Protein-coupled Cannabinoid Receptors Research Articles

Cannabinoid receptor type 1 activation causes a water diuresis by inducing an acute central diabetes insipidus in mice.

Cannabis and synthetic cannabinoid consumption are increasing worldwide. Cannabis contains numerous phytocannabinoids that act on the G protein-coupled cannabinoid receptor type 1 (CB1R) and cannabinoid receptor type 2 expressed throughout the body, including the kidney. Essentially every organ, including the kidney, produces endocannabinoids, which are endogenous ligands to these receptors. Cannabinoids acutely increase urine output in rodents and humans, thus potentially influencing total body water and electrolyte homeostasis. As the kidney collecting duct (CD) regulates total body water, acid/base, and electrolyte balance through specific functions of principal cells (PCs) and intercalated cells (ICs), we examined the cell-specific immunolocalization of CB1R in the mouse CD. Antibodies against either the C-terminus or N-terminus of CB1R consistently labeled aquaporin 2 (AQP2)-negative cells in the cortical and medullary CD and thus presumably ICs. Given the well-established role of ICs in urinary acidification, we used a clearance approach in mice that were acid loaded with 280 mM NH4Cl for 7 days and nonacid-loaded mice treated with the cannabinoid receptor agonist WIN55,212-2 (WIN) or a vehicle control. Although WIN had no effect on urinary acidification, these WIN-treated mice had less apical + subapical AQP2 expression in PCs compared with controls and developed acute diabetes insipidus associated with the excretion of large volumes of dilute urine. Mice maximally concentrated their urine when WIN and 1-desamino-8-d-arginine vasopressin [desmopressin (DDAVP)] were coadministered, consistent with central rather than nephrogenic diabetes insipidus. Although ICs express CB1R, the physiological role of CB1R in this cell type remains to be determined.NEW & NOTEWORTHY The CB1R agonist WIN55,212-2 induces central diabetes insipidus in mice. This research integrates existing knowledge regarding the diuretic effects of cannabinoids and the influence of CB1R on vasopressin secretion while adding new mechanistic insights about total body water homeostasis. Our findings provide a deeper understanding about the potential clinical impact of cannabinoids on human physiology and may help identify targets for novel therapeutics to treat water and electrolyte disorders such as hyponatremia and volume overload.

Selected cannabis terpenes synergize with THC to produce increased CB1 receptor activation

The cannabis plant exerts its pharmaceutical activity primarily by the binding of cannabinoids to two G protein-coupled cannabinoid receptors, CB1 and CB2. The role that cannabis terpenes play in this activation has been considered and debated repeatedly, based on only limited experimental results. In the current study we used a controlled in-vitro heterologous expression system to quantify the activation of CB1 receptors by sixteen cannabis terpenes individually, by tetrahydrocannabinol (THC) alone and by THC-terpenes mixtures. The results demonstrate that all terpenes, when tested individually, activate CB1 receptors, at about 10–50% of the activation by THC alone. The combination of some of these terpenes with THC significantly increases the activity of the CB1 receptor, compared to THC alone. In some cases, several fold. Importantly, this amplification is evident at terpene to THC ratios similar to those in the cannabis plant, which reflect very low terpene concentrations. For some terpenes, the activation obtained by THC- terpene mixtures is notably greater than the sum of the activations by the individual components, suggesting a synergistic effect. Our results strongly support a modulatory effect of some of the terpenes on the interaction between THC and the CB1 receptor. As the most effective terpenes are not necessarily the most abundant ones in the cannabis plant, reaching “whole plant” or “full spectrum” composition is not necessarily an advantage. For enhanced therapeutic effects, desired compositions are attainable by enriching extracts with selected terpenes. These compositions adjust the treatment for various desired medicinal and personal needs.

Microglial Cannabinoid CB2 Receptors in Pain Modulation.

Pain, especially chronic pain, can strongly affect patients' quality of life. Cannabinoids ponhave been reported to produce potent analgesic effects in different preclinical pain models, where they primarily function as agonists of Gi/o protein-coupled cannabinoid CB1 and CB2 receptors. The CB1 receptors are abundantly expressed in both the peripheral and central nervous systems. The central activation of CB1 receptors is strongly associated with psychotropic adverse effects, thus largely limiting its therapeutic potential. However, the CB2 receptors are promising targets for pain treatment without psychotropic adverse effects, as they are primarily expressed in immune cells. Additionally, as the resident immune cells in the central nervous system, microglia are increasingly recognized as critical players in chronic pain. Accumulating evidence has demonstrated that the expression of CB2 receptors is significantly increased in activated microglia in the spinal cord, which exerts protective consequences within the surrounding neural circuitry by regulating the activity and function of microglia. In this review, we focused on recent advances in understanding the role of microglial CB2 receptors in spinal nociceptive circuitry, highlighting the mechanism of CB2 receptors in modulating microglia function and its implications for CB2 receptor- selective agonist-mediated analgesia.

Cannabinoid and planar cell polarity signaling converges to direct placentation

Directed trophoblast migration toward the maternal mesometrial pole is critical for placentation and pregnancy success. Trophoblasts replace maternal arterial endothelial cells to increase blood supply to the placenta. Inferior trophoblast invasion results in pregnancy complications including preeclampsia, intrauterine growth restriction, miscarriage, and preterm delivery. The maternal chemotactic factors that direct trophoblast migration and the mechanism by which trophoblasts respond to these factors are not clearly understood. Here, we show that invasive trophoblasts deficient in Vangl2, a core planar cell polarity (PCP) component, fail to invade in maternal decidua, and this deficiency results in middle-gestational fetal demise. Previously, we have shown that tightly regulated endocannabinoids via G protein-coupled cannabinoid receptor CB1 are critical to the invasion of trophoblasts called spiral artery trophoblast giant cells (SpA-TGCs). We find that CB1 directly interacts with VANGL2. Trophoblast stem cells devoid of Cnr1 and/or Vangl2 show compromised cell migration. To study roles of VANGL2 and CB1 in trophoblast invasion in vivo, we conditionally deleted Cnr1 (coding CB1) and Vangl2 in progenitors of SpA-TGCs using trophoblast-specific protein alpha (Tpbpa)-Cre. We observed that signaling mediated by VANGL2 and CB1 restrains trophoblasts from random migration by keeping small GTPases quiescent. Our results show that organized PCP in trophoblasts is indispensable for their directed movement and that CB1 exerts its function by direct interaction with membrane proteins other than its canonical G protein-coupled receptor role.

Roles of Endocannabinoids and Endocannabinoid-Like Molecules in Energy Homeostasis and Metabolic Regulation: A Nutritional Perspective.

The endocannabinoid system is involved in signal transduction in mammals. It comprises principally G protein-coupled cannabinoid receptors and their endogenous agonists, called endocannabinoids, as well as the enzymes and transporters responsible for the metabolism of endocannabinoids. Two arachidonic acid-containing lipid molecules, arachidonoylethanolamide (anandamide) and 2-arachidonoylglycerol, function as endocannabinoids. N-acylethanolamines and monoacylglycerols, in which the arachidonic acid chain is replaced with a saturated or monounsaturated fatty acid, are not directly involved in the endocannabinoid system but exhibit agonistic activities for other receptors. These endocannabinoid-like moleculesinclude palmitoylethanolamide, oleoylethanolamide (OEA), and 2-oleoylglycerol. Endocannabinoids stimulate feeding behavior and the anabolism of lipids and glucose, while OEA suppresses appetite. Both central and peripheral systems are included in these nutritional and metabolic contexts. Therefore, they have potential in the treatment and prevention of obesity. We outline the structure, metabolism, and biological activities of endocannabinoids and related molecules, and focus on their involvement in energy homeostasis and metabolic regulation.

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.

Influence of Cannabinoid Receptor Deficiency on Parameters Involved in Blood Glucose Regulation in Mice.

Cannabinoids are known to influence hormone secretion of pancreatic islets via G protein-coupled cannabinoid receptor type 1 and 2 (CB1 and CB2). The present study was designed to further investigate the impact of cannabinoid receptors on the parameters involved in insulin secretion and blood glucose recognition. To this end, CB1 and CB2 receptor knockout mice (10–12 week old, both sexes) were characterised at basal state and compared to wild-type mice. The elimination of cannabinoid receptor signalling resulted in alterations of blood glucose concentrations, body weights and insulin levels. Changes were dependent on the deleted receptor type and on the sex. Analyses at mRNA and protein levels provided evidence for the impact of cannabinoid receptor deficiency on the glucose sensing apparatus in the pancreas. Both receptor knockout mouse lines showed decreased mRNA and protein amounts of glucose transporters Glut1 and Glut2, combined with alterations in immunostaining. In addition, pancreatic glucokinase expression was elevated and immunohistochemical labelling was modified in the pancreatic islets. Taken together, CB1 and CB2 signalling pathways seem to influence glucose sensing in β-cells by affecting glucose transporters and glucokinase. These alterations were more pronounced in CB2 knockout mice, resulting in higher blood glucose and lower plasma insulin levels.

Interference with the Cannabinoid Receptor CB1R Results in Miswiring of GnRH3 and AgRP1 Axons in Zebrafish Embryos.

The G protein-coupled cannabinoid receptors type 1 (CB1R) and type 2 (CB2R), and their endocannabinoid (eCBs) ligands, have been implicated in several aspects of brain wiring during development. Here we aim to assess whether interfering with CB1R affects development, neuritogenesis and pathfinding of GnRH and AgRP neurons, forebrain neurons that control respectively reproduction and appetite. We pharmacologically and genetically interfered with CB1R in zebrafish strains with fluorescently labeled GnRH3 and the AgRP1 neurons. By applying CB1R antagonists we observed a reduced number of GnRH3 neurons, fiber misrouting and altered fasciculation. Similar phenotypes were observed by CB1R knockdown. Interfering with CB1R also resulted in a reduced number, misrouting and poor fasciculation of the AgRP1 neuron’s axonal projections. Using a bioinformatic approach followed by qPCR validation, we have attempted to link CB1R functions with known guidance and fasciculation proteins. The search identified stathmin-2, a protein controlling microtubule dynamics, previously demonstrated to be coexpressed with CB1R and now shown to be downregulated upon interference with CB1R in zebrafish. Together, these results raise the likely possibility that embryonic exposure to low doses of CB1R-interfering compounds could impact on the development of the neuroendocrine systems controlling sexual maturation, reproduction and food intake.

3110 A Case of Reflux Esophagitis Mimicking Globus Sensation in a Patient With Cannabis Abuse

INTRODUCTION: Cannabis is a widely used drug of abuse in the United States (US). It is mostly commonly abused by young individuals and is known for it's psychedelic effects. Cannabis is also utilized by the medical field for a number of medicinal purposes. We present a case of a young male who presented with sudden onset dysphagia and had a significant history of marijuana abuse for several years. CASE DESCRIPTION/METHODS: 28 year old Caucasian male with a past medical history of ADHD presented with dysphagia. The symptoms started 2 days ago. He felt like the food was stuck in his throat. Every time he attempted to drink water or liquids it regurgitated. He also reported several episodes of nausea and vomiting since the onset of his symptoms. Review of systems was negative for odynophagia, fever, drooling, weight loss, chest pain, diarrhea, constipation or acid reflux. Denied any allergies, surgical history or family history. He denied alcohol or cigarette smoking. He agreed to smoking marijunana 2-3 times per day for over 4 years. Physical examination revealed a healthy non obese male with blood pressure and pulse of 120/80 mmhg and 86 per minute respectively. Rest of his examination was unremarkable. Upper endoscopy was performed on the day of admission which revealed LA Grade D esophagitis with multiple superficial ulcers extending 5 cm from GE junction. Patient was started on proton pump inhibitors twice daily. His symptoms improved and was eventually discharged. Biopsies were suggestive of changes consistent with chronic reflux esophagitis. DISCUSSION: Tetrahydrocannabinol (THC) is the active ingredient derived from Cannabis Sativum. THC works by stimulating the G protein-coupled cannabinoid receptors (CBR) receptors. The presence of CBRs in the esophagus, specifically those affecting transient lower esophageal sphincter relaxations (TLESRs), offers a potential therapeutic target for treating GERD. Using Delta (9)-THC, Beaumont et al. demonstrated decreased rate of TLESRs in healthy volunteers who received 10 and 20 mg of Δ9-THC on three occasions a week apart. However, in the high dose of ΔDelta (9)-THC (20 mg) group, central activity led to increased nausea, vomiting and reflux symptoms. With ongoing discussions for the legalization of recreational cannabis use in US, we may encounter an increase in the number of reflux esophagitis cases. With this in mind, additional studies would be beneficial to determine potential risk factors for developing severe reflux esophagitis with extensive marijuana use.

Novel CB1-ligands maintain homeostasis of the endocannabinoid system in ω3- and ω6-long-chain-PUFA deficiency

Mammalian ω3- and ω6-PUFAs are synthesized from essential fatty acids (EFAs) or supplied by the diet. PUFAs are constitutive elements of membrane architecture and precursors of lipid signaling molecules. EFAs and long-chain (LC)-PUFAs are precursors in the synthesis of endocannabinoid ligands of Gi/o protein-coupled cannabinoid receptor (CB)1 and CB2 in the endocannabinoid system, which critically regulate energy homeostasis as the metabolic signaling system in hypothalamic neuronal circuits and behavioral parameters. We utilized the auxotrophic fatty acid desaturase 2-deficient (fads2−/−) mouse, deficient in LC-PUFA synthesis, to follow the age-dependent dynamics of the PUFA pattern in the CNS-phospholipidome in unbiased dietary studies of three cohorts on sustained LC-PUFA-free ω6-arachidonic acid- and DHA-supplemented diets and their impact on the precursor pool of CB1 ligands. We discovered the transformation of eicosa-all cis-5,11,14-trienoic acid, uncommon in mammalian lipidomes, into two novel endocannabinoids, 20:35,11,14-ethanolamide and 2-20:35,11,14-glycerol. Their function as ligands of CB1 has been characterized in HEK293 cells. Labeling experiments excluded Δ8-desaturase activity and proved the position specificity of FADS2. The fads2−/− mutant might serve as an unbiased model in vivo in the development of novel CB1 agonists and antagonists.

Δ9-tetrahydrocannabivarin impairs epithelial calcium transport through inhibition of TRPV5 and TRPV6.

Compounds extracted from the cannabis plant, including the psychoactive Δ9-tetrahydrocannabinol (THC) and related phytocannabinoids, evoke multiple diverse biological actions as ligands of the G protein-coupled cannabinoid receptors CB1 and CB2. In addition, there is increasing evidence that phytocannabinoids also have non-CB targets, including several ion channels of the transient receptor potential superfamily. We investigated the effects of six non-THC phytocannabinoids on the epithelial calcium channels TRPV5 and TRPV6, and found that one of them, Δ9-tetrahydrocannabivarin (THCV), exerted a strong and concentration-dependent inhibitory effect on mammalian TRPV5 and TRPV6 and on the single zebrafish orthologue drTRPV5/6. Moreover, THCV attenuated the drTRPV5/6-dependent ossification in zebrafish embryos in vivo. Oppositely, 11-hydroxy-THCV (THCV-OH), a product of THCV metabolism in mammals, stimulated drTRPV5/6-mediated Ca2+ uptake and ossification. These results identify the epithelial calcium channels TRPV5 and TRPV6 as novel targets of phytocannabinoids, and suggest that THCV-containing products may modulate TRPV5- and TRPV6-dependent epithelial calcium transport.

Cannabinoids modulate the affective component of chronic pain

Cannabis sativa preparations have probably been used since millennia for pain relief and are still an important part of our analgesic pharmacopeia. This presentation will summarize our current understanding about the analgesic mechanism of cannabinoids, from molecular mechanisms to the physiological effects. The psychoactive ingredient of C. sativa, 9-tetrahydrocannabinol (THC) activates two different G protein-coupled cannabinoid receptors, CB1 and CB2, both of which mediate different aspects of cannabinoid analgesic effects. CB2 receptors are mostly localized on immune cells and activated microglia. The analgesic effects of CB2 activation are mainly due to anti-inflammatory mechanisms, but probably also through the modulation of algogenic mediators. CB1 is prominently expressed in the central nervous system and is well-known as an important mediator of synaptic plasticity. Activation of CB1 on neurons modulates pain responses at different level, including the processing of nociceptive stimuli and the regulation of descending pain control systems. I will also show evidence that endocannabinoid signaling is involved in the modulation of the affective component of pain.

Learning-induced modulation of the effect of endocannabinoids on inhibitory synaptic transmission.

are key modulators that regulate central brain functions and behaviors, including learning and memory. At the cellular and molecular levels, endocannabinoids are potent modulators of excitatory and inhibitory synaptic function. Most effects of cannabinoids are thought to be mediated via G protein-coupled cannabinoid receptors. In particular, cannabinoids released from postsynaptic neurons are suggested to act as retrograde messengers, activating presynaptic type-1 cannabinoid receptors (CB1Rs), thereby inducing suppression of synaptic release. Another central mechanism of cannabinoid-induced action requires activation of astroglial CB1Rs. CB1Rs are also implicated in self-modulation of cortical neurons. Rats that are trained in a particularly difficult olfactory-discrimination task show a dramatic increased ability to acquire memories of new odors. The memory of the acquired high-skill acquisition, termed "rule learning" or "learning set," lasts for many months. Using this behavioral paradigm, we show a novel function of action for CB1Rs, supporting long-term memory by maintaining persistent enhancement of inhibitory synaptic transmission. Long-lasting enhancement of inhibitory synaptic transmission is blocked by a CB1R inverse agonist. This effect is mediated by a novel purely postsynaptic mechanism, obtained by enhancing the single GABAA channel conductance that is PKA dependent. The significant role that CB1R has in maintaining learning-induced long-term strengthening of synaptic inhibition suggests that endocannabinoids have a key role in maintaining long-term memory by enhancing synaptic inhibition. NEW & NOTEWORTHY In this study we show a novel function and mechanism of action for cannabinoids in neurons, mediated by activation of type-1 cannabinoid receptors, supporting long-term memory by maintaining persistent enhancement of inhibitory synaptic transmission on excitatory neurons. This effect is mediated by a novel purely postsynaptic mechanism, obtained by enhancing the single GABAA channel conductance that is PKA dependent. Thus we report for the first time that endocannabinoids have a key role maintaining learning-induced synaptic modification.

(S)Pot on Mitochondria: Cannabinoids Disrupt Cellular Respiration to Limit Neuronal Activity

Classical views posit G protein-coupled cannabinoid receptor 1s (CB1Rs) at the cell surface with cytosolic Giα-mediated signal transduction. Hebert-Chatelain etal. (2016) instead place CB1Rs at mitochondria limiting neuronal respiration by soluble adenylyl cyclase-dependent modulation of complex I activity. Thus, neuronal bioenergetics link to synaptic plasticity and, globally, learning and memory.

Application of Strep-Tactin XT for affinity purification of Twin-Strep-tagged CB2, a G protein-coupled cannabinoid receptor

Human cannabinoid receptor CB2 belongs to the class A of G protein-coupled receptor (GPCR). CB2 is predominantly expressed in membranes of cells of immune origin and is implicated in regulation of metabolic pathways of inflammation, neurodegenerative disorders and pain sensing. High resolution structural studies of CB2 require milligram quantities of purified, structurally intact protein. While we previously reported on the methodology for expression of the recombinant CB2 and its stabilization in a functional state, here we describe an efficient protocol for purification of this protein using the Twin-Strep-tag/Strep-Tactin XT system.To improve the affinity of interaction of the recombinant CB2 with the resin, the double repeat of the Strep-tag (a sequence of eight amino acids WSHPQFEK), named the Twin-Strep-tag was attached either to the N- or C-terminus of CB2 via a short linker, and the recombinant protein was expressed in cytoplasmic membranes of E. coli as a fusion with the N-terminal maltose binding protein (MBP). The CB2 was isolated at high purity from dilute solutions containing high concentrations of detergents, glycerol and salts, by capturing onto the Strep-Tactin XT resin, and was eluted from the resin under mild conditions upon addition of biotin.Surface plasmon resonance studies performed on the purified protein demonstrate the high affinity of interaction between the Twin-Strep-tag fused to the CB2 and Strep-Tactin XT with an estimated Kd in the low nanomolar range. The affinity of binding did not vary significantly in response to the position of the tag at either N- or C-termini of the fusion. The binding capacity of the resin was several-fold higher for the tag located at the N-terminus of the protein as opposed to the C-terminus- or middle of the fusion. The variation in the length of the linker between the double repeats of the Strep-tag from 6 to 12 amino acid residues did not significantly affect the binding.The novel purification protocol reported here enables efficient isolation of a recombinant GPCR expressed at low titers in host cells. This procedure is suitable for preparation of milligram quantities of stable isotope-labelled receptor for high-resolution NMR studies.

Differential regulation of NMDAR and NMDAR-mediated metaplasticity by anandamide and 2-AG in the hippocampus.

Endocannabinoids (eCBs), including AEA and 2-AG, are endogenous signaling mediators involved in many physiological and pathological events. The G protein-coupled cannabinoid receptor 1 (CB1 R) is an important target for eCBs, however, additional non-CB1 receptor targets have also been identified. Although recent evidence suggests that NMDA receptor function may be regulated by eCBs, the underlying mechanisms remain poorly characterized. Using acutely isolated CA1 neurons and slices from the hippocampus, we found that both AEA and 2-AG potentiate NMDAR-mediated currents independently of CB1 receptors (CB1 Rs) and via distinct signaling pathways. Potentiation by AEA requires the activation of TRPV1 channels. In contrast, potentiation by 2-AG requires the sequential activation of PKC and Src. Additionally, in hippocampal slices, we found that both AEA and 2-AG induce NMDAR-mediated metaplasticity and facilitate the induction of subsequent LTD independently of CB1 Rs. Enhanced LTD by AEA, but not 2-AG, was dependent on TRPV1 channels. Our findings reveal previously unrecognized non-CB1 R-dependent signaling cascades through which the two major eCBs regulate NMDA receptor function and consequently synaptic plasticity.

Cnr2 Deficiency Confers Resistance to Inflammation-Induced Preterm Birth in Mice

Infection-induced inflammation, frequently associated with increased production of proinflammatory cytokines, is considered a significant contributor to preterm birth. A G protein-coupled cannabinoid receptor 2 (CB2), encoded by Cnr2, is expressed in various immune cells and was shown to modulate immune responses. We show here that Cnr2, but not Cnr1, deficient mice are resistant to lipopolysaccharide (LPS)-driven preterm birth and suppression of serum progesterone levels. After LPS challenge, Cnr2(-/-) mice exhibited increased serum levels of IL-10 with decreased IL-6 levels. These changes were associated with reduced LPS-induced Ptgs2 expression at the maternal-conceptus interface on day 16 of pregnancy. LPS stimulation of Cnr2(-/-) dendritic cells in vitro resulted in increased IL-10 with reduced IL-6 production and correlated with increased cAMP accumulation. Collectively, our results suggest that increased IL-10 production occurring via augmented cAMP accumulation represents a potential mechanism for the resistance of Cnr2(-/-) mice to LPS-induced preterm birth. These results may have clinical relevance, because currently, there are limited options to prevent preterm birth.

Control of Inhibition by the Direct Action of Cannabinoids on GABAAReceptors

Cannabinoids are known to regulate inhibitory synaptic transmission via activation of presynaptic G protein-coupled cannabinoid CB1 receptors (CB1Rs). Additionally, recent studies suggest that cannabinoids can also directly interact with recombinant GABAA receptors (GABAARs), potentiating currents activated by micromolar concentrations of γ-aminobutyric acid (GABA). However, the impact of this direct interaction on GABAergic inhibition in central nervous system is unknown. Here we report that currents mediated by recombinant GABAARs activated by high (synaptic) concentrations of GABA as well as GABAergic inhibitory postsynaptic currents (IPSCs) at neocortical fast spiking (FS) interneuron to pyramidal neuron synapses are suppressed by exogenous and endogenous cannabinoids in a CB1R-independent manner. This IPSC suppression may account for disruption of inhibitory control of pyramidal neurons by FS interneurons. At FS interneuron to pyramidal neuron synapses, endocannabinoids induce synaptic low-pass filtering of GABAAR-mediated currents evoked by high-frequency stimulation. The CB1R-independent suppression of inhibition is synapse specific. It does not occur in CB1R containing hippocampal cholecystokinin-positive interneuron to pyramidal neuron synapses. Furthermore, in contrast to synaptic receptors, the activity of extrasynaptic GABAARs in neocortical pyramidal neurons is enhanced by cannabinoids in a CB1R-independent manner. Thus, cannabinoids directly interact differentially with synaptic and extrasynaptic GABAARs, providing a potent novel context-dependent mechanism for regulation of inhibition.

The G Protein‐Coupled Receptor 55 Ligand l‐α‐Lysophosphatidylinositol Exerts Microglia‐Dependent Neuroprotection After Excitotoxic Lesion

Searching for chemical agents and molecular targets protecting against secondary neuronal damage reflects one major issue in neuroscience. Cannabinoids limit neurodegeneration by activation of neuronal G protein-coupled cannabinoid receptor 1 (CB1 ) and microglial G protein-coupled cannabinoid receptor 2 (CB2 ). However, pharmacological experiments with CB1 /CB2 -deficient mice unraveled the existence of further, so-called non-CB1 /non-CB2 G protein-coupled receptor (GPR) subtypes. GPR55, whose function in the brain is still poorly understood, represents a novel target for various cannabinoids. Here, we investigated whether GPR55 reflects a potential beneficial target in neurodegeneration by using the excitotoxicity in vitro model of rat organotypic hippocampal slice cultures (OHSC). l-α-Lysophosphatidylinositol (LPI), so far representing the most selective agonist for GPR55, protected dentate gyrus granule cells and reduced the number of activated microglia after NMDA (50 µM) induced lesions. The relevance of GPR55 activation for LPI-mediated neuroprotection was determined by using Gpr55 siRNA. Microglia seems to mediate the observed neuroprotection since their depletion in OHSC attenuated the beneficial effects of LPI. Moreover, LPI alone induced microglia chemotaxis but conversely significantly attenuated ATP triggered microglia migration. These effects seemed to be independent from intracellular Ca(2+) and p38 or p44/p42 MAPK phosphorylation. In conclusion, this study unmasked a yet unknown role for GPR55 in neuroprotection driven by LPI-mediated modulation of microglia function.

Brain regional cannabinoid CB1 receptor signalling and alternative enzymatic pathways for 2-arachidonoylglycerol generation in brain sections of diacylglycerol lipase deficient mice

Endocannabinoids are the endogenous ligands of the G protein-coupled cannabinoid receptors. The principal brain endocannabinoid, 2-arachidonoylglycerol (2-AG), is enzymatically produced by postsynaptic neurons and then activates presynaptic CB1 receptors in a retrograde manner. The primary pathway for 2-AG generation is believed to be conversion from the diacylglycerols (DAGs) by two sn-1-specific lipases, DAGLα and DAGLβ. Previous studies with DAGL-deficient mice indicated that DAGLα is the major enzyme needed for retrograde synaptic 2-AG signalling. The current study investigated whether the CB1 receptor-mediated Gi/o protein activity is altered in brain cryosections of DAGL-deficient mice when compared to wild-type mice and whether the sn-1-specific DAGLs are able to generate 2-AG in brain cryosections. Functional autoradiography indicated that brain regional CB1 receptor-Gi/o-activity largely remained unaltered in DAGLα-knockout and DAGLβ-knockout mice when compared to wild-type littermates. Following comprehensive pharmacological blockade of 2-AG hydrolysis, brain sections generated sufficient amounts of 2-AG to activate CB1 receptors throughout the regions endowed with these receptors. As demonstrated by LC/MS/MS, this pool of 2-AG was generated via tetrahydrolipstatin-sensitive enzymatic pathways distinct from DAGLα or DAGLβ. We conclude that in addition to the sn-1-specific DAGLs, additional 2-AG generating enzymatic pathways are active in brain sections.