Underlying mechanisms of long-term potentiation during the inhibition of the cannabinoid CB1 and GABAB receptors in the dentate gyrus of hippocampus.

BACKGROUND: The release of various neurotransmitters and thereby the excitability of neuronal circuits are regulated by the endocannabinoid system in an activity-dependent manner. Hippocampal long-term potentiation (LTP) is augmented in cannabinoid type 1 (CB1) receptor-deficient mice. CB1 receptors exist on GABAergic axon terminals in the hippocampus. In our previous work, we showed that CB1 antagonists increased the population spike (PS) amplitude, field excitatory post-synaptic potential (fEPSP), and the LTP induction in the dentate gyrus (DG) of the rat hippocampus while the GABAB antagonist decreased these parameters. Determining the underlying mechanisms of the pre- and/or postsynaptic locus of LTP expression is of great importance. In this study, we investigated whether LTP alteration acutely caused by CB1 and GABAB receptor antagonists (AM251 and CGP55845, respectively) happens at the postsynaptic or presynaptic regions, or at both. Therefore, the paired-pulse ratio (PPR) was assessed prior to and following the LTP induction in the studied groups. METHODS: Male Wistar rats were randomly assigned to the groups of control, AM251, CGP55845, CGP55845 + AM251. A high-frequency stimulation (HFS) of the perforant path (PP) was used to induce LTP in the DG region. RESULTS: Statistical analysis revealed that AM251 produced significant increase in excitatory postsynaptic potential (EPSP) slope and amplitude of PS. Conversely, administration of CGP55845 produced decrease in slope of EPSP. The current results indicated that the PPR was not influenced by LTP induction in the presence of AM251 or CGP55845 either alone or their combination. CONCLUSIONS: It can be concluded that the site causing LTP expression is, at least in part, the postsynaptic site because PPR was not influenced by LTP induction in the presence of AM251 or CGP55845 either alone or their combination.

The anabolic steroid nandrolone alters cannabinoid self-administration and brain CB1 receptor density and function.

Clinical and pre-clinical observations indicate that anabolic-androgenic steroids can induce neurobiological changes that alter the rewarding effects of drugs of abuse. In this study, we investigated the effect of the anabolic steroid nandrolone on the rewarding properties of the cannabinoid CB1 receptor agonist WIN55,212-2 (WIN) in rats. Lister Hooded male rats were treated intramuscularly with nandrolone (15mg/kg) or vehicle for 14 consecutive days, and then allowed to self-administer WIN (12.5µg/kg/infusion) intravenously. After reaching stable drug intake, self-administration behavior was extinguished to examine drug- and cue-induced reinstatement of cannabinoid-seeking behavior. Other behavioral parameters presumed to influence drug-taking and drug-seeking behaviors were examined to gain more insight into the behavioral specificity of nandrolone treatment. Finally, animals were sacrificed for analysis of CB1 receptor density and function in selected brain areas. We found that nandrolone-treated rats self-administered up to 2 times more cannabinoid than vehicle-treated rats, but behaved similarly to control rats when tested for drug- and cue-induced reinstatement of cannabinoid-seeking behavior. Enhanced cannabinoid intake by nandrolone-treated rats was not accompanied by changes in locomotor activity, sensorimotor gating, or memory function. However, our molecular data show that after chronic WIN self-administration nandrolone-treated rats display altered CB1 receptor density and function in selected brain areas. We hypothesize that increased cannabinoid self-administration in nandrolone-treated rats results from a nandrolone-induced decrease in reward function, which rats seem to compensate by voluntarily increasing their cannabinoid intake. Altogether, our findings corroborate the hypothesis that chronic exposure to anabolic-androgenic steroids induces dysfunction of the reward pathway in rats and might represent a potential risk factor for abuse of cannabis and other drugs in humans.

Cocaine-induced endocannabinoid release modulates behavioral and neurochemical sensitization in mice.

The endocannabinoid system has been implicated in the development of synaptic plasticity induced by several drugs abused by humans, including cocaine. However, there remains some debate about the involvement of cannabinoid receptors/ligands in cocaine-induced plasticity and corresponding behavioral actions. Here, we show that a single cocaine injection in Swiss-Webster mice produces behavioral and neurochemical alterations that are under the control of the endocannabinoid system. This plasticity may be the initial basis for changes in brain processes leading from recreational use of cocaine to its abuse and ultimately to dependence. Locomotor activity was monitored with photobeam cell detectors, and accumbens shell/core microdialysate dopamine levels were monitored by high-performance liquid chromatography with electrochemical detection. Development of single-trial cocaine-induced behavioral sensitization, measured as increased distance traveled in sensitized mice compared to control mice, was paralleled by a larger stimulation of extracellular dopamine levels in the core but not the shell of the nucleus accumbens. Both the behavioral and neurochemical effects were reversed by CB1 receptor blockade produced by rimonabant pre-treatments. Further, both behavioral and neurochemical cocaine sensitization were facilitated by pharmacological blockade of endocannabinoid metabolism, achieved by inhibiting the fatty acid amide hydrolase enzyme. In conclusion, our results suggest that a single unconditioned exposure to cocaine produces sensitization through neuronal alterations that require regionally specific release of endocannabinoids. Further, the present results suggest that endocannabinoids play a primary role from the earliest stage of cocaine use, mediating the inception of long-term brain-adaptive responses, shaping central pathways and likely increasing vulnerability to stimulant abuse disorders.

Basolateral amygdala cannabinoid CB1 receptors mediate the antinociceptive activity of harmaline in adolescent male mice.

Evidence suggests a clear role for the amygdala endocannabinoid system in pain processing. Harmaline has been also known as the main nociceptive agent extracted from the Peganum harmala plant. In this study, the role of basolateral amygdala (BLA) cannabinoid CB1 receptors in pain sensitivity of harmaline-treated mice were assessed using tail-flick and hot plate methods in adolescent male NMRI mice. Intraperitoneal administration of two higher doses of harmaline (6 and 8 mg/kg) increased tail-flick latency, suggesting an antinociceptive activity. The same result was observed for the higher dose of harmaline in the hot plate test. Intra-BLA microinjection of CB1 receptor agonist ACPA (1 and 1.5 ng/mouse) or (1.5 ng/mouse) enhanced the ineffective dose-response of harmaline on pain threshold in the tail-flick or hot plate tests, respectively. Microinjection of two higher doses of CB1 receptor antagonist AM251 (0.5 and 1 ng/mouse) attenuated the antinociceptive activity of harmaline (8 ng/mouse) in both tail-flick and hot plate tests. Meanwhile, ACPA and AM251 did not alter latency to withdraw from the noxious stimulus in both tests, by themselves. It should be noted that the analgesic dose of the drugs alone or in combination did not affect locomotor activity. The obtained results highlight that BLA CB1 receptors mediate the antinociceptive activity of harmaline.

The Impact of CB1 Receptor on Inflammation in Skeletal Muscle Cells.

BACKGROUND: Various factors trigger the inflammatory response and cytokine activation in skeletal muscle. Inflamed muscle will exhibit significant levels of inflammation and cytokine activity. Interleukin-6 (IL-6), a pro-inflammatory cytokine, exerts pleiotropic effects on skeletal muscle. Endocannabinoid produced by all cell types binds to a class of G protein-coupled receptors, in particular cannabinoid CB1 receptors, to induce skeletal muscle actions. OBJECTIVE: The purpose of this research was to discover whether activation of cannabinoid CB1 receptors in L6 skeletal muscle cells may promote IL-6 gene expression. MATERIALS AND METHODS: L6 skeletal muscle cells were cultured in 25 cm2 flasks and quantitative reverse transcription-polymerase chain reaction (probe-based) utilised to quantify IL-6 gene expression levels among different treatment settings. RESULTS: Arachidonyl-2'-chloroethylamide (ACEA) 10 nM, a persistent selective CB1 receptor agonist, promotes IL-6 gene expression in a time-dependent manner. Rimonabant 100 nM, a selective cannabinoid CB1 receptor antagonist, blocks the impact of ACEA. However, insulin does not change IL-6 gene expression. CONCLUSION: For the first time, a unique link between ACEA and IL-6 up-regulation has been established; IL-6 up-regulation generated by ACEA is mediated in skeletal muscle through cannabinoid CB1 receptor activation. As a result, cannabinoid CB1 receptors may be useful pharmaceutical targets in the treatment of inflammation and related disorders in skeletal muscle tissues.

The Impact of CB1 Receptor on Nuclear Receptors in Skeletal Muscle Cells.

Cannabinoids are abundant signaling compounds; their influence predominantly arises via engagement with the principal two G-protein-coupled cannabinoid receptors, CB1 and CB2. One suggested theory is that cannabinoids regulate a variety of physiological processes within the cells of skeletal muscle. Earlier publications have indicated that expression of CB1 receptor mRNA and protein has been recognized within myotubes and tissues of skeletal muscle from both murines and humans, thus representing a potentially significant pathway which plays a role in the control of skeletal muscular activities. The part played by CB1 receptor activation or inhibition with respect to these functions and relevant to targets in the periphery, especially skeletal muscle, is not fully delineated. Thus, the aim of the current research was to explore the influence of CB1 receptor stimulation and inhibition on downstream signaling of the nuclear receptor, NR4A, which regulates the immediate impacts of arachidonyl-2'-chloroethylamide (ACEA) and/or rimonabant in the cells of skeletal muscle. Murine L6 skeletal muscle cells were used in order to clarify additional possible molecular signaling pathways which contribute to alterations in the CB1 receptor. Skeletal muscle cells have often been used; it is well-documented that they express cannabinoid receptors. Quantitative real-time probe-based polymerase chain reaction (qRT-PCR) assays are deployed in order to assess the gene expression characteristics of CB1 receptor signaling. In the current work, it is demonstrated that skeletal muscle cells exhibit functional expression of CB1 receptors. This can be deduced from the qRT-PCR assays; triggering CB1 receptors amplifies both NR4A1 and NR4A3 mRNA gene expression. The impact of ACEA is inhibited by the selective CB1 receptor antagonist, rimonabant. The present research demonstrated that 10 nM of ACEA notably amplified mRNA gene expression of NR4A1 and NR4A3; this effect was suppressed by the addition of 100 nM rimonabant. Furthermore, the CB1 receptor antagonist led to the downregulation of mRNA gene expression of NR4A1, NR4A2 and NR4A3. In conclusion, in skeletal muscle, CB1 receptors were recognized to be important moderators of NR4A1 and NR4A3 mRNA gene expression; these actions may have possible clinical benefits. Thus, in skeletal muscle cells, a possible physiological expression of CB1 receptors was identified. It is as yet unknown whether these CB1 receptors contribute to pathways underlying skeletal muscle biological function and disease processes. Further research is required to fully delineate their role(s).

Pharmacological characterisation of the CB1 receptor antagonist activity of cannabidiol in the rat vas deferens bioassay.

Cannabidiol is increasingly considered for treatment of a wide range of medical conditions. Binding studies suggest that cannabidiol binds to CB1 receptors. In the rat isolated vas deferens bioassay, a single electrical pulse causes a biphasic contraction from nerve-released ATP and noradrenaline. WIN 55,212-2 acts on prejunctional CB1 receptors to inhibit release of these transmitters. In this bioassay, we tested whether cannabidiol and SR141716 were acting as competitive antagonists of this receptor. Monophasic contractions mediated by ATP or noradrenaline in the presence of prazosin or NF449 (P2X1 inhibitor), respectively, were measured to a single electrical pulse delivered every 30 min. Following treatment with cannabidiol (10-100 µM) or SR141716 (0.003-10 µM), cumulative concentrations of WIN 55,212-2 (0.001-30 µM) were applied followed by a single electrical pulse. The WIN 55,212-2 concentration-contraction curve EC50 values were applied to global regression analysis to determine the pKB. The antagonist potency of cannabidiol at the CB1 receptor in the rat vas deferens bioassay matched the reported receptor binding affinity. Cannabidiol was a competitive antagonist of WIN 55,212-2 with pKB values of 5.90 when ATP was the effector transmitter and 5.29 when it was noradrenaline. Similarly, SR141716 was a competitive antagonist with pKB values of 8.39 for ATP and 7.67 for noradrenaline as the active transmitter. Cannabidiol's low micromolar CB1 antagonist pKB values suggest that at clinical blood levels (1-3 µM) it may act as a CB1 antagonist at prejunctional neuronal sites with more potency when ATP is the effector than for noradrenaline.

Medial Prefrontal Cortical Cannabinoid CB1 Receptors Mediate Morphine-Dextromethorphan Cross State-Dependent Memory: The Involvement of BDNF/cFOS Signaling Pathways.

The present study set out to assess the possible role of the medial prefrontal cortex (mPFC) cannabinoid CB1 receptors and BDNF/cFOS signaling pathways in morphine-dextromethorphan (DXM) cross state-dependent memory (SDM) using male Wistar rats. Changes on the levels of BDNF and cFOS proteins in the PFC were examined by Western blot analysis. Present results revealed that levels of BDNF and cFOS proteins were significantly increased in the animals that were trained in the passive avoidance apparatus. Intraperitoneal injection of morphine (6 mg/kg, i.p.) after training impaired memory which was associated with decreases in the levels of both proteins. Moreover, the injection of a cannabinoid CB1 receptor agonist, ACPA, or a selective CB1 receptor antagonist, AM-251, into the mPFC prior to testing had no effect on memory retrieval by itself and also on morphine-induced memory loss. Pre-test administration of DXM (a NMDA receptors antagonist, 30 mg/kg, i.p.) impaired memory retrieval and attenuated BDNF levels. Moreover, DXM administration (pre-test) prevented morphine-induced memory loss and increased the levels of both proteins, suggesting morphine-DXM cross-SDM. Interestingly, pre-test intra-mPFC injections of ACPA inhibited cross-SDM between the drugs which was associated with an elevation of BDNF expression in the PFC. Additionally, pre-test administration of an ineffective dose of DXM (10 mg/kg, i.p.) could not reverse morphine-induced memory loss, while pre-test intra-mPFC injections of AM-251 potentiated morphine-DXM cross-SDM. Taken together, it can be concluded that mPFC through CB1cannabinoid receptors has a critical role in morphine-DXM cross-SDM which may be associated with the PFC BDNF/cFOS signaling pathway.

Gene-environment interactions affect long-term depression (LTD) through changes in dopamine receptor affinity in Snap25 deficient mice.

Genes and environmental conditions interact in the development of cognitive capacities and each plays an important role in neuropsychiatric disorders such as attention deficit/hyperactivity disorder (ADHD) and schizophrenia. Multiple studies have indicated that the gene for the SNARE protein SNAP-25 is a candidate susceptibility gene for ADHD, as well as schizophrenia, while maternal smoking is a candidate environmental risk factor for ADHD. We utilized mice heterozygous for a Snap25 null allele and deficient in SNAP-25 expression to model genetic effects in combination with prenatal exposure to nicotine to explore genetic and environmental interactions in synaptic plasticity and behavior. We show that SNAP-25 deficient mice exposed to prenatal nicotine exhibit hyperactivity and deficits in social interaction. Using a high frequency stimulus electrophysiological paradigm for long-term depression (LTD) induction, we examined the roles of dopaminergic D2 receptors (D2Rs) and cannabinoid CB1 receptors (CB1Rs), both critical for LTD induction in the striatum. We found that prenatal exposure to nicotine in Snap25 heterozygote null mice produced a deficit in the D2R-dependent induction of LTD, although CB1R regulation of plasticity was not impaired. We also show that prenatal nicotine exposure altered the affinity and/or receptor coupling of D2Rs, but not the number of these receptors in heterozygote null Snap25 mutants. These results refine the observations made in the coloboma mouse mutant, a proposed mouse model of ADHD, and illustrate how gene×environmental influences can interact to perturb neural functions that regulate behavior.

Interleukin-1ß alters the sensitivity of cannabinoid CB1 receptors controlling glutamate transmission in the striatum.

Proinflammatory cytokines such as tumor necrosis factor-α and interleukin-1ß (IL1ß) regulate both excitatory and inhibitory synaptic transmission in the central nervous system. The interaction between IL1ß and endocannabinoid system (ECS) is also emerging, based on the evidence that IL1ß effects on striatal spontaneous excitatory and inhibitory postsynaptic currents are regulated by transient receptor potential vanilloid 1 (TRPV1) channels, members of the ECS. Furthermore, IL1ß has also been shown to control the sensitivity of cannabinoid CB1 receptors controlling GABA transmission (CB1Rs(GABA)) in the striatum. To better detail the synaptic action of IL1ß, and to clarify its complex interaction with the ECS, here we investigated the possible interplay between IL1ß and CB1Rs controlling glutamate transmission (CB1Rs(glu)), other critical elements of the ECS. Our results show that the sensitivity of CB1Rs(glu) is fully blocked in the presence of IL1ß in corticostriatal brain slices, and that the protein kinase C/TRPV1 pathway is involved in this effect. IL1ß failed to modulate the sensitivity of glutamate synapses to the stimulation of GABAB receptors. We also provided evidence that IL1ß-CB1Rs(GABA) but not IL1ß-CB1Rs(glu) interaction is under the control of the brain-derived neurotrophic factor (BDNF)/trkB signaling and of lipid raft composition, because BDNF gene partial deletion, pharmacological blockade of trkB and membrane cholesterol removal with methyl-ß-cyclodextrin all blocked IL1ß-mediated inhibition of CB1Rs(GABA) but left unaltered the sensitivity of CB1Rs(glu) to this cytokine. Our results provide further evidence that synaptic transmission and the ECS are regulated by IL1ß in the striatum.