Endogenous cannabinoids are required for MC4R-mediated control of energy homeostasis.

Hypothalamic regulation of feeding and energy expenditure is a fundamental and evolutionarily conserved neurophysiological process critical for survival. Dysregulation of these processes, due to environmental or genetic causes, can lead to a variety of pathological conditions ranging from obesity to anorexia. Melanocortins and endogenous cannabinoids (eCBs) have been implicated in the regulation of feeding and energy homeostasis; however, the interaction between these signaling systems is poorly understood. Here, we show that the eCB 2-arachidonoylglycerol (2-AG) regulates the activity of melanocortin 4 receptor (MC4R) cells in the paraventricular nucleus of the hypothalamus (PVNMC4R) via inhibition of afferent GABAergic drive. Furthermore, the tonicity of eCBs signaling is inversely proportional to energy state, and mice with impaired 2-AG synthesis within MC4R neurons weigh less, are hypophagic, exhibit increased energy expenditure, and are resistant to diet-induced obesity. These mice also exhibit MC4R agonist insensitivity, suggesting that the energy state-dependent, 2-AG-mediated suppression of GABA input modulates PVNMC4R neuron activity to effectively respond to the MC4R natural ligands to regulate energy homeostasis. Furthermore, post-developmental disruption of PVN 2-AG synthesis results in hypophagia and death. These findings illustrate a functional interaction at the cellular level between two fundamental regulators of energy homeostasis, the melanocortin and eCB signaling pathways in the hypothalamic feeding circuitry.

Monoacylglycerol Lipase Inhibition Using JZL184 Attenuates Paw Inflammation and Functional Deficits in a Mouse Model of Inflammatory Arthritis.

Background: Patients with rheumatoid arthritis (RA) experience joint swelling and cartilage destruction resulting in chronic pain, functional disability, and compromised joint function. Current RA treatments, including glucocorticoid receptor agonists, produce adverse side effects and lack prolonged treatment efficacy. Cannabinoids (i.e., cannabis-like signaling molecules) exert anti-inflammatory and analgesic effects with limited side effects compared to traditional immunosuppressants, making them excellent targets for the development of new arthritic therapeutics. Monoacylglycerol lipase (MAGL) inhibition reduces inflammation in mouse models of acute inflammation, through cannabinoid receptor dependent and independent pathways. The current study investigated the efficacy of inhibiting synthetic and catabolic enzymes that regulate the endocannabinoid 2-arachidonoylglycerol (2-AG) in blocking paw inflammation, pain-related behaviors, and functional loss caused by collagen-induced arthritis (CIA). Methods: Male DB1A mice subjected to CIA were administered the glucocorticoid agonist dexamethasone (DEX), MAGL inhibitor JZL184 (8 or 40 mg/kg, s.c.), alone or in combination, or diacylglycerol lipase ß (DAGLß) inhibitor KT109 (40 mg/kg, s.c.). CIA-induced deficits were assayed by arthritic clinical scoring, paw thickness measurements, and behavioral tests of pain and paw function. Results: DEX or dual administration with JZL184 reduced paw thickness and clinical scores, and JZL184 dose-dependently attenuated grip strength and balance beam deficits caused by CIA. Traditional measures of pain-induced behaviors (hyperalgesia and allodynia) were inconsistent. The antiarthritic effects of JZL184 (40 mg/kg) were largely blocked by coadministration of the CB2 antagonist SR144528, and the DAGLß inhibitor KT109 had no effect on CIA, indicating that these effects likely occurred through CB2 activation. Conclusions: MAGL inhibition reduced paw inflammation and pain-depressed behavioral signs of arthritis, likely through an endocannabinoid mechanism requiring CB2. These data support the development of MAGL as a target for therapeutic treatment of inflammatory arthritis.

Granulocyte Colony-Stimulating Factor Enhances Brain Repair Following Traumatic Brain Injury Without Requiring Activation of Cannabinoid Receptors.

Introduction: Treatment of traumatic brain injury (TBI) with granulocyte colony-stimulating factor (G-CSF) has been shown to enhance brain repair by direct neurotrophic actions on neural cells and by modulating the inflammatory response. Administration of cannabinoids after TBI has also been reported to enhance brain repair by similar mechanisms. Objectives: The primary objective of this study was to test the hypothesis that G-CSF mediates brain repair by interacting with the endocannabinoid system. Methods and Results: (i) Mice that underwent controlled cortical impact (CCI) were treated with G-CSF for 3 days either alone or in the presence of selective cannabinoid receptor 1 (CB1-R) or cannabinoid receptor 2 (CB2-R) agonists and antagonists. The trauma resulted in decreased expression of CB1-R and increased expression of CB2-R in the cortex, striatum, and hippocampus. Cortical and striatal levels of the major endocannabinoid ligand, 2-arachidonoyl-glycerol, were also increased by the CCI. Administration of the hematopoietic cytokine, G-CSF, following TBI, resulted in mitigation or reversal of trauma-induced CB1-R downregulation and CB2-R upregulation in the three brain regions. Treatment with CB1-R agonist (WIN55) or CB2-R agonist (HU308) mimicked the effects of G-CSF. (ii) Pharmacological blockade of CB1-R or CB2-R was not effective in preventing G-CSF's mitigation or reversal of trauma-induced alterations in these receptors. Conclusions: These results suggest that cellular and molecular mechanisms that mediate subacute effects of G-CSF do not depend on activation of CB1 or CB2 receptors. Failure of selective CB receptor antagonists to prevent the effects of G-CSF in this model has to be accepted with caution. CB receptor antagonists can interact with other CB and non-CB receptors. Investigation of the role of CB receptors in this TBI model will require studies with CB1-R and in CB2-R knockout mice to avoid nonspecific interaction of CB receptor agents with other receptors.

Impact of circadian rhythmicity and sleep restriction on circulating endocannabinoid (eCB) N-arachidonoylethanolamine (anandamide).

OBJECTIVE: The endocannabinoid (eCB) system is involved in diverse aspects of human physiology and behavior but little is known about the impact of circadian rhythmicity on the system. The two most studied endocannabinoids, AEA (ananamide) and 2-AG (2-arachidonoylglycerol), can be measured in peripheral blood however the functional relevance of peripheral eCB levels is not clear. Having previously detailed the 24-h profile of serum 2-AG, here we report the 24-h serum profile of AEA to determine if these two endocannabinoids vary in parallel across the biological day including a nocturnal 8.5-h sleep period. Further, we assessed and compared the effect of a physiological challenge, in the form of sleep restriction to 4.5-h, on these two profiles. METHODS: In this randomized crossover study, we examined serum concentrations of AEA across a 24-h period in fourteen young adults. Congeners of AEA, the structural analogs oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) were simultaneously assayed. Prior to 24-h blood sampling, each participant was exposed to two nights of normal (8.5 h) or restricted sleep (4.5 h). The two sleep conditions were separated by at least one month. In both sleep conditions, during the period of blood sampling, each individual ate the same high-carbohydrate meal at 0900, 1400, and 1900. RESULTS: Mean 24-h concentrations of AEA were 0.697 ±â€¯0.11 pmol/ml. A reproducible biphasic 24-h profile of AEA was observed with a first peak occurring during early sleep (0200) and a second peak in the mid-afternoon (1500) while a nadir was detected in the mid-morning (1000). The 24-h profiles for both OEA and PEA followed a similar pattern to that observed for AEA. AEA, OEA, and PEA levels were not affected by sleep restriction at any time of day, contrasting with the elevation of early afternoon levels previously observed for 2-AG. CONCLUSIONS: The 24-h rhythm of AEA is markedly different from that of 2-AG, being of lesser amplitude and biphasic, rather than monophasic. These observations suggest distinct regulatory pathways of the two eCB and indicate that time of day needs to be carefully controlled in studies attempting to delineate their relative roles. Moreover, unlike 2-AG, AEA is not altered by sleep restriction, suggesting that physiological perturbations may affect AEA and 2-AG differently. Similar 24-h profiles were observed for OEA and PEA following normal and restricted sleep, further corroborating the validity of the wave-shape and lack of response to sleep loss observed for the AEA profile. Therapeutic approaches involving agonism or antagonism of peripheral eCB signaling will likely need to be tailored according to time of day.

Integrating endocannabinoid signaling in the regulation of anxiety and depression.

Brain endogenous cannabinoid (eCB) signaling seems to harmonize appropriate behavioral responses, which are essential for the organism's long-term viability and homeostasis. Dysregulation of eCB signaling contributes to negative emotional states and increased stress responses. An understanding of the underlying neural cell populations and neural circuit regulation will enable the development of therapeutic strategies to mitigate behavioral maladaptation and provide insight into the influence of eCB on the neural circuits involved in anxiety and depression. This review focuses on recent evidence that has added a new layer of complexity to the idea of targeting the eCB system for therapeutic benefits in neuropsychiatric disease and on the future research direction of neural circuit modulation.

Are the persistent effects of "gate control" stimulation on nociception a form of generalization of habituation that is endocannabinoid-dependent?

Repetitive activation of non-nociceptive afferents is known to attenuate nociceptive signaling. However, the functional details of how this modulatory process operates are not understood and this has been a barrier in using such stimuli to effectively treat chronic pain. The present study tests the hypothesis that the ability of repeated non-nociceptive stimuli to reduce nociception is a form of generalized habituation from the non-nociceptive stimulus-response pathway to the nociceptive pathway. Habituation training, using non-nociceptive mechanosensory stimuli, did reduce responses to nociceptive thermal stimulation. This generalization of habituation to nociceptive stimuli required endocannabinoid-mediated neuromodulation, although disrupting of endocannabinoid signaling did not affect "direct" habituation of to the non-nociceptive stimulus. Surprisingly, the reduced response to nociceptive stimuli following habituation training was very long-lasting (3-8 days). This long-term habituation required endocannabinoid signaling during the training/acquisition phase, but endocannabinoids were not required for post-training retention phase. The implications of these results are that applying principles of habituation learning could potentially improve anti-nociceptive therapies utilizing repeated non-nociceptive stimulation such as transcutaneous nerve stimulation (TENS), spinal cord stimulation (SCS), or electro-acupuncture.

The endocannabinoid 2-arachidonoylglycerol regulates oligodendrocyte progenitor cell migration.

While the endocannabinoid 2-arachidonoylglycerol (2-AG) is thought to enhance the proliferation and differentiation of oligodendrocyte progenitor cells (OPCs) in vitro, less is known about how endogenous 2-AG may influence the migration of these cells. When we assessed this in Agarose drop and Boyden chemotaxis chamber assays, inhibiting the sn-1-diacylglycerol lipases α and ß (DAGLs) that are responsible for 2-AG synthesis significantly reduced the migration of OPCs stimulated by platelet-derived growth factor-AA (PDGF) and basic fibroblast growth factor (FGF). Likewise, antagonists of the CB1 and CB2 cannabinoid receptors (AM281 and AM630, respectively) produced a similar inhibition of OPC migration. By contrast, increasing the levels of endogenous 2-AG by blocking its degradation (impairing monoacylglycerol lipase activity with JZL-184) significantly increased OPC migration, as did agonists of the CB1, CB2 or CB1/CB2 cannabinoid receptors. This latter effect was abolished by selective CB1 or CB2 antagonists, strongly suggesting that cannabinoid receptor activation specifically potentiates OPC chemotaxis and chemokinesis in response to PDGF/FGF. Furthermore, the chemoattractive activity of these cannabinoid receptor agonists on OPCs was even evident in the absence of PDGF/FGF. In cultured brain slices prepared from the corpus callosum of postnatal rat brains, DAGL or cannabinoid receptor inhibition substantially diminished the in situ migration of Sox10+ OPCs. Overall, these results reveal a novel function of endogenous 2-AG in PDGF and FGF induced OPC migration, highlighting the importance of the endocannabinoid system in regulating essential steps in oligodendrocyte development.

Metabolomic assessment of arsenite toxicity and novel biomarker discovery in early development of zebrafish embryos.

CONTEXT: Arsenic poisoning commonly occurs through exposure to water contaminated with arsenic and causes long-term symptoms. Of all the arsenic derivatives, arsenite is the one of the most toxic compounds. However, the toxicity of arsenite during developmental stages is still unclear. OBJECTIVE: In this study, we performed a metabolomic analysis of arsenite responses in embryonic zebrafish. MATERIALS AND METHODS: Embryonic zebrafish were used as an animal model in this study. They were exposed to sodium arsenite under different concentrations (0.5, 1.0, 2.0, and 5.0 mg/L) in 24 h, 48 h and 72 h post fertilization. Changes in morphology were observed through a light microscope. Changes in metabolomics were identified using an ultraperformance liquid chromatography quadrupole time-of-flight system. RESULTS: The IC50 range was 0.75 ±â€¯0.25 mg/L. Compared with the control group, the embryonic lethality rate decreased to 33.3% under 1.0 mg/L of arsenite treatment, whereas it decreased to 20.0% under 2.0 mg/L of arsenite treatment. Numerous body axis curvatures were also observed under treatment with 2.0 and 5.0 mg/L of arsenic. Pericardium and yolk sac edema were randomly discovered and found to worsen over time. Moreover, the 10 metabolites with the highest variable importance in projection score were identified as potential biomarkers for arsenic exposure. CONCLUSION: Arsenic exposure not only leads to a change in the morphology of embryonic zebrafish but also disturbs the metabolism of zebrafish in early developmental stages.

Functional Relevance of Endocannabinoid-Dependent Synaptic Plasticity in the Central Nervous System.

The endocannabinoid (eCB) signaling system plays a key role in short-term and long-term synaptic plasticity in brain regions involved in various neural functions ranging from action selection to appetite control. This review will explore the role of eCBs in shaping neural circuit function to regulate behaviors. In particular, we will discuss the behavioral consequences of eCB mediated long-term synaptic plasticity in different brain regions. This review brings together evidence from in vitro and ex vivo studies and points out the need for more in vivo studies.

Cell-Autonomous Excitation of Midbrain Dopamine Neurons by Endocannabinoid-Dependent Lipid Signaling.

The major endocannabinoid in the mammalian brain is the bioactive lipid 2-arachidonoylglycerol (2-AG). The best-known effects of 2-AG are mediated by G-protein-coupled cannabinoid receptors. In principle, 2-AG could modify neuronal excitability by acting directly on ion channels, but such mechanisms are poorly understood. Using a preparation of dissociated mouse midbrain dopamine neurons to isolate effects on intrinsic excitability, we found that 100 nM 2-AG accelerated pacemaking and steepened the frequency-current relationship for burst-like firing. In voltage-clamp experiments, 2-AG reduced A-type potassium current (IA) through a cannabinoid receptor-independent mechanism mimicked by arachidonic acid, which has no activity on cannabinoid receptors. Activation of orexin, neurotensin, and metabotropic glutamate Gq/11-linked receptors mimicked the effects of exogenous 2-AG and their actions were prevented by inhibiting the 2-AG-synthesizing enzyme diacylglycerol lipase α. The results show that 2-AG and related lipid signaling molecules can directly tune neuronal excitability in a cell-autonomous manner by modulating IA.

Harnessing the Endocannabinoid 2-Arachidonoylglycerol to Lower Intraocular Pressure in a Murine Model.

PURPOSE: Cannabinoids, such as Δ9-THC, act through an endogenous signaling system in the vertebrate eye that reduces IOP via CB1 receptors. Endogenous cannabinoid (eCB) ligand, 2-arachidonoyl glycerol (2-AG), likewise activates CB1 and is metabolized by monoacylglycerol lipase (MAGL). We investigated ocular 2-AG and its regulation by MAGL and the therapeutic potential of harnessing eCBs to lower IOP. METHODS: We tested the effect of topical application of 2-AG and MAGL blockers in normotensive mice and examined changes in eCB-related lipid species in the eyes and spinal cord of MAGL knockout (MAGL-/-) mice using high performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). We also examined the protein distribution of MAGL in the mouse anterior chamber. RESULTS: 2-Arachidonoyl glycerol reliably lowered IOP in a CB1- and concentration-dependent manner. Monoacylglycerol lipase is expressed prominently in nonpigmented ciliary epithelium. The MAGL blocker KML29, but not JZL184, lowered IOP. The ability of CB1 to lower IOP is not desensitized in MAGL-/- mice. Ocular monoacylglycerols, including 2-AG, are elevated in MAGL-/- mice but, in contrast to the spinal cord, arachidonic acid and prostaglandins are not changed. CONCLUSIONS: Our data confirm a central role for MAGL in metabolism of ocular 2-AG and related lipid species, and that endogenous 2-AG can be harnessed to reduce IOP. The MAGL blocker KML29 has promise as a therapeutic agent, while JZL184 may have difficulty crossing the cornea. These data, combined with the relative specificity of MAGL for ocular monoacylglycerols and the lack of desensitization in MAGL-/- mice, suggest that the development of an optimized MAGL blocker offers therapeutic potential for treatment of elevated IOP.

The Role of the Endocannabinoid System in the Brain-Gut Axis.

The actions of cannabis are mediated by receptors that are part of an endogenous cannabinoid system. The endocannabinoid system (ECS) consists of the naturally occurring ligands N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG), their biosynthetic and degradative enzymes, and the cannabinoid (CB) receptors CB1 and CB2. The ECS is a widely distributed transmitter system that controls gut functions peripherally and centrally. It is an important physiologic regulator of gastrointestinal motility. Polymorphisms in the gene encoding CB1 (CNR1) have been associated with some forms of irritable bowel syndrome. The ECS is involved in the control of nausea and vomiting and visceral sensation. The homeostatic role of the ECS also extends to the control of intestinal inflammation. We review the mechanisms by which the ECS links stress and visceral pain. CB1 in sensory ganglia controls visceral sensation, and transcription of CNR1 is modified through epigenetic processes under conditions of chronic stress. These processes might link stress with abdominal pain. The ECS is also involved centrally in the manifestation of stress, and endocannabinoid signaling reduces the activity of hypothalamic-pituitary-adrenal pathways via actions in specific brain regions, notably the prefrontal cortex, amygdala, and hypothalamus. Agents that modulate the ECS are in early stages of development for treatment of gastrointestinal diseases. Increasing our understanding of the ECS will greatly advance our knowledge of interactions between the brain and gut and could lead to new treatments for gastrointestinal disorders.

Diacylglycerol lipase disinhibits VTA dopamine neurons during chronic nicotine exposure.

Chronic nicotine exposure (CNE) alters synaptic transmission in the ventral tegmental area (VTA) in a manner that enhances dopaminergic signaling and promotes nicotine use. The present experiments identify a correlation between enhanced production of the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) and diminished release of the inhibitory neurotransmitter GABA in the VTA following CNE. To study the functional role of on-demand 2-AG signaling in GABAergic synapses, we used 1,2,3-triazole urea compounds to selectively inhibit 2-AG biosynthesis by diacylglycerol lipase (DAGL). The potency and selectivity of these inhibitors were established in rats in vitro (rat brain proteome), ex vivo (brain slices), and in vivo (intracerebroventricular administration) using activity-based protein profiling and targeted metabolomics analyses. Inhibition of DAGL (2-AG biosynthesis) rescues nicotine-induced VTA GABA signaling following CNE. Conversely, enhancement of 2-AG signaling in naïve rats by inhibiting 2-AG degradation recapitulates the loss of nicotine-induced GABA signaling evident following CNE. DAGL inhibition reduces nicotine self-administration without disrupting operant responding for a nondrug reinforcer or motor activity. Collectively, these findings provide a detailed characterization of selective inhibitors of rat brain DAGL and demonstrate that excessive 2-AG signaling contributes to a loss of inhibitory GABAergic constraint of VTA excitability following CNE.

An Introduction to the Endogenous Cannabinoid System.

The endocannabinoid system (ECS) is a widespread neuromodulatory system that plays important roles in central nervous system development, synaptic plasticity, and the response to endogenous and environmental insults. The ECS comprises cannabinoid receptors, endogenous cannabinoids (endocannabinoids), and the enzymes responsible for the synthesis and degradation of the endocannabinoids. The most abundant cannabinoid receptors are the CB1 cannabinoid receptors; however, CB2 cannabinoid receptors, transient receptor potential channels, and peroxisome proliferator activated receptors are also engaged by some cannabinoids. Exogenous cannabinoids, such as tetrahydrocannabinol, produce their biological effects through their interactions with cannabinoid receptors. The best-studied endogenous cannabinoids are 2-arachidonoyl glycerol and arachidonoyl ethanolamide (anandamide). Despite similarities in chemical structure, 2-arachidonoyl glycerol and anandamide are synthesized and degraded by distinct enzymatic pathways, which impart fundamentally different physiologic and pathophysiologic roles to these two endocannabinoids. As a result of the pervasive social use of cannabis and the involvement of endocannabinoids in a multitude of biological processes, much has been learned about the physiologic and pathophysiologic roles of the ECS. This review provides an introduction to the ECS with an emphasis on its role in synaptic plasticity and how the ECS is perturbed in schizophrenia.

Endogenous 2-Arachidonoylglycerol Alleviates Cyclooxygenases-2 Elevation-Mediated Neuronal Injury From SO2 Inhalation via PPARγ Pathway.

Although the health effects of sulfur dioxide (SO2) pollution in the atmospheric environment are not new, epidemiological studies and parallel experimental investigations indicate that acute SO2 exposure causes glutamate-mediated excitotoxicity and even contributes to the outcome of cerebral ischemia. Additionally, the free radical-related inflammatory responses are responsible for neuronal insults and consequent brain disorders. However, few medications are available for preventing the inflammatory responses and relieving the subsequent harmful insults from SO2 inhalation. Here, we show that endocannabinoid 2-arachidonoylglycerol (2-AG) prevents neurotoxicity from SO2 inhalation by suppressing cyclooxygenase-2 (COX-2) overexpression, and this action appears to be mediated via cannabinoid receptor 1 (CB1)-dependent mitogen-activated protein kinase/nuclear factor κB (NF-κB) signaling pathways. Furthermore, CB1-dependent peroxisome proliferator activated receptor γ (PPARγ) expression was an important modulator of the 2-AG-mediated resolution on NF-κB-coupled COX-2 elevation in response to SO2 neuroinflammation. This finding provides evidence of a possible therapeutic effect of endogenous 2-AG regulation for protecting against neurological dysfunction from SO2 inhalation in polluted areas.

Modulation of sweet taste sensitivities by endogenous leptin and endocannabinoids in mice.

KEY POINTS: Potential roles of endogenous leptin and endocannabinoids in sweet taste were examined by using pharmacological antagonists and mouse models including leptin receptor deficient (db/db) and diet-induced obese (DIO) mice. Chorda tympani (CT) nerve responses of lean mice to sweet compounds were increased after administration of leptin antagonist (LA) but not affected by administration of cannabinoid receptor antagonist (AM251). db/db mice showed clear suppression of CT responses to sweet compounds after AM251, increased endocannabinoid levels in the taste organ, and enhanced expression of a biosynthesizing enzyme of endocannabinoids in taste cells. The effect of LA was gradually decreased and that of AM251 was increased during the course of obesity in DIO mice. These findings suggest that circulating leptin, but not local endocannabinoids, is a dominant modulator for sweet taste in lean mice and endocannabinoids become more effective modulators of sweet taste under conditions of deficient leptin signalling. ABSTRACT: Leptin is an anorexigenic mediator that reduces food intake by acting on hypothalamic receptor Ob-Rb. In contrast, endocannabinoids are orexigenic mediators that act via cannabinoid CB1 receptors in hypothalamus, limbic forebrain, and brainstem. In the peripheral taste system, leptin administration selectively inhibits behavioural, taste nerve and taste cell responses to sweet compounds. Opposing the action of leptin, endocannabinoids enhance sweet taste responses. However, potential roles of endogenous leptin and endocannabinoids in sweet taste remain unclear. Here, we used pharmacological antagonists (Ob-Rb: L39A/D40A/F41A (LA), CB1 : AM251) and examined the effects of their blocking activation of endogenous leptin and endocannabinoid signalling on taste responses in lean control, leptin receptor deficient db/db, and diet-induced obese (DIO) mice. Lean mice exhibited significant increases in chorda tympani (CT) nerve responses to sweet compounds after LA administration, while they showed no significant changes in CT responses after AM251. In contrast, db/db mice showed clear suppression of CT responses to sweet compounds after AM251, increased endocannabinoid (2-arachidonoyl-sn-glycerol (2-AG)) levels in the taste organ, and enhanced expression of a biosynthesizing enzyme (diacylglycerol lipase α (DAGLα)) of 2-AG in taste cells. In DIO mice, the LA effect was gradually decreased and the AM251 effect was increased during the course of obesity. Taken together, our results suggest that circulating leptin, but not local endocannabinoids, may be a dominant modulator for sweet taste in lean mice; however, endocannabinoids may become more effective modulators of sweet taste under conditions of deficient leptin signalling, possibly due to increased production of endocannabinoids in taste tissue.

Enhanced endocannabinoid-mediated modulation of rostromedial tegmental nucleus drive onto dopamine neurons in Sardinian alcohol-preferring rats.

The progressive predominance of rewarding effects of addictive drugs over their aversive properties likely contributes to the transition from drug use to drug dependence. By inhibiting the activity of DA neurons in the VTA, GABA projections from the rostromedial tegmental nucleus (RMTg) are well suited to shift the balance between drug-induced reward and aversion. Since cannabinoids suppress RMTg inputs to DA cells and CB1 receptors affect alcohol intake in rodents, we hypothesized that the endocannabinoid system, by modulating this pathway, might contribute to alcohol preference. Here we found that RMTg afferents onto VTA DA neurons express CB1 receptors and display a 2-arachidonoylglycerol (2-AG)-dependent form of short-term plasticity, that is, depolarization-induced suppression of inhibition (DSI). Next, we compared rodents with innate opposite alcohol preference, the Sardinian alcohol-preferring (sP) and alcohol-nonpreferring (sNP) rats. We found that DA cells from alcohol-naive sP rats displayed a decreased probability of GABA release and a larger DSI. This difference was due to the rate of 2-AG degradation. In vivo, we found a reduced RMTg-induced inhibition of putative DA neurons in sP rats that negatively correlated with an increased firing. Finally, alcohol failed to enhance RMTg spontaneous activity and to prolong RMTg-induced silencing of putative DA neurons in sP rats. Our results indicate functional modifications of RMTg projections to DA neurons that might impact the reward/aversion balance of alcohol attributes, which may contribute to the innate preference observed in sP rats and to their elevated alcohol intake.

Endocannabinoid modulation by FAAH and monoacylglycerol lipase within the analgesic circuitry of the periaqueductal grey.

BACKGROUND AND PURPOSE: Endogenous cannabinoids (endocannabinoids) in the periaqueductal grey (PAG) play a vital role in mediating stress-induced analgesia. This analgesic effect of endocannabinoids is enhanced by pharmacological inhibition of their degradative enzymes. However, the specific effects of endocannabinoids and the inhibitors of their degradation are largely unknown within this pain-modulating region. EXPERIMENTAL APPROACH: In vitro electrophysiological recordings were conducted from PAG neurons in rat midbrain slices. The effects of the major endocannabinoids and their degradation inhibitors on inhibitory GABAergic synaptic transmission were examined. KEY RESULTS: Exogenous application of the endocannabinoid, anandamide (AEA), but not 2-arachidonoylglycerol (2-AG), produced a reduction in inhibitory GABAergic transmission in PAG neurons. This AEA-induced suppression of inhibition was enhanced by the fatty acid amide hydrolase (FAAH) inhibitor, URB597, whereas a 2-AG-induced suppression of inhibition was unmasked by the monoacylglycerol lipase (MGL) inhibitor, JZL184. In addition, application of the CB1 receptor antagonist, AM251, facilitated the basal GABAergic transmission in the presence of URB597 and JZL184, which was further enhanced by the dual FAAH/MGL inhibitor, JZL195. CONCLUSIONS AND IMPLICATIONS: Our results indicate that AEA and 2-AG act via disinhibition within the PAG, a cellular action consistent with analgesia. These actions of AEA and 2-AG are tightly regulated by their respective degradative enzymes, FAAH and MGL. Furthermore, individual or combined inhibition of FAAH and/or MGL enhanced tonic disinhibition within the PAG. Therefore, the current findings support the therapeutic potential of FAAH and MGL inhibitors as a novel pharmacotherapy for pain.

Elevation of endogenous anandamide impairs LTP, learning, and memory through CB1 receptor signaling in mice.

In rodents, many exogenous and endogenous cannabinoids, such as anandamide (AEA) and 2-arachidonyl glycerol (2-AG), have been shown to play an important role in certain hippocampal memory processes. However, the mechanisms by which endogenous AEA regulate this processes are not well understood. Here the effects of AEA on long-term potentiation (LTP), hippocampal-dependent learning and memory tasks, pERK1/2, pCaMKIV, and pCREB signaling events in both cannabinoid receptor type 1 (CB1R) wild-type (WT) and knockout (KO) mice were assessed following administration of URB597, an inhibitor of the fatty acid amide hydrolase (FAAH). Acute administration of URB597 enhanced AEA levels without affecting the levels of 2-AG or CB1R in the hippocampus and neocortex as compared to vehicle. In hippocampal slices, URB597 impaired LTP in CB1R WT but not in KO littermates. URB597 impaired object recognition, spontaneous alternation and spatial memory in the Y-maze test in CB1R WT mice but not in KO mice. Furthermore, URB597 enhanced ERK phosphorylation in WT without affecting total ERK levels in WT or KO mice. URB597 impaired CaMKIV and CREB phosphorylation in WT but not in KO mice. CB1R KO mice have a lower pCaMKIV/CaMKIV ratio and higher pCREB/CREB ratio as compared to WT littermates. Our results indicate that pharmacologically elevated AEA impair LTP, learning and memory and inhibit CaMKIV and CREB phosphorylation, via the activation of CB1Rs. Collectively, these findings also suggest that pharmacological elevation of AEA beyond normal concentrations is also detrimental for the underlying physiological responses.

Systems biology analysis of the endocannabinoid system reveals a scale-free network with distinct roles for anandamide and 2-arachidonoylglycerol.

We represented the endocannabinoid system (ECS) as a biological network, where ECS molecules are the nodes (123) and their interactions the links (189). ECS network follows a scale-free topology, which confers robustness against random damage, easy navigability, and controllability. Network topological parameters, such as clustering coefficient (i.e., how the nodes form clusters) of 0.0009, network diameter (the longest shortest path among all pairs of nodes) of 12, averaged number of neighbors (the mean number of connections per node) of 3.073, and characteristic path length (the expected distance between two connected nodes) of 4.715, suggested that molecular messages are transferred through the ECS network quickly and specifically. Interestingly, ∼75% of nodes are located on, or are active at the level of, the cell membrane. The hubs of ECS network are anandamide (AEA) and 2-arachidonoylglycerol (2-AG), which have also the highest value of betweeness centrality, and their removal causes network collapse into multiple disconnected components. Importantly, AEA is a ubiquitous player while 2-AG plays more restricted actions. Instead, the product of their degradation, arachidonic acid, and their hydrolyzing enzyme, fatty acid amide hydrolase, FAAH, have a marginal impact on ECS network, indeed their removal did not significantly affect its topology.