follicle-stimulating hormone (FSH) and luteinizing hormone (LH), estrogen, testosterone, and affects spermatogenesis in males. Δ9-tetrahydrocannabinol (THC) and other phytocannabinoids from Cannabis sativa affect a variety of physiological processes by altering, or under certain conditions hijacking, the ECB system. Therefore, phytocannabinoids, in particular THC, may modify the homeostasis of the HPG axis by altering CB receptor signaling and cause deficits in reproductive function. While the ability of phytocannabinoids, THC and/or cannabidiol (CBD), to reduce pain and inflammation provides promising opportunities for therapeutic intervention for genitourinary and degenerative disorders, important questions remain regarding their unwanted long-term effects. It is nevertheless clear that the therapeutic potential of modulating the ECS calls for further scientific and clinical investigation.

Fatty acid amide hydrolase inhibition alleviates anxiety-like symptoms in a rat model used to study post-traumatic stress disorder.

BACKGROUND AND AIM: Post-traumatic stress disorder (PTSD), a chronic debilitating condition that affects nearly 5-10% of American adults, is treated with a handful of FDA-approved drugs that provide at best symptomatic relief and exert multiple side effects. Preclinical and clinical evidence shows that inhibitors of the enzyme fatty acid amide hydrolase (FAAH), which deactivates the endocannabinoid anandamide, exhibit anxiolytic-like properties in animal models. In the present study, we investigated the effects of two novel brain-permeable FAAH inhibitors - the compounds ARN14633 and ARN14280 - in a rat model of predator stress-induced long-term anxiety used to study PTSD. METHODS: We exposed male Sprague-Dawley rats to 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), a volatile constituent of fox feces, and assessed anxiety-like behaviors in the elevated plus maze (EPM) test seven days later. We measured FAAH activity using a radiometric assay and brain levels of FAAH substrates by liquid chromatography/tandem mass spectrometry. RESULTS: Rats challenged with TMT developed persistent (≥ 7 days) anxiety-like symptoms in the EPM test. Intraperitoneal administration of ARN14633 or ARN14280 1 h before testing suppressed TMT-induced anxiety-like behaviors with median effective doses (ED50) of 0.23 and 0.33 mg/kg, respectively. The effects were negatively correlated (ARN14663: R2 = 0.455; ARN14280: R2 = 0.655) with the inhibition of brain FAAH activity and were accompanied by increases in brain FAAH substrate levels. CONCLUSIONS: The results support the hypothesis that FAAH-regulated lipid signaling serves important regulatory functions in the response to stress and confirm that FAAH inhibitors may be useful for the management of PTSD.

Adolescent exposure to low-dose Δ9-tetrahydrocannabinol depletes the ovarian reserve in female mice.

Cannabis use by adolescents is widespread, but its effects on the ovaries remain largely unknown. Δ9-tetrahydrocannabinol (THC) exerts its pharmacological effects by activating, and in some conditions hijacking, cannabinoid receptors (CBRs). We hypothesized that adolescent exposure to THC affects ovarian function in adulthood. Peripubertal female C57BL/6N mice were given THC (5 mg/kg) or its vehicle, once daily by intraperitoneal injection. Some mice received THC from postnatal day (PND) 30-33 and their ovaries were harvested PND34; other mice received THC from PND30-43, and their ovaries were harvested PND70. Adolescent treatment with THC depleted ovarian primordial follicle numbers by 50% at PND70, 4 weeks after the last dose. The treatment produced primordial follicle activation, which persisted until PND70. THC administration also caused DNA damage in primary follicles and increased PUMA protein expression in oocytes of primordial and primary follicles. Both CB1R and CB2R were expressed in oocytes and theca cells of ovarian follicles. Enzymes involved in the formation (N-acylphosphatidylethanolamine phospholipase D) or deactivation (fatty acid amide hydrolase) of the endocannabinoid anandamide were expressed in granulosa cells of ovarian follicles and interstitial cells. Levels of mRNA for CBR1 were significantly increased in ovaries after adolescent THC exposure, and upregulation persisted for at least 4 weeks. Our results support that adolescent exposure to THC may cause aberrant activation of the ovarian endocannabinoid system in female mice, resulting in substantial loss of ovarian reserve in adulthood. Relevance of these findings to women who frequently used cannabis during adolescence warrants investigation.

Adolescent exposure to low-dose THC disrupts energy balance and adipose organ homeostasis in adulthood.

One of cannabis' most iconic effects is the stimulation of hedonic high-calorie eating-the "munchies"-yet habitual cannabis users are, on average, leaner than non-users. We asked whether this phenotype might result from lasting changes in energy balance established during adolescence, when use of the drug often begins. We found that daily low-dose administration of cannabis' intoxicating constituent, Δ9-tetrahydrocannabinol (THC), to adolescent male mice causes an adult metabolic phenotype characterized by reduced fat mass, increased lean mass and utilization of fat as fuel, partial resistance to diet-induced obesity and dyslipidemia, enhanced thermogenesis, and impaired cold- and ß-adrenergic receptor-stimulated lipolysis. Further analyses revealed that this phenotype is associated with molecular anomalies in the adipose organ, including ectopic overexpression of muscle-associated proteins and heightened anabolic processing. Thus, adolescent exposure to THC may promote an enduring "pseudo-lean" state that superficially resembles healthy leanness but might in fact be rooted in adipose organ dysfunction.

Frequent Low-Dose Δ9-Tetrahydrocannabinol in Adolescence Disrupts Microglia Homeostasis and Disables Responses to Microbial Infection and Social Stress in Young Adulthood.

BACKGROUND: During adolescence, microglia are actively involved in neocortical maturation while concomitantly undergoing profound phenotypic changes. Because the teenage years are also a time of experimentation with cannabis, we evaluated whether adolescent exposure to the drug's psychotropic constituent, Δ9-tetrahydrocannabinol (THC), might persistently alter microglia function. METHODS: We administered THC (5 mg/kg, intraperitoneal) once daily to male and female mice from postnatal day (PND) 30 to PND44 and examined the transcriptome of purified microglia in adult animals (PND70 and PND120) under baseline conditions or following either of two interventions known to recruit microglia: lipopolysaccharide injection and repeated social defeat. We used high-dimensional mass cytometry by time-of-flight to map brain immune cell populations after lipopolysaccharide challenge. RESULTS: Adolescent THC exposure produced in mice of both sexes a state of microglial dyshomeostasis that persisted until young adulthood (PND70) but receded with further aging (PND120). Key features of this state included broad alterations in genes involved in microglia homeostasis and innate immunity along with marked impairments in the responses to lipopolysaccharide- and repeated social defeat-induced psychosocial stress. The endocannabinoid system was also dysfunctional. The effects of THC were prevented by coadministration of either a global CB1 receptor inverse agonist or a peripheral CB1 neutral antagonist and were not replicated when THC was administered in young adulthood (PND70-84). CONCLUSIONS: Daily low-intensity CB1 receptor activation by THC during adolescence may disable critical functions served by microglia until young adulthood with potentially wide-ranging consequences for brain and mental health.

Diacylglycerol lipase-alpha and -beta control neurite outgrowth in neuro-2a cells through distinct molecular mechanisms.

The endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG) is produced through hydrolysis of 1,2-diacyl-sn-glycerol (DAG), which is catalyzed by DAG lipase (DGL). Two DGL isoforms have been molecularly cloned, but their respective roles in endocannabinoid signaling have not been fully elucidated. Here, we report that DGL-α and DGL-ß may contribute to all-trans-retinoic acid (RA)-induced neurite outgrowth in neuroblastoma Neuro-2a cells through distinct mechanisms. RA-induced differentiation of Neuro-2a cells was associated with elevations of cellular 2-AG levels and DGL activity, which were accompanied by temporally separated transcription of DGL-α and DGL-ß mRNA. Knockdown of either DGL-α or DGL-ß expression attenuated neurite outgrowth, which indicates that both isoforms contribute to neuritogenesis. Immunostaining experiments showed that DGL-ß is localized to perinuclear lipid droplets, whereas DGL-α is found on plasma membranes. After RA-induced differentiation, both DGL-α- and DGL-ß-green fluorescent protein were distributed also in neurites but in distinguishable patterns. Overexpression of either DGL-α or DGL-ß increased the number of neurite-bearing cells, but DGL-ß caused substantially larger morphological changes than DGL-α did. Finally, the cannabinoid-1 antagonist rimonabant (1 µM) inhibited DGL-α-induced neuritogenesis, whereas it had no such effect on DGL-ß-induced morphological differentiation. The results indicate that RA-induced DGL expression is required for neurite outgrowth of Neuro-2a cells. The findings further suggest that DGL-α and -ß may regulate neurite outgrowth by engaging temporally and spatially distinct molecular pathways.

Ceramide induces serotonin release from RBL-2H3 mast cells through calcium mediated activation of phospholipase A2.

Ceramide has been suggested to function as a mediator of exocytosis in response to the addition of a calcium ionophore from PC12 cells. Here, we show that although cell-permeable C(6)-ceramide or a calcium ionophore alone did not increase either the degranulation of serotonin or the release of arachidonic acid (AA) from RBL-2H3 cells, their combined effect significantly stimulated these processes in a time- and dose-dependent manner. This effect was inhibited by the presence of an exogenous calcium chelator and significantly suppressed by the CERK inhibitor (K1) and phospholipase A(2) (PLA(2)) inhibitors. Moreover, cytosolic PLA(2) GIVA (cPLA(2) GIVA) siRNA-transfected RBL-2H3 cells showed a lower level of serotonin release than scramble siRNA-transfected cells. Little is known about the regulation of degranulation proximal to the activation of cytosolic phospholipase A(2) GIVA, the initial rate-limiting step in RBL-2H3 cells. In this study, we suggest that CERK, ceramide-1-phosphate, and PLA(2) are involved in degranulation in a calcium-dependent manner. Inhibition of p44/p42 mitogen-activated protein kinase partially decreased the AA release, but did not affect degranulation. Furthermore, treatment of the cells with AA (ω-6, C20:4), not linoleic acid (ω-6, C18:2) or α-linolenic acid (ω-6, C18:3), induced degranulation. Taken together, these results suggest that ceramide is involved in mast cell degranulation via the calcium-mediated activation of PLA(2).