A microbiome-dependent gut-brain pathway regulates motivation for exercise.

Exercise exerts a wide range of beneficial effects for healthy physiology1. However, the mechanisms regulating an individual's motivation to engage in physical activity remain incompletely understood. An important factor stimulating the engagement in both competitive and recreational exercise is the motivating pleasure derived from prolonged physical activity, which is triggered by exercise-induced neurochemical changes in the brain. Here, we report on the discovery of a gut-brain connection in mice that enhances exercise performance by augmenting dopamine signalling during physical activity. We find that microbiome-dependent production of endocannabinoid metabolites in the gut stimulates the activity of TRPV1-expressing sensory neurons and thereby elevates dopamine levels in the ventral striatum during exercise. Stimulation of this pathway improves running performance, whereas microbiome depletion, peripheral endocannabinoid receptor inhibition, ablation of spinal afferent neurons or dopamine blockade abrogate exercise capacity. These findings indicate that the rewarding properties of exercise are influenced by gut-derived interoceptive circuits and provide a microbiome-dependent explanation for interindividual variability in exercise performance. Our study also suggests that interoceptomimetic molecules that stimulate the transmission of gut-derived signals to the brain may enhance the motivation for exercise.

The endocannabinoid system impacts seizures in a mouse model of Dravet syndrome.

Dravet syndrome is a catastrophic childhood epilepsy with multiple seizure types that are refractory to treatment. The endocannabinoid system regulates neuronal excitability so a deficit in endocannabinoid signaling could lead to hyperexcitability and seizures. Thus, we sought to determine whether a deficiency in the endocannabinoid system might contribute to seizure phenotypes in a mouse model of Dravet syndrome and whether enhancing endocannabinoid tone is anticonvulsant. Scn1a+/- mice model the clinical features of Dravet syndrome: hyperthermia-induced seizures, spontaneous seizures and reduced survival. We examined whether Scn1a+/- mice exhibit deficits in the endocannabinoid system by measuring brain cannabinoid receptor expression and endocannabinoid concentrations. Next, we determined whether pharmacologically enhanced endocannabinoid tone was anticonvulsant in Scn1a+/- mice. We used GAT229, a positive allosteric modulator of the cannabinoid (CB1) receptor, and ABX-1431, a compound that inhibits the degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG). The Scn1a+/- phenotype is strain-dependent with mice on a 129S6/SvEvTac (129) genetic background having no overt phenotype and those on an F1 (129S6/SvEvTac x C57BL/6J) background exhibiting a severe epilepsy phenotype. We observed lower brain cannabinoid CB1 receptor expression in the seizure-susceptible F1 compared to seizure-resistant 129 strain, suggesting an endocannabinoid deficiency might contribute to seizure susceptibility. GAT229 and ABX-1431 were anticonvulsant against hyperthermia-induced seizures. However, subchronic ABX1431 treatment increased spontaneous seizure frequency despite reducing seizure severity. Cnr1 is a putative genetic modifier of epilepsy in the Scn1a+/- mouse model of Dravet syndrome. Compounds that increase endocannabinoid tone could be developed as novel treatments for Dravet syndrome.

Parkinson's disease related alterations in cannabinoid transmission.

Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic (DAergic) neurons of the substantia nigra pars compacta (SNc) by neurodegeneration. Recent findings in animal models of PD propose tonic inhibition of the remaining DA neurons through GABA release from reactive glial cells. Movement dysfunctions could be ameliorated by promotion of activity in dormant DA cells. The endocannabinoid system (ECS) is extensively present in basal ganglia (BG) and is known as an indirect modulator of DAergic neurotransmission, thus drugs designed to target this system have shown promising therapeutic potential in PD patients. Interestingly, down/up-regulation of cannabinoid receptors (CBRs) varies across the different stages of PD, suggesting that some of the motor/ non-motor deficits may be related to changes in CBRs. Determination of the profile of changes of these receptors across the different stages of PD as well as their neural distribution within the BG could improve understanding of PD and identify pathways important in disease pathobiology. In this review, we focus on temporal and spatial alterations of CBRs during PD in the BG. At present, as inconclusive, but suggestive results have been obtained, future investigations should be conducted to extend preclinical studies examining CBRs changes within each stage in controlled clinical trials in order to determine the potential of targeting CBRs in management of PD.

On the Biomedical Properties of Endocannabinoid Degradation and Reuptake Inhibitors: Pre-clinical and Clinical Evidence.

The endocannabinoid system (ECS) is composed of endogenous cannabinoids; components involved in their synthesis, transport, and degradation; and an expansive variety of cannabinoid receptors. Hypofunction or deregulation of the ECS is related to pathological conditions. Consequently, endogenous enhancement of endocannabinoid levels and/or regulation of their metabolism represent promising therapeutic approaches. Several major strategies have been suggested for the modulation of the ECS: (1) blocking endocannabinoids degradation, (2) inhibition of endocannabinoid cellular uptake, and (3) pharmacological modulation of cannabinoid receptors as potential therapeutic targets. Here, we focused in this review on degradation/reuptake inhibitors over cannabinoid receptor modulators in order to provide an updated synopsis of contemporary evidence advancing mechanisms of endocannabinoids as pharmacological tools with therapeutic properties for the treatment of several disorders. For this purpose, we revisited the available literature and reported the latest advances regarding the biomedical properties of fatty acid amide hydrolase and monoacylglycerol lipase inhibitors in pre-clinical and clinical studies. We also highlighted anandamide and 2-arachidonoylglycerol reuptake inhibitors with promising results in pre-clinical studies using in vitro and animal models as an outlook for future research in clinical trials.

mGluR5-Mediated eCB Signaling in the Nucleus Accumbens Controls Vulnerability to Depressive-Like Behaviors and Pain After Chronic Social Defeat Stress.

Stress contributes to major depressive disorder (MDD) and chronic pain, which affect a significant portion of the global population, but researchers have not clearly determined how these conditions are initiated or amplified by stress. The chronic social defeat stress (CSDS) model is a mouse model of psychosocial stress that exhibits depressive-like behavior and chronic pain. We hypothesized that metabotropic glutamate receptor 5 (mGluR5) expressed in the nucleus accumbens (NAc) normalizes the depressive-like behaviors and pain following CSDS. Here, we show that CSDS induced both pain and social avoidance and that the level of mGluR5 decreased in susceptible mice. Overexpression of mGluR5 in the NAc shell and core prevented the development of depressive-like behaviors and pain in susceptible mice, respectively. Conversely, depression-like behaviors and pain were exacerbated in mice with mGluR5 knockdown in the NAc shell and core, respectively, compared to control mice subjected to 3 days of social defeat stress. Furthermore, (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), an mGluR5 agonist, reversed the reduction in the level of the endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) in the NAc of susceptible mice, an effect that was blocked by 3-((2-methyl-1, 3-thiazol-4-yl) ethynyl) pyridine hydrochloride (MTEP), an mGluR5 antagonist. In addition, the injection of CHPG into the NAc shell and core normalized depressive-like behaviors and pain, respectively, and these effects were inhibited by AM251, a cannabinoid type 1 receptor (CB1R) antagonist. Based on these results, mGluR5-mediated eCB production in the NAc relieves stress-induced depressive-like behaviors and pain.

Cocaine-induced increases in motivation require 2-arachidonoylglycerol mobilization and CB1 receptor activation in the ventral tegmental area.

A wide body of evidence supports an integral role for mesolimbic dopamine (DA) in motivated behavior. In brief, drugs that increase DA in mesolimbic terminal regions, like cocaine, enhance motivation, while drugs that decrease DA concentration reduce motivation. Data from our laboratory and others shows that phasic activation of mesolimbic DA requires signaling at cannabinoid type-1 (CB1) receptors in the ventral tegmental area (VTA), and systemic delivery of CB1 receptor antagonists reduces DA cell activity and attenuates motivated behaviors. Recent findings demonstrate that cocaine mobilizes the endocannabinoid 2-arachidonoylglycerol (2-AG) in the VTA to cause phasic activation of DA neurons and terminal DA release. It remains unclear, however, if cocaine-induced midbrain 2-AG signaling contributes to the motivation-enhancing effects of cocaine. To examine this, we trained male and female rats on a progressive ratio (PR) task for a food reinforcer. Each rat underwent a series of tests in which they were pretreated with cocaine alone or in combination with systemic or intra-VTA administration of the CB1 receptor antagonist rimonabant or the 2-AG synthesis inhibitor tetrahydrolipstatin (THL). Cocaine increased motivation, measured by augmented PR breakpoints, while rimonabant dose-dependently decreased motivation. Importantly, intra-VTA administration of rimonabant or THL, at doses that did not decrease breakpoints on their own, blocked systemic cocaine administration from increasing breakpoints in male and female rats. These data suggest that cocaine-induced increases in motivation require 2-AG signaling at CB1 receptors in the VTA and may provide critical insight into cannabinoid-based pharmacotherapeutic targets for the successful treatment of substance abuse.

Inhibition of 2-Arachidonoylglycerol Metabolism Alleviates Neuropathology and Improves Cognitive Function in a Tau Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common cause of dementia, which affects more than 5 million individuals in the USA. Unfortunately, no effective therapies are currently available to prevent development of AD or to halt progression of the disease. It has been proposed that monoacylglycerol lipase (MAGL), the key enzyme degrading the endocannabinoid 2-arachidonoylglycerol (2-AG) in the brain, is a therapeutic target for AD based on the studies using the APP transgenic models of AD. While inhibition of 2-AG metabolism mitigates ß-amyloid (Aß) neuropathology, it is still not clear whether inactivation of MAGL alleviates tauopathies as accumulation and deposition of intracellular hyperphosphorylated tau protein are the neuropathological hallmark of AD. Here we show that JZL184, a potent MAGL inhibitor, significantly reduced proinflammatory cytokines, astrogliosis, phosphorylated GSK3ß and tau, cleaved caspase-3, and phosphorylated NF-kB while it elevated PPARγ in P301S/PS19 mice, a tau mouse model of AD. Importantly, tau transgenic mice treated with JZL184 displayed improvements in spatial learning and memory retention. In addition, inactivation of MAGL ameliorates deteriorations in expression of synaptic proteins in P301S/PS19 mice. Our results provide further evidence that MAGL is a promising therapeutic target for AD.

Interactions between the nitrergic and the endocannabinoid system in rats exposed to the elevated T-maze.

OBJECTIVE: The aim of this study was to test the hypothesis that synthesis of nitric oxide (NO) and activation of CB1 receptors have opposite effects in a behavioural animal model of panic and anxiety. METHODS: To test the hypothesis, male Wistar rats were exposed to the elevated T-maze (ETM) model under the following treatments: L-Arginine (L-Arg) was administered before treatment with WIN55,212-2, a CB1 receptor agonist; AM251, a CB1 antagonist, was administered before treatment with L-Arg. All treatments were by intraperitoneal route. RESULTS: The CB1 receptor agonist, WIN55,212-2 (1 mg/kg), induced an anxiolytic-like effect, which was prevented by pretreatment with an ineffective dose of L-Arg (1 mg/kg). Administration of AM251 (1 mg/kg), a CB1 antagonist before treatment with L-Arg (1 mg/kg) did not produce anxiogenic-like responses. CONCLUSION: Altogether, this study suggests that the anxiolytic-like effect of cannabinoids may occur through modulation of NO signalling.

Endogenous opioid and cannabinoid systems modulate the muscle pain: A pharmacological study into the peripheral site.

The participation of the peripheral opioid and cannabinoid endogenous systems in modulating muscle pain and inflammation has not been fully explored. Thus, the aim of this study was to investigate the involvement of these endogenous systems during muscular-tissue hyperalgesia induced by inflammation. Hyperalgesia was induced by carrageenan injection into the tibialis anterior muscles of male Wistar rats. We padronized an available Randal-Sellito test adaptation to evaluate nociceptive behavior elicited by mechanical insult in muscles. Western blot analysis was performed to evaluate the expression levels of opioid and cannabinoid receptors in the dorsal root ganglia. The non-selective opioid peptide receptor antagonist (naloxone) and the selective mu opioid receptor MOP (clocinnamox) and kappa opioid receptor KOP (nor-binaltorphimine) antagonists were able to intensify carrageenan-induced muscular hyperalgesia. On the other hand, the selective delta opioid receptor (DOP) antagonist (naltrindole) did not present any effect on nociceptive behavior. Moreover, the selective inhibitor of aminopeptidases (Bestatin) provoked considerable dose-dependent analgesia when intramuscularly injected into the hyperalgesic muscle. The CB1 receptor antagonist (AM251), but not the CB2 receptor antagonist (AM630), intensified muscle hyperalgesia. All irreversible inhibitors of anandamide hydrolase (MAFP), the inhibitor for monoacylglycerol lipase (JZL184) and the anandamide reuptake inhibitor (VDM11) decreased carrageenan-induced hyperalgesia in muscular tissue. Lastly, MOP, KOP and CB1 expression levels in DRG were baseline even after muscular injection with carrageenan. The endogenous opioid and cannabinoid systems participate in peripheral muscle pain control through the activation of MOP, KOP and CB1 receptors.

Endocannabinoid System as a Promising Therapeutic Target in Inflammatory Bowel Disease - A Systematic Review.

Inflammatory bowel disease (IBD) is a general term used to describe a group of chronic inflammatory conditions of the gastrointestinal tract of unknown etiology, including two primary forms: Crohn's disease (CD) and ulcerative colitis (UC). The endocannabinoid system (ECS) plays an important role in modulating many physiological processes including intestinal homeostasis, modulation of gastrointestinal motility, visceral sensation, or immunomodulation of inflammation in IBD. It consists of cannabinoid receptors (CB1 and CB2), transporters for cellular uptake of endocannabinoid ligands, endogenous bioactive lipids (Anandamide and 2-arachidonoylglycerol), and the enzymes responsible for their synthesis and degradation (fatty acid amide hydrolase and monoacylglycerol lipase), the manipulation of which through agonists and antagonists of the system, shows a potential therapeutic role for ECS in inflammatory bowel disease. This review summarizes the role of ECS components on intestinal inflammation, suggesting the advantages of cannabinoid-based therapies in inflammatory bowel disease.

Biosynthesis of N-Docosahexanoylethanolamine from Unesterified Docosahexaenoic Acid and Docosahexaenoyl-Lysophosphatidylcholine in Neuronal Cells.

We investigated the synthesis of N-docosahexaenoylethanolamine (synaptamide) in neuronal cells from unesterified docosahexaenoic acid (DHA) or DHA-lysophosphatidylcholine (DHA-lysoPC), the two major lipid forms that deliver DHA to the brain, in order to understand the formation of this neurotrophic and neuroprotective metabolite of DHA in the brain. Both substrates were taken up in Neuro2A cells and metabolized to N-docosahexaenoylphosphatidylethanolamine (NDoPE) and synaptamide in a time- and concentration-dependent manner, but unesterified DHA was 1.5 to 2.4 times more effective than DHA-lysoPC at equimolar concentrations. The plasmalogen NDoPE (pNDoPE) amounted more than 80% of NDoPE produced from DHA or DHA-lysoPC, with 16-carbon-pNDoPE being the most abundant species. Inhibition of N-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD) by hexachlorophene or bithionol significantly decreased the synaptamide production, indicating that synaptamide synthesis is mediated at least in part via NDoPE hydrolysis. NDoPE formation occurred much more rapidly than synaptamide production, indicating a precursor-product relationship. Although NDoPE is an intermediate for synaptamide biosynthesis, only about 1% of newly synthesized NDoPE was converted to synaptamide, possibly suggesting additional biological function of NDoPE, particularly for pNDoPE, which is the major form of NDoPE produced.

Hippocampal 2-Arachidonoyl Glycerol Signaling Regulates Time-of-Day- and Stress-Dependent Effects on Rat Short-Term Memory.

BACKGROUND: Cannabinoids induce biphasic effects on memory depending on stress levels. We previously demonstrated that different stress intensities, experienced soon after encoding, impaired rat short-term recognition memory in a time-of-day-dependent manner, and that boosting endocannabinoid anandamide (AEA) levels restored memory performance. Here, we examined if two different stress intensities and time-of-day alter hippocampal endocannabinoid tone, and whether these changes modulate short-term memory. METHODS: Male Sprague-Dawley rats were subjected to an object recognition task and exposed, at two different times of the day (i.e., morning or afternoon), to low or high stress conditions, immediately after encoding. Memory retention was assessed 1 hr later. Hippocampal AEA and 2-arachidonoyl glycerol (2-AG) content and the activity of their primary degrading enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), were measured soon after testing. RESULTS: Consistent with our previous findings, low stress impaired 1-hr memory performance only in the morning, whereas exposure to high stress impaired memory independently of testing time. Stress exposure decreased AEA levels independently of memory alterations. Interestingly, exposure to high stress decreased 2-AG content and, accordingly, increased MAGL activity, selectively in the afternoon. Thus, to further evaluate 2-AG's role in the modulation of short-term recognition memory, rats were given bilateral intra-hippocampal injections of the 2-AG hydrolysis inhibitor KML29 immediately after training, then subjected to low or high stress conditions and tested 1 hr later. CONCLUSIONS: KML29 abolished the time-of-day-dependent impairing effects of stress on short-term memory, ameliorating short-term recognition memory performance.

COX-2 Inhibition Antagonizes Intra-Accumbens 2-Arachidonoylglycerol-Mediated Reduction in Ethanol Self-Administration in Rats.

BACKGROUND: Ethanol (EtOH) self-administration is particularly sensitive to the modulation of CB1 signaling in the nucleus accumbens (NAc) shell, and EtOH consumption increases extracellular levels of the endogenous cannabinoid CB1 receptor agonist 2-arachidonoyl glycerol (2-AG) in this brain region. Stimulation of CB1 receptor with agonists increases EtOH consumption, suggesting that EtOH-induced increases in 2-AG might sustain motivation for EtOH intake. METHODS: In order to further explore this hypothesis, we analyzed the alterations in operant EtOH self-administration induced by intra-NAc shell infusions of 2-AG itself, the CB1 inverse agonist SR141716A, the 2-AG clearance inhibitor URB602, anandamide, and the cyclooxygenase-2 (COX-2) inhibitor nimesulide. RESULTS: Surprisingly, self-administration of 10% EtOH was dose-dependently reduced by either intra-NAc shell SR141716A or 2-AG infusions. Similar effects were found by intra-NAc shell infusions of URB602, suggesting again a role for accumbal 2-AG on the modulation of EtOH intake. Intra-NAc shell anandamide did not alter EtOH self-administration, pointing to a specific role for 2-AG in the modulation of EtOH self-administration. Finally, the inhibitory effect of intra-NAc shell 2-AG on EtOH intake was significantly reversed by pretreatment with nimesulide, suggesting that oxidative metabolites of 2-AG might mediate these inhibitory effects on operant self-administration. CONCLUSIONS: We propose that 2-AG signaling in the NAc exerts an inhibitory influence on EtOH consumption through a non-CB1 receptor mechanism involving the COX-2 pathway.

Druggable Targets in Endocannabinoid Signaling.

Cannabis and cannabinoid-based extracts have long been utilized for their perceived therapeutic value, and support for the legalization of cannabis for medicinal purposes continues to increase worldwide. Since the discovery of Δ9-tetrahydrocannabinol (THC) as the primary psychoactive component of cannabis over 50 years ago, substantial effort has been directed toward detection of endogenous mediators of cannabinoid activity. The discovery of anandamide and 2-arachidonoylglycerol as two endogenous lipid mediators of cannabinoid-like effects (endocannabinoids) has inspired exponential growth in our understanding of this essential pathway, as well as the pathological conditions that result from dysregulated endocannabinoid signaling. This review examines current knowledge of the endocannabinoid system including metabolic enzymes involved in biosynthesis and degradation and their receptors, and evaluates potential druggable targets for therapeutic intervention.

The endocannabinoid signaling pathway as an emerging target in pharmacotherapy, earmarking mitigation of destructive events in rheumatoid arthritis.

Rheumatoid arthritis is an inflammatory autoimmune disease, characterized by synovial proliferation, destruction to articular cartilage and severe pain. The cannabinoids obtained from Cannabis sativa exhibited their actions via cannabinoid-1 and -2 receptors, which also provides a platform for endocannabinoids to act. The endocannabinoid system comprises endocannabinoid molecules involved in signaling processes, along with G-protein coupled receptors and enzymes associated with ligand biosynthesis, activation and degradation. The action of endocannabinoid system in immune system regulation, via primary CB2 activation, followed by inhibition of production of pro-inflammatory cytokines, auto-antibodies and MMPs, FLSs proliferation and T-cell mediated immune response, are elaborated as potential therapeutic regimes in rheumatoid arthritis. The involvement of endocannabinoid system in immune cells like, B cells, T cells and macrophages, as well as regulatory actions on sensory noniceptors to ameliorate pain is significantly highlighted in the review, elaborating the actions of endocannabinoid signaling in mitigating the disease events. The review also focuses on enhancement of endocannabinoid tone, either by inhibiting the degradation enzymes, like FAAH, MAGL, COX, CytP450, LOX, etc. or by retarding cellular uptake processes. Moreover, the review portrays the optimizing role of endocannabinoid system, in abbreviating the symptoms and complications of rheumatoid arthritis in patients and mitigating inflammation, pain and immune mediated effects significantly.

The endocannabinoid receptors CB1 and CB2 affect the regenerative potential of adipose tissue MSCs.

Human adipose tissue includes large quantities of mesenchymal stromal cells (atMSCs), which represent an abundant cell source for therapeutic applications in the field of regenerative medicine. Adipose tissue secrets various soluble factors including endocannabinoids, and atMSCs express the cannabinoid receptors CB1 and CB2. This indicates that adipose tissue possesses an endocannabinoid system (ECS). The ECS is also ascribed great significance for wound repair, e.g. by modulating inflammation. However, the exact effects of CB1/CB2 activation in human atMSCs have not been investigated, yet. In the present study, we stimulated human atMSCs with increasing concentrations (1-30 µM) of the unspecific cannabinoid receptor ligand WIN55,212-2 and the specific CB2 agonist JWH-133, either alone or co-applied with the receptor antagonist Rimonabant (CB1) or AM 630 (CB2). We investigated the effects on metabolic activity, cell number, differentiation and cytokine release, which are important processes during tissue regeneration. WIN decreased metabolic activity and cell number, which was reversed by Rimonabant. This suggests a CB1 dependent mechanism, whereas the number of atMSCs was increased after CB2 ligation. WIN and JWH increased the release of VEGF, TGF-ß1 and HGF. Adipogenesis was enhanced by WIN, which could be reversed by blocking CB1. There was no effect on osteogenesis, and only WIN increased chondrogenic differentiation. Our results indicate that definite activation of the cannabinoid receptors exerted different effects in atMSCs, which could be of specific value in cell-based therapy for wound regeneration.

Endocannabinoid modulating drugs improve anxiety but not the expression of conditioned fear in a rodent model of post-traumatic stress disorder.

The endocannabinoid (eCB) system is a potential target for the treatment of symptoms of post-traumatic stress disorder (PTSD). Similar to clinical PTSD, approximately 25-30% of rats that undergo cued fear conditioning exhibit impaired extinction learning. In addition to extinction-resistant fear, these "weak extinction" (WE) rats show persistent anxiety-like behaviors. The goal of the present study was to test the hypothesis that behavioural differences between WE animals and those presenting normal extinction patterns (strong extinction; SE) could be mediated by the eCB system. Rats undergoing fear conditioning/extinction and fear recall sessions were initially segregated in weak and strong-extinction groups. Two weeks later, animals underwent a fear recall session followed by a novelty-suppressed feeding (NSF) test. In acute experiments, WE rats were injected with either the fatty acid amide hydrolase (FAAH) inhibitor URB597 or the CB1 agonist WIN55,212-2 1 h prior to long-term recall and NSF testing. SE animals were injected with the inverse CB1 receptor agonist AM251. In chronic experiments, WE and SE rats were given daily injections of URB597 or AM251 between short and long-term recall sessions. We found that acute administration of WIN55,212-2 but not URB597 reduced anxiety-like behaviour in WE rats. In contrast, AM251 was anxiogenic in SE animals. Neither treatment was effective in altering freezing expression during fear recall. The chronic administration of AM251 to SE or URB597 to WE did not alter fear or anxiety-like behaviour or changed the expression of FAAH and CB1. Together, these results suggest that systemic manipulations of the eCB system may alter anxiety-like behaviour but not the behavioural expression of an extinction-resistant associative fear memory.

Endogenous cannabinoid modulation of restraint stress-induced analgesia in thermal nociception.

It is thought that endogenous cannabinoids have a role in the analgesia induced by specific forms of stress. We examined if the role of endogenous cannabinoids is also dependent upon the mode of nociception, and whether this could be altered by drugs which block their enzymatic degradation. In C57BL/6 mice, restraint stress produced analgesia in the hot-plate and plantar tests, two thermal pain assays that engage distinct supraspinal and spinal nociceptive pathways. Stress-induced analgesia in the hot-plate test was abolished by pre-treatment with the opioid receptor antagonist naltrexone but was unaffected by the cannabinoid receptor antagonist 1-(2,4-Dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide (AM281). By contrast, stress-induced analgesia in the plantar test was abolished by pre-treatment with naltrexone plus AM281, but not by either antagonist individually. Remarkably, inhibiting the breakdown of endocannabinoids, with the dual fatty acid amide hydrolase and monoacylglycerol lipase inhibitor JZL195, rescued stress-induced analgesia in the hotplate test when endogenous opioid signalling was blocked by naltrexone. Furthermore, JZL195 recruited analgesia induced by sub-threshold restraint stress in both thermal pain assays. These findings indicate the role of endocannabinoids in stress-induced analgesia differs with the type of thermal pain behaviour. However, by inhibiting their breakdown, endocannabinoids can be recruited to substitute for endogenous opioid signalling when their activity is blocked, indicating a degree of redundancy between opioid and cannabinoid systems. Together these data suggest targeting endocannabinoid breakdown could provide an alternative, or adjuvant to mainstream analgesics such as opioids.

Astrocytic Endocannabinoids Mediate Hippocampal Transient Heterosynaptic Depression.

Astrocytes are highly dynamic cells that modulate synaptic transmission within a temporal domain of seconds to minutes in physiological contexts such as Long-Term Potentiation (LTP) and Heterosynaptic Depression (HSD). Recent studies have revealed that astrocytes also modulate a faster form of synaptic activity (milliseconds to seconds) known as Transient Heterosynaptic Depression (tHSD). However, the mechanism underlying astrocytic modulation of tHSD is not fully understood. Are the traditional gliotransmitters ATP or glutamate released via hemichannels/vesicles or are other, yet, unexplored pathways involved? Using various approaches to manipulate astrocytes, including the Krebs cycle inhibitor fluoroacetate, connexin 43/30 double knockout mice (hemichannels), and inositol triphosphate type-2 receptor knockout mice, we confirmed early reports demonstrating that astrocytes are critical for tHSD. We also confirmed the importance of group II metabotropic glutamate receptors (mGluRs) in astrocytic modulation of tHSD using a group II agonist. Using dominant negative SNARE mice, which have disrupted glial vesicle function, we also found that vesicular release of gliotransmitters and activation of adenosine A1 receptors are not required for tHSD. As astrocytes can release lipids upon receptor stimulation, we asked if astrocyte-derived endocannabinoids are involved in tHSD. Interestingly, a cannabinoid receptor 1 (CB1R) antagonist blocked and an inhibitor of the endogenous endocannabinoid 2-arachidonyl glycerol (2-AG) degradation potentiates tHSD in hippocampal slices. Taken together, this study provides the first evidence for group II mGluR-mediated astrocytic endocannabinoids in transiently suppressing presynaptic neurotransmitter release associated with the phenomenon of tHSD.

Disrupting the endocannabinoid system in early adolescence negatively impacts sociability.

Animal models suggest that the endocannabinoid system (eCS) helps regulate various aspects of social behavior, including play behavior and social reward, during adolescence. Properly tuned endocannabinoid signaling may be a critical developmental component in the emergence of normal adult sociability. In the current experiment, we attempted to pharmacologically disrupt endocannabinoid tone during early adolescence, and then measure the behavioral effects at two subsequent time points. 36 male and 36 female Long Evans rats received daily injections of one of three treatments between post-natal day (PND) 25-39: 1) vehicle treatment, 2) 0.4 mg/kg CP55,940 (a potent CB1/CB2 receptor agonist), or 3) 0.5 mg/kg AM251 (a CB1 receptor antagonist/inverse agonist). Both soon after treatment (PND 40-44) and several weeks later (PND 66-70), subjects were tested in an elevated plus maze (EPM) for anxiety and in a three-chambered apparatus for sociability. For the latter test, the number of entries into each chamber and the amount of time spent investigating each target were measured. Analyses revealed significant main effects of both sex and age on sociability: males expressed greater sociability compared to females, and sociability was higher in adolescence than adulthood. Most importantly, drug treatment (both CP55,940 and AM251) attenuated sociability in adolescence without having a significant effect on anxiety in the EPM. However, this effect did not persist into adulthood. These results indicate that pharmacological disruption of endocannabinoid tone - through either chronic agonism or antagonism of cannabinoid receptors - during early adolescence has a detrimental effect on sociability. This effect may be caused by transient, compensatory alterations in the eCS.