Anandamide in the dorsal periaqueductal gray inhibits sensory input without a correlation to sympathoexcitation.

There is growing literature supporting cannabinoids as a potential therapeutic for pain conditions. The development of chronic pain has been associated with reduced concentrations of the endogenous cannabinoid anandamide (AEA) in the midbrain dorsal periaqueductal gray (dPAG), and microinjections of synthetic cannabinoids into the dPAG are antinociceptive. Therefore, the goal of this study was to examine the role of the dPAG in cannabinoid-mediated sensory inhibition. Given that cannabinoids in the dPAG also elicit sympathoexcitation, a secondary goal was to assess coordination between sympathetic and antinociceptive responses. AEA was microinjected into the dPAG while recording single unit activity of wide dynamic range (WDR) dorsal horn neurons (DHNs) evoked by high intensity mechanical stimulation of the hindpaw, concurrently with renal sympathetic nerve activity (RSNA), in anesthetized male rats. AEA microinjected into the dPAG decreased evoked DHN activity (n = 24 units), for half of which AEA also elicited sympathoexcitation. AEA actions were mediated by cannabinoid 1 receptors as confirmed by local pretreatment with the cannabinoid receptor antagonist AM281. dPAG microinjection of the synaptic excitant DL-homocysteic acid (DLH) also decreased evoked DHN activity (n = 27 units), but in all cases this was accompanied by sympathoexcitation. Thus, sensory inhibition elicited from the dPAG is not exclusively linked with sympathoexcitation, suggesting discrete neuronal circuits. The rostrocaudal location of sites may affect evoked responses as AEA produced sensory inhibition without sympathetic effects at 86 % of caudal compared to 25 % of rostral sites, supporting anatomically distinct neurocircuits. These data indicate that spatially selective manipulation of cannabinoid signaling could provide analgesia without potentially harmful autonomic activation.

Sex and stressor modality influence acute stress-induced dynamic changes in corticolimbic endocannabinoid levels in adult Sprague Dawley rats.

Research over the past few decades has established a role for the endocannabinoid system in contributing to the neural and endocrine responses to stress exposure. The two endocannabinoid ligands, anandamide (AEA) and 2-arachidonoyl glycerol (2-AG), both play roles in regulating the stress response and both exhibit dynamic changes in response to stress exposure. Most of this previous research, however, was conducted in male rodents. Given that, especially in rodents, the stress response is influenced by sex, an understanding of how these dynamic responses of endocannabinoids in response to stress is influenced by sex could provide insight into sex differences of the acute stress response. We exposed adult, Sprague Dawley rats to different commonly utilized acute stress modalities, specifically restraint, swim and foot shock stress. Thirty minutes following stress onset, we excised the amygdala, hippocampus and medial prefrontal cortex, corticolimbic brain regions involved in the stress response, to measure endocannabinoid levels. When AEA levels were altered in response to restraint and swim stress, they were reduced, whereas exposure to foot shock stress led to an increase in the amygdala. 2-AG levels, when they were altered by stress exposure were only increased, specifically in males in the amygdala following swim stress, and in the hippocampus and medial prefrontal cortex overall following foot shock stress. This increase in 2-AG levels following stress only in males was the only sex difference found in stress-induced changes in endocannabinoid levels. There were no consistent sex differences observed. Collectively, these data contribute to our further understanding of the interactions between stress and endocannabinoid function.

Endocannabinoids as potential biomarkers: It's all about pre-analytics.

INTRODUCTION: Arachidonoyl ethanolamide (AEA) and 2-arachidonoyl glycerol (2-AG) are central lipid mediators of the endocannabinoid system. They are highly relevant due to their involvement in a wide variety of inflammatory, metabolic or malign diseases. Further elucidation of their modes of action and use as biomarkers in an easily accessible matrix, like blood, is restricted by their susceptibility to deviations during blood sampling and physiological co-dependences, which results in high variability of reported concentrations in low ng/mL ranges. OBJECTIVES: The objective of this review is the identification of critical parameters during the pre-analytical phase and proposal of minimum requirements for reliable determination of endocannabinoids (ECs) in blood samples. METHODS: Reported physiological processes influencing the EC concentrations were put into context with published pre-analytical research and stability data from bioanalytical method validation. RESULTS: The cause for variability in EC concentrations is versatile. In part, they are caused by inter-individual factors like sex, metabolic status and/or diurnal changes. Nevertheless, enzymatic activity in freshly drawn blood samples is the main reason for changing concentrations of AEA and 2-AG, besides additional non-enzymatic isomerization of the latter. CONCLUSION: Blood samples for EC analyses require immediate processing at low temperatures (>0 °C) to maintain sample integrity. Standardization of the respective blood tube or anti-coagulant, sampling time point, applied centrifugal force and complete processing time can further decrease variability caused by sample handling. Nevertheless, extensive characterization of study participants is needed to reduce distortion of clinical data caused by co-variables and facilitate research on the endocannabinoid system.

Cannabidiol and the corticoraphe circuit in post-traumatic stress disorder.

Post-Traumatic Stress Disorder (PTSD), characterized by re-experiencing, avoidance, negative affect, and impaired memory processing, may develop after traumatic events. PTSD is complicated by impaired plasticity and medial prefrontal cortex (mPFC) activity, hyperactivity of the amygdala, and impaired fear extinction. Cannabidiol (CBD) is a promising candidate for treatment due to its multimodal action that enhances plasticity and calms hyperexcitability. CBD's mechanism in the mPFC of PTSD patients has been explored extensively, but literature on the mechanism in the dorsal raphe nucleus (DRN) is lacking. Following the PRISMA guidelines, we examined current literature regarding CBD in PTSD and overlapping symptomologies to propose a mechanism by which CBD treats PTSD via corticoraphe circuit. Acute CBD inhibits excess 5-HT release from DRN to amygdala and releases anandamide (AEA) onto amygdala inputs. By first reducing amygdala and DRN hyperactivity, CBD begins to ameliorate activity disparity between mPFC and amygdala. Chronic CBD recruits the mPFC, creating harmonious corticoraphe signaling. DRN releases enough 5-HT to ameliorate mPFC hypoactivity, while the mPFC continuously excites DRN 5-HT neurons via glutamate. Meanwhile, AEA regulates corticoraphe activity to stabilize signaling. AEA prevents DRN GABAergic interneurons from inhibiting 5-HT release so the DRN can assist the mPFC in overcoming its hypoactivity. DRN-mediated restoration of mPFC activity underlies CBD's mechanism on fear extinction and learning of stress coping.

Targeting the endocannabinoid system for management of HIV-associated neuropathic pain: A systematic review.

Human immunodeficiency virus (HIV) infection and antiretroviral therapy can independently induce HIV-associated neuropathic pain (HIV-NP). There is a dearth of drugs or therapeutic modalities that can alleviate HIV-NP. Smoked cannabis has been reported to improve pain measures in patients with neuropathic pain. Cannabis, phytocannabinoids, and the endocannabinoids such N-arachidonoylethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG), produce some of their effects via cannabinoid receptors (CBRs). Endocannabinoids are degraded by various enzymes such as fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase. We searched PubMed, Google Scholar, clinicaltrials.gov and clinicaltrialsregister.eu using various key words and their combinations for published papers that studied HIV-NP and cannabis, cannabinoids, or endocannabinoids up to 27th December 2020. All original research articles that evaluated the efficacy of molecules that modulate the endocannabinoid system (ECS) for the prevention and/or treatment of pain in HIV-NP animal models and patients with HIV-NP were included. The PubMed search produced a total of 117 articles, whereas the Google Scholar search produced a total of 9467 articles. Amongst the 13 articles that fulfilled the inclusion criteria 11 articles were found in both searches whereas 2 articles were found in Google Scholar only. The clinicaltrials.gov and clinicaltrialsregister.eu searches produced five registered trials of which three were completed and with results. Ten preclinical studies found that the endocannabinoids (2-AG and AEA), synthetic mixed CB1R/CB2R agonist WIN 55,212-2, a CB2R-selective phytocannabinoid ß-caryophyllene, synthetic CB2R-selective agonists (AM1710, JWH015, JWH133 and Gp1a, but not HU308); FAAH inhibitors (palmitoylallylamide, URB597 and PF-3845) and a drug combination of indomethacin plus minocycline, which produces its effects in a CBR-dependent manner, either prevented the development of and/or attenuated established HIV-NP. Two clinical trials demonstrated greater efficacy of smoked cannabis over placebo in alleviating HIV-NP, whereas another clinical trial demonstrated that cannabidivarin, a cannabinoid that does not activate CBRs, did not reduce HIV-NP. The available preclinical results suggest that targeting the ECS for prevention and treatment of HIV-NP is a plausible therapeutic option. Clinical evidence shows that smoked cannabis alleviates HIV-NP. Further research is needed to find out if non-psychoactive drugs that target the ECS and are delivered by other routes than smoking could be useful as treatment options for HIV-NP.

Perinatal maternal high-fat diet induces early obesity and sex-specific alterations of the endocannabinoid system in white and brown adipose tissue of weanling rat offspring.

Perinatal maternal high-fat (HF) diet programmes offspring obesity. Obesity is associated with overactivation of the endocannabinoid system (ECS) in adult subjects, but the role of the ECS in the developmental origins of obesity is mostly unknown. The ECS consists of endocannabinoids, cannabinoid receptors (cannabinoid type-1 receptor (CB1) and cannabinoid type-2 receptor (CB2)) and metabolising enzymes. We hypothesised that perinatal maternal HF diet would alter the ECS in a sex-dependent manner in white and brown adipose tissue of rat offspring at weaning in parallel to obesity development. Female rats received standard diet (9 % energy content from fat) or HF diet (29 % energy content from fat) before mating, during pregnancy and lactation. At weaning, male and female offspring were killed for tissue harvest. Maternal HF diet induced early obesity, white adipocyte hypertrophy and increased lipid accumulation in brown adipose tissue associated with sex-specific changes of the ECS's components in weanling rats. In male pups, maternal HF diet decreased CB1 and CB2 protein in subcutaneous adipose tissue. In female pups, maternal HF diet increased visceral and decreased subcutaneous CB1. In brown adipose tissue, maternal HF diet increased CB1 regardless of pup sex. In addition, maternal HF diet differentially changed oestrogen receptor across the adipose depots in male and female pups. The ECS and oestrogen signalling play an important role in lipogenesis, adipogenesis and thermogenesis, and we observed early changes in their targets in adipose depots of the offspring. The present findings provide insights into the involvement of the ECS in the developmental origins of metabolic disease induced by inadequate maternal nutrition in early life.

Prolonged activation of human islet cannabinoid receptors in vitro induces adaptation but not dysfunction.

BACKGROUND: Although in vivo studies have implicated endocannabinoids in metabolic dysfunction, little is known about direct, chronic activation of the endocannabinoid system (ECS) in human islets. Therefore, this study investigated the effects of prolonged exposure to cannabinoid agonists on human islet gene expression and function. METHODS: Human islets were maintained for 2 and 5 days in the absence or presence of CB1r (ACEA) or CB2r (JWH015) agonists. Gene expression was quantified by RT-PCR, hormone levels by radioimmunoassay and apoptosis by caspase activities. RESULTS: Human islets express an ECS, with mRNAs encoding the biosynthetic and degrading enzymes NAPE-PLD, FAAH and MAGL being considerably more abundant than DAGLα, an enzyme involved in 2-AG synthesis, or CB1 and CB2 receptor mRNAs. Prolonged activation of CB1r and CB2r altered expression of mRNAs encoding ECS components, but did not have major effects on islet hormone secretion. JWH015 enhanced insulin and glucagon content at 2 days, but had no effect after 5 days. Treatment with ACEA or JWH015 for up to 5 days did not have marked effects on islet viability, as assessed by morphology and caspase activities. CONCLUSIONS: Maintenance of human islets for up to 5 days in the presence of CB1 and CB2 receptor agonists causes modifications in ECS element gene expression, but does not have any major impact on islet function or viability. GENERAL SIGNIFICANCE: These data suggest that the metabolic dysfunction associated with over-activation of the ECS in obesity and diabetes in humans is unlikely to be secondary to impaired islet function.

Interactions between dietary oil treatments and genetic variants modulate fatty acid ethanolamides in plasma and body weight composition.

Fatty acid ethanolamides (FAE), a group of lipid mediators derived from long-chain fatty acids (FA), mediate biological activities including activation of cannabinoid receptors, stimulation of fat oxidation and regulation of satiety. However, how circulating FAE levels are influenced by FA intake in humans remains unclear. The objective of the present study was to investigate the response of six major circulating FAE to various dietary oil treatments in a five-period, cross-over, randomised, double-blind, clinical study in volunteers with abdominal obesity. The treatment oils (60 g/12 552 kJ per d (60 g/3000 kcal per d)) provided for 30 d were as follows: conventional canola oil, high oleic canola oil, high oleic canola oil enriched with DHA, flax/safflower oil blend and corn/safflower oil blend. Two SNP associated with FAE degradation and synthesis were studied. Post-treatment results showed overall that plasma FAE levels were modulated by dietary FA and were positively correlated with corresponding plasma FA levels; minor allele (A) carriers of SNP rs324420 in gene fatty acid amide hydrolase produced higher circulating oleoylethanolamide (OEA) (P=0·0209) and docosahexaenoylethanolamide (DHEA) levels (P=0·0002). In addition, elevated plasma DHEA levels in response to DHA intake tended to be associated with lower plasma OEA levels and an increased gynoid fat mass. In summary, data suggest that the metabolic and physiological responses to dietary FA may be influenced via circulating FAE. Genetic analysis of rs324420 might help identify a sub-population that appears to benefit from increased consumption of DHA and oleic acid.

The involvement of TRPV1 in emesis and anti-emesis.

Diverse transmitter systems (e.g. acetylcholine, dopamine, endocannabinoids, endorphins, glutamate, histamine, 5-hydroxytryptamine, substance P) have been implicated in the pathways by which nausea and vomiting are induced and are targets for anti-emetic drugs (e.g. 5-hydroxytryptamine3 and tachykinin NK1 antagonists). The involvement of TRPV1 in emesis was discovered in the early 1990s and may have been overlooked previously as TRPV1 pharmacology was studied in rodents (mice, rats) lacking an emetic reflex. Acute subcutaneous administration of resiniferatoxin in the ferret, dog and Suncus murinus revealed that it had "broad-spectrum" anti-emetic effects against stimuli acting via both central (vestibular system, area postrema) and peripheral (abdominal vagal afferents) inputs. One of several hypotheses discussed here is that the anti-emetic effect is due to acute depletion of substance P (or another peptide) at a critical site (e.g. nucleus tractus solitarius) in the central emetic pathway. Studies in Suncus murinus revealed a potential for a long lasting (one month) effect against the chemotherapeutic agent cisplatin. Subsequent studies using telemetry in the conscious ferret compared the anti-emetic, hypothermic and hypertensive effects of resiniferatoxin (pungent) and olvanil (non-pungent) and showed that the anti-emetic effect was present (but reduced) with olvanil which although inducing hypothermia it did not have the marked hypertensive effects of resiniferatoxin. The review concludes by discussing general insights into emetic pathways and their pharmacology revealed by these relatively overlooked studies with TRPV1 activators (pungent an non-pungent; high and low lipophilicity) and antagonists and the potential clinical utility of agents targeted at the TRPV1 system.

2-Arachidonoylglycerol enhances platelet formation from human megakaryoblasts.

Platelets modulate vascular system integrity, and their loss is critical in haematological pathologies and after chemotherapy. Therefore, identification of molecules enhancing platelet production would be useful to counteract thrombocytopenia. We have previously shown that 2-arachidonoylglycerol (2-AG) acts as a true agonist of platelets, as well as it commits erythroid precursors toward the megakaryocytic lineage. Against this background, we sought to further interrogate the role of 2-AG in megakaryocyte/platelet physiology by investigating terminal differentiation, and subsequent thrombopoiesis. To this end, we used MEG-01 cells, a human megakaryoblastic cell line able to produce in vitro platelet-like particles. 2-AG increased the number of cells showing ruffled surface and enhanced surface expression of specific megakaryocyte/platelet surface antigens, typical hallmarks of terminal megakaryocytic differentiation and platelet production. Changes in cytoskeleton modeling also occurred in differentiated megakaryocytes and blebbing platelets. 2-AG acted by binding to CB1 and CB2 receptors, because specific antagonists reverted its effect. Platelets were split off from megakaryocytes and were functional: they contained the platelet-specific surface markers CD61 and CD49, whose levels increased following stimulation with a natural agonist like collagen. Given the importance of 2-AG for driving megakaryopoiesis and thrombopoiesis, not surprisingly we found that its hydrolytic enzymes were tightly controlled by classical inducers of megakaryocyte differentiation. In conclusion 2-AG, by triggering megakaryocyte maturation and platelet release, may have clinical efficacy to counteract thrombocytopenia-related diseases.

Astroglial CB1 cannabinoid receptors regulate leptin signaling in mouse brain astrocytes.

Type-1 cannabinoid (CB1) and leptin (ObR) receptors regulate metabolic and astroglial functions, but the potential links between the two systems in astrocytes were not investigated so far. Genetic and pharmacological manipulations of CB1 receptor expression and activity in cultured cortical and hypothalamic astrocytes demonstrated that cannabinoid signaling controls the levels of ObR expression. Lack of CB1 receptors also markedly impaired leptin-mediated activation of signal transducers and activators of transcription 3 and 5 (STAT3 and STAT5) in astrocytes. In particular, CB1 deletion determined a basal overactivation of STAT5, thereby leading to the downregulation of ObR expression, and leptin failed to regulate STAT5-dependent glycogen storage in the absence of CB1 receptors. These results show that CB1 receptors directly interfere with leptin signaling and its ability to regulate glycogen storage, thereby representing a novel mechanism linking endocannabinoid and leptin signaling in the regulation of brain energy storage and neuronal functions.

Progesterone reverts LPS-reduced FAAH activity in murine peripheral blood mononuclear cells by a receptor-mediated fashion.

Increased anandamide concentrations are associated with pregnancy failure. Anandamide levels are regulated by the fatty acid amide hydrolase (FAAH). The aim of the study was to investigate the role of progesterone (P) on FAAH modulation in murine peripheral blood mononuclear cells (PBMC) under septic conditions. We observed that in vivo administration of LPS to non-pregnant (NP) mice decreased FAAH activity of PBMC while in pregnant mice no changes in FAAH activity were observed. NP animals administered with P had a similar response to LPS as the pregnant animals. Also, NP mice injected with P antagonist and P showed that the effect of P on LPS-reduced FAAH activity was impaired. Furthermore, LPS produced a decrease in the ratio of PR-B/PR-A in NP animals. Our results showed that, in our model the endotoxin decreased PBMC's FAAH activity and this condition was reverted by P in a receptor-mediated fashion.