The endogenous cannabinoid system protects against colonic inflammation.

Excessive inflammatory responses can emerge as a potential danger for organisms' health. Physiological balance between pro- and anti-inflammatory processes constitutes an important feature of responses against harmful events. Here, we show that cannabinoid receptors type 1 (CB1) mediate intrinsic protective signals that counteract proinflammatory responses. Both intrarectal infusion of 2,4-dinitrobenzene sulfonic acid (DNBS) and oral administration of dextrane sulfate sodium induced stronger inflammation in CB1-deficient mice (CB1(-/-)) than in wild-type littermates (CB1(+/+)). Treatment of wild-type mice with the specific CB1 antagonist N-(piperidino-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-pyrazole-3-carboxamide (SR141716A) mimicked the phenotype of CB1(-/-) mice, showing an acute requirement of CB1 receptors for protection from inflammation. Consistently, treatment with the cannabinoid receptor agonist R(-)-7-hydroxy-Delta(6)-tetra-hydrocannabinol-dimethylheptyl (HU210) or genetic ablation of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH) resulted in protection against DNBS-induced colitis. Electrophysiological recordings from circular smooth muscle cells, performed 8 hours after DNBS treatment, revealed spontaneous oscillatory action potentials in CB1(-/-) but not in CB1(+/+) colons, indicating an early CB1-mediated control of inflammation-induced irritation of smooth muscle cells. DNBS treatment increased the percentage of myenteric neurons expressing CB1 receptors, suggesting an enhancement of cannabinoid signaling during colitis. Our results indicate that the endogenous cannabinoid system represents a promising therapeutic target for the treatment of intestinal disease conditions characterized by excessive inflammatory responses.

Corticotropin-releasing hormone-mediated induction of intracellular signaling pathways and brain-derived neurotrophic factor expression is inhibited by the activation of the endocannabinoid system.

CRH receptor (CRHR) 1 and the cannabinoid receptor 1 (CB1) are both G protein-coupled receptors. Activation of CRHR1 leads to increases in cAMP production and phosphorylation of the transcription factor cAMP response element-binding protein (CREB). In contrast, CB1 is negatively coupled to the cAMP signaling cascade. In this study, we analyzed a putative interaction between these two systems focusing on the regulation of the expression of brain-derived neurotrophic factor (BDNF), a CREB-regulated gene. In situ hybridization revealed coexpression of CRHR1 and CB1 receptors in the granular layer of the cerebellum. Therefore, we analyzed the effects of CRH and the CB1 agonist WIN-55,212-2 on BDNF expression in primary cerebellar neurons from rats and mice. We observed that application of CRH for 48 h led to an increase in BDNF mRNA and protein levels. This effect was inhibited by WIN-55,212-2. At the level of intracellular signaling, short-term application of WIN-55,212-2 inhibited CRH-induced cAMP accumulation and CREB phosphorylation. Pharmacological analysis demonstrated that the CRHR1 antagonist R121919, the protein kinase A inhibitor H89, and the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester inhibited CRH-mediated BDNF expression. Moreover, depolarization-induced BDNF synthesis was also inhibited by long-term application of WIN-55,212-2 in wild-type mice but not in CB1-deficient mice. Thus, these data highlight an interaction between the CRH and the cannabinoid system in the regulation of BDNF expression by influencing cAMP and Ca2+ signaling pathways.

Coexpression of the cannabinoid receptor type 1 with the corticotropin-releasing hormone receptor type 1 in distinct regions of the adult mouse forebrain.

The high abundance of the cannabinoid receptor type 1 (CB1) in the brain and the discovery of its endogenous ligands possessing neuromodulatory activities suggest an important potential of the endocannabinoid system to influence the functions of other receptor systems in the brain, including the corticotropin releasing hormone (CRH) system. Several studies evidenced a cross-talk between these two receptor systems. In trying to detail functional interactions between CB1 and the CRH receptor type 1 (CRHR1), we performed double-label-in situ hybridisation on mouse forebrain sections to localise the transcripts encoding the two receptors at a cellular level. Colocalisation of both receptor mRNAs was only detected in low CB1-expressing cells, which are mainly principal projecting neurons, whereas high CB1-expressing cells, which are considered to be mostly GABAergic did not contain mRNA encoding CRHR1. CB1 is differentially coexpressed with CRHR1 in olfactory regions, in several cortical and limbic structures, and in some hypothalamic and thalamic nuclei. These observations suggest a complex mechanism underlying the mutual interrelation and modulation of the two receptor systems. In particular, high levels of coexpressing cells in cortical and limbic areas may relate to cognitive functions, such as working memory, emotional and declarative learning. Colocalisation of CB1 and CRHR1 in hypothalamic regions strongly suggests functional interactions regarding the neuroendocrine homeostasis, including feeding behaviour.

CB1 cannabinoid receptors modulate kinase and phosphatase activity during extinction of conditioned fear in mice.

Cannabinoid receptors type 1 (CB1) play a central role in both short-term and long-term extinction of auditory-cued fear memory. The molecular mechanisms underlying this function remain to be clarified. Several studies indicated extracellular signal-regulated kinases (ERKs), the phosphatidylinositol 3-kinase with its downstream effector AKT, and the phosphatase calcineurin as potential molecular substrates of extinction behavior. To test the involvement of these kinase and phosphatase activities in CB1-dependent extinction of conditioned fear behavior, conditioned CB1-deficient mice (CB1(-/-)) and wild-type littermates (CB1(+/+)) were sacrificed 30 min after recall of fear memory, and activation of ERKs, AKT, and calcineurin was examined by Western blot analysis in different brain regions. As compared with CB1(+/+), the nonreinforced tone presentation 24 h after auditory-cued fear conditioning led to lower levels of phosphorylated ERKs and/or calcineurin in the basolateral amygdala complex, ventromedial prefrontal cortex, dorsal hippocampus, and ventral hippocampus of CB1(-/-). In contrast, higher levels of phosphorylated p44 ERK and calcineurin were observed in the central nucleus of the amygdala of CB1(-/-). Phosphorylation of AKT was more pronounced in the basolateral amygdala complex and the dorsal hippocampus of CB1(-/-). We propose that the endogenous cannabinoid system modulates extinction of aversive memories, at least in part via regulation of the activity of kinases and phosphatases in a brain structure-dependent manner.

Neuroprotective properties of cannabinoids against oxidative stress: role of the cannabinoid receptor CB1.

Neuroprotective effects have been described for many cannabinoids in several neurotoxicity models. However, the exact mechanisms have not been clearly understood yet. In the present study, antioxidant neuroprotective effects of cannabinoids and the involvement of the cannabinoid receptor 1 (CB1) were analysed in detail employing cell-free biochemical assays and cultured cells. As it was reported for oestrogens that the phenolic group is a lead structure for antioxidant neuroprotective effects, eight compounds were classified into three groups. Group A: phenolic compounds that do not bind to CB1. Group B: non-phenolic compounds that bind to CB1. Group C: phenolic compounds that bind to CB1. In the biochemical assays employed, a requirement of the phenolic lead structure for antioxidant activity was shown. The effects paralleled the protective potential of group A and C compounds against oxidative neuronal cell death using the mouse hippocampal HT22 cell line and rat primary cerebellar cell cultures. To elucidate the role of CB1 in neuroprotection, we established stably transfected HT22 cells containing CB1 and compared the protective potential of cannabinoids with that observed in the control transfected HT22 cell line. Furthermore, oxidative stress experiments were performed in cultured cerebellar granule cells, which were derived either from CB1 knock-out mice or from control wild-type littermates. The results strongly suggest that CB1 is not involved in the cellular antioxidant neuroprotective effects of cannabinoids.

Involvement of brain-derived neurotrophic factor in cannabinoid receptor-dependent protection against excitotoxicity.

Cannabinoid type 1 (CB1) receptors play a central role in the protection against excitotoxicity induced by treatment of mice with kainic acid (KA). As inactivation of CB1 receptor function in mice blocks KA-induced increase of brain-derived neurotrophic factor (BDNF) mRNA levels in hippocampus, the notion was put forward that BDNF might be a mediator, at least in part, of CB1 receptor-dependent neuroprotection [Marsicano et al. (2003) Science, 302, 84-88]. To assess this signalling cascade in more detail, organotypic hippocampal slice cultures were used, as this in vitro system conserves morphological and functional properties of the hippocampus. Here, we show that both genetic ablation of CB1 receptors and pharmacological blockade with the specific CB1 receptor antagonist SR141716A increased the susceptibility of the in vitro cultures to KA-induced excitotoxicity, leading to extensive neuronal death. Next, we found that the application of SR141716A to hippocampal cultures from wild-type mice abolished the KA-induced increase in BDNF protein levels. Therefore, we tried to rescue these organotypic cultures from neuronal death by exogenously applied BDNF. Indeed, BDNF was sufficient to prevent KA-induced neuronal death after blockade of CB1 receptor signalling. In conclusion, our results strongly suggest that BDNF is a key mediator in CB1 receptor-dependent protection against excitotoxicity, and further underline the physiological importance of the endogenous cannabinoid system in neuroprotection.

The endogenous cannabinoid system controls extinction of aversive memories.

Acquisition and storage of aversive memories is one of the basic principles of central nervous systems throughout the animal kingdom. In the absence of reinforcement, the resulting behavioural response will gradually diminish to be finally extinct. Despite the importance of extinction, its cellular mechanisms are largely unknown. The cannabinoid receptor 1 (CB1) and endocannabinoids are present in memory-related brain areas and modulate memory. Here we show that the endogenous cannabinoid system has a central function in extinction of aversive memories. CB1-deficient mice showed strongly impaired short-term and long-term extinction in auditory fear-conditioning tests, with unaffected memory acquisition and consolidation. Treatment of wild-type mice with the CB1 antagonist SR141716A mimicked the phenotype of CB1-deficient mice, revealing that CB1 is required at the moment of memory extinction. Consistently, tone presentation during extinction trials resulted in elevated levels of endocannabinoids in the basolateral amygdala complex, a region known to control extinction of aversive memories. In the basolateral amygdala, endocannabinoids and CB1 were crucially involved in long-term depression of GABA (gamma-aminobutyric acid)-mediated inhibitory currents. We propose that endocannabinoids facilitate extinction of aversive memories through their selective inhibitory effects on local inhibitory networks in the amygdala.

CB1 cannabinoid receptors and on-demand defense against excitotoxicity.

Abnormally high spiking activity can damage neurons. Signaling systems to protect neurons from the consequences of abnormal discharge activity have been postulated. We generated conditional mutant mice that lack expression of the cannabinoid receptor type 1 in principal forebrain neurons but not in adjacent inhibitory interneurons. In mutant mice,the excitotoxin kainic acid (KA) induced excessive seizures in vivo. The threshold to KA-induced neuronal excitation in vitro was severely reduced in hippocampal pyramidal neurons of mutants. KA administration rapidly raised hippocampal levels of anandamide and induced protective mechanisms in wild-type principal hippocampal neurons. These protective mechanisms could not be triggered in mutant mice. The endogenous cannabinoid system thus provides on-demand protection against acute excitotoxicity in central nervous system neurons.