Fatty acid-binding proteins transport N-acylethanolamines to nuclear receptors and are targets of endocannabinoid transport inhibitors.

N-acylethanolamines (NAEs) are bioactive lipids that engage diverse receptor systems. Recently, we identified fatty acid-binding proteins (FABPs) as intracellular NAE carriers. Here, we provide two new functions for FABPs in NAE signaling. We demonstrate that FABPs mediate the nuclear translocation of the NAE oleoylethanolamide, an agonist of nuclear peroxisome proliferator-activated receptor α (PPARα). Antagonism of FABP function through chemical inhibition, dominant-negative approaches, or shRNA-mediated knockdown reduced PPARα activation, confirming a requisite role for FABPs in this process. In addition, we show that NAE analogs, traditionally employed as inhibitors of the putative endocannabinoid transmembrane transporter, target FABPs. Support for the existence of the putative membrane transporter stems primarily from pharmacological inhibition of endocannabinoid uptake by such transport inhibitors, which are widely employed in endocannabinoid research despite lacking a known cellular target(s). Our approach adapted FABP-mediated PPARα signaling and employed in vitro binding, arachidonoyl-[1-(14)C]ethanolamide ([(14)C]AEA) uptake, and FABP knockdown to demonstrate that transport inhibitors exert their effects through inhibition of FABPs, thereby providing a molecular rationale for the underlying physiological effects of these compounds. Identification of FABPs as targets of transport inhibitors undermines the central pharmacological support for the existence of an endocannabinoid transmembrane transporter.

Endocannabinoid signaling directs differentiation of trophoblast cell lineages and placentation.

In most mammals, placentation is critical for fetal development and pregnancy success. Exposure to marijuana during pregnancy has adverse effects, but whether the placenta is a target of cannabinoid/endocannabinoid signaling is not known. Using mice as a model system, we found that the endocannabinoid system is present in the ectoplacental cone and spongiotrophoblast cells. We also observed that aberrant endocannabinoid signaling confers premature trophoblast stem cell differentiation, and defective trophoblast development and invasion. These defects are reflected in retarded fetal development and compromised pregnancy outcome. Because the endocannabinoid system is conserved in mice and humans, our study suggests that endocannabinoid signaling is critical to placentation and pregnancy success in humans and implicates its potential significance in stem cell biology.

Characterization of NTera2/D1 cells as a model system for the investigation of cannabinoid function in human neurons and astrocytes.

The limited availability and potential to culture primary human brain cells means that there is still a need for cell lines that reliably model human neurons and glial cells. The human-derived NTera2/D1 (NT2) cell line is a promising tool from which both neuronal (NT2N) and astrocytic (NT2A) cells can be derived in vitro. Here we have investigated the potential to use this cell model to investigate the endocannabinoid system in the CNS. Through immunocytochemical characterization with a range of neuronal and glial markers, we found that these cell lines differentiate into cells with immature neuronal and astrocytic phenotypes, respectively. By real-time PCR, immunocytochemistry, and functional inhibition of cAMP accumulation, the cannabinoid 1 receptors were identified only on NT2N cells, consistent with high levels of expression of this receptor in neuronal cells of the CNS. No evidence of cannabinoid 2 receptor expression was found on any of the NT2 cell types. Both the precursors and the differentiated NT2N and NT2A cells demonstrated mRNA expression for the key enzymes involved in endocannabinoid synthesis and degradation. This work establishes a cannabinergic phenotype in NT2N and NT2A cells, providing an alternative human derived renewable cell model for investigation of cannabinoid receptor function and endocannabinoid synthesis and metabolism in the CNS.

Investigations of the human endocannabinoid system in two subcutaneous adipose tissue depots in lean subjects and in obese subjects before and after weight loss.

CONTEXT: Endocannabinoids (ECs) have a role in obesity by affecting appetite and through peripheral effects. Obesity is associated with a dysregulation of the endocannabinoid system (ECS). OBJECTIVE: We aimed to determine the ECS in subcutaneous adipose tissue (AT) in obese subject and investigate the influence of diet-induced weight loss on this system. DESIGN: The obese study participants underwent a 12 weeks diet regimen resulting in 10-12% weight loss. All study participants underwent fasting blood samples and AT biopsies from abdomen and gluteal region, the obese subjects both before and after weight loss. SETTING AND PARTICIPANTS: A total of 21 healthy obese individuals (10 men/11 women, age 39.5 ± 1.6 years, body mass index (BMI): 37.5 ± 0.8 kg m(-2)) and 21 age- and gender-matched lean subjects (BMI: 23.8 ± 0.4 kg m(-2)) were studied. MAIN OUTCOME MEASURES: The activity of ECS in AT was determined by measuring arachidonoyl glycerol (2-AG) and N-arachidonoylethanolamine/anandamide in AT by mass spectrometry and gene expressions of enzymes and receptors involved in the ECS. RESULTS: The EC, 2-AG was reduced in obese individuals in the gluteal AT depot (P<0.01). Moreover, 2-AG increased in both depots in the obese subjects following weight loss (P<0.05). The gene expression of the CB1 was either not affected by the obese state (in the gluteal AT depot) or reduced (in the abdominal depot, P<0.05) and significantly affected by weight loss. The expression of the degrading enzymes FAAH, FAAH2, MGL and MGL2 was differently affected by obesity, AT depot and weight loss. CONCLUSION: We found reduced levels of 2-AG in subcutaneous AT in obesity, which increased after weight loss. In abdominal AT, the low CB1 expression was normalised after weight loss, whereas in gluteal AT the CB1 expression was reduced after weight loss. These findings support the concept of a dysregulated ECS in AT in association with obesity.

Cannabinoid exposure during zebra finch sensorimotor vocal learning persistently alters expression of endocannabinoid signaling elements and acute agonist responsiveness.

BACKGROUND: Previously we have found that cannabinoid treatment of zebra finches during sensorimotor stages of vocal development alters song patterns produced in adulthood. Such persistently altered behavior must be attributable to changes in physiological substrates responsible for song. We are currently working to identify the nature of such physiological changes, and to understand how they contribute to altered vocal learning. One possibility is that developmental agonist exposure results in altered expression of elements of endocannabinoid signaling systems. To test this hypothesis we have studied effects of the potent cannabinoid receptor agonist WIN55212-2 (WIN) on endocannabinoid levels and densities of CB1 immunostaining in zebra finch brain. RESULTS: We found that late postnatal WIN treatment caused a long-term global disregulation of both levels of the endocannabinoid, 2-arachidonyl glycerol (2-AG) and densities of CB1 immunostaining across brain regions, while repeated cannabinoid treatment in adults produced few long-term changes in the endogenous cannabinoid system. CONCLUSIONS: Our findings indicate that the zebra finch endocannabinoid system is particularly sensitive to exogenous agonist exposure during the critical period of song learning and provide insight into susceptible brain areas.

An endocannabinoid system is localized to the hypophysial pars tuberalis of Syrian hamsters and responds to photoperiodic changes.

The hypophysial pars tuberalis (PT), an important interface between neuroendocrine brain centers (hypothalamus, pineal organ) and the pars distalis (PD) of the hypophysis, plays a central role in regulating seasonal reproduction and prolactin release. However, the signaling molecules that transmit photoperiodic information from the PT to the PD and control prolactin release (the so-called "tuberalins") have not yet been identified, despite an intense search for more than three decades. Here, we demonstrate an endocannabinoid system in the PT of the Syrian hamster, a photoperiodic species. By means of in situ hybrization, the PT was found to express N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD), fatty acid amide hydrolase (FAAH), sn-1-selective diacylglycerol lipases (DAGLalpha and DAGLbeta), and monoacylglycerol lipase (MAGL), enzymes involved in endocannabinoid synthesis and degradation. The expression of NAPE-PLD, FAAH, and DAGLalpha was confirmed by immunohistochemistry. Expression and protein levels of DAGLs controlling the synthesis of 2-arachidonoyl glycerol (2-AG), a major endocannabinoid, were upregulated in the PT of Syrian hamsters kept under long-day conditions. Consequently, 2-AG levels were increased in the PT of these hamsters. A primary target of 2-AG, the cannabinoid receptor 1 (CB1), was expressed in the PD. Double-immunolabeling revealed that most of the CB1-immunoreactive cells in the PD were folliculostellate cells that were also immunoreactive for S-100 protein. Thus, the PT comprises an endocannabinoid system, and 2-AG may act as a photoperiodic messenger from the PT to the PD for the regulation of hypophysial hormonal secretion.

Variations in critical morphine biosynthesis genes and their potential to influence human health.

Endogenous morphine has been detected in human tissues from the vascular, immune and nervous systems. The genes/enzymes (CYP2D6, COMT and PNMT) that are involved in the biosynthesis of morphine have variations that affect their functionality. Some of these variations are the result of single nucleotide polymorphisms of DNA sequences. This review highlights some of the functional differences in the critical enzymes required for the biosynthesis of morphine that may affect human health. These variations have been shown to change the way animals react to stressors, perceive pain and behave. The presence of morphine signaling in almost all organ systems suggests that it is most likely playing a role in maintaining the health and promoting the normal functioning of these physiological systems.

Enzymatic machinery for endocannabinoid biosynthesis associated with calcium stores in glutamatergic axon terminals.

Endocannabinoids are regarded as retrograde signaling molecules at various types of synapses throughout the CNS. The lipid derivatives anandamide and 2-arachidonoylglycerol (2-AG) are generally thought to be the key molecular players in this process. Previous anatomical and electrophysiological studies provided compelling evidence that the biosynthetic enzyme of 2-AG is indeed localized in the postsynaptic plasma membrane, whereas its target, the CB1 cannabinoid receptor, and the enzyme responsible for its inactivation are both found presynaptically. This molecular architecture of 2-AG signaling is a conserved feature of most synapses and supports the retrograde signaling role of 2-AG. Conversely, the molecular and neuroanatomical organization of synaptic anandamide signaling remains largely unknown. In contrast to its predicted role in retrograde signaling, here we show that N-acylphosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD), a biosynthetic enzyme of anandamide and its related bioactive congeners, the N-acylethanolamines (NAEs), is concentrated presynaptically in several types of hippocampal excitatory axon terminals. Furthermore, high-resolution quantitative immunogold labeling demonstrates that this calcium-sensitive enzyme is localized predominantly on the intracellular membrane cisternae of axonal calcium stores. Finally, the highest density of NAPE-PLD is found in mossy terminals of granule cells, which do not express CB1 receptors. Together, these findings suggest that anandamide and related NAEs are also present at glutamatergic synapses, but the sites of their synthesis and action are remarkably different from 2-AG, indicating distinct physiological roles for given endocannabinoids in the regulation of synaptic neurotransmission and plasticity.

Downregulation of the CB1 cannabinoid receptor and related molecular elements of the endocannabinoid system in epileptic human hippocampus.

Endocannabinoid signaling is a key regulator of synaptic neurotransmission throughout the brain. Compelling evidence shows that its perturbation leads to development of epileptic seizures, thus indicating that endocannabinoids play an intrinsic protective role in suppressing pathologic neuronal excitability. To elucidate whether long-term reorganization of endocannabinoid signaling occurs in epileptic patients, we performed comparative expression profiling along with quantitative electron microscopic analysis in control (postmortem samples from subjects with no signs of neurological disorders) and epileptic (surgically removed from patients with intractable temporal lobe epilepsy) hippocampal tissue. Quantitative PCR measurements revealed that CB(1) cannabinoid receptor mRNA was downregulated to one-third of its control value in epileptic hippocampus. Likewise, the cannabinoid receptor-interacting protein-1a mRNA was decreased, whereas 1b isoform levels were unaltered. Expression of diacylglycerol lipase-alpha, an enzyme responsible for 2-arachidonoylglycerol synthesis, was also reduced by approximately 60%, whereas its related beta isoform levels were unchanged. Expression level of N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D and fatty acid amide hydrolase, metabolic enzymes of anandamide, and 2-arachidonoylglycerol's degrading enzyme monoacylglycerol lipase did not change. The density of CB(1) immunolabeling was also decreased in epileptic hippocampus, predominantly in the dentate gyrus, where quantitative electron microscopic analysis did not reveal changes in the ratio of CB(1)-positive GABAergic boutons, but uncovered robust reduction in the fraction of CB(1)-positive glutamatergic axon terminals. These findings show that a neuroprotective machinery involving endocannabinoids is impaired in epileptic human hippocampus and imply that downregulation of CB(1) receptors and related molecular components of the endocannabinoid system may facilitate the deleterious effects of increased network excitability.

Altered responses of dopamine D3 receptor null mice to excitotoxic or anxiogenic stimuli: Possible involvement of the endocannabinoid and endovanilloid systems.

Dopamine and the endocannabinoids, anandamide and 2-arachidonoylglycerol, interact at several levels in the brain, with the involvement of both cannabinoid CB(1) receptors and transient receptor potential vanilloid type-1 (TRPV1) channels (which are alternative anandamide receptors). Using pharmacological, immunohistochemical and analytical approaches, we investigated the response of dopamine D(3) receptor null (D3R((-/-))) mice in models of epilepsy and anxiety, in relation to their brain endocannabinoid and endovanilloid tone. Compared to wild-type mice, D3R((-/-)) mice exhibited a delayed onset of clonic seizures, enhanced survival time, reduced mortality rate and more sensitivity to anticonvulsant effects of diazepam after intraperitoneal administration of picrotoxin (7 mg/kg), and a less anxious-like behaviour in the elevated plus maze test. D3R((-/-)) mice also exhibited different endocannabinoid and TRPV1, but not CB(1), levels in the hippocampus, nucleus accumbens, amygdala and striatum. Given the role played by CB(1) and TRPV1 in neuroprotection and anxiety, and based on data obtained here with pharmacological tools, we suggest that the alterations of endocannabinoid and endovanilloid tone found in D3R((-/-)) mice might account for part of their altered responses to excitotoxic and anxiogenic stimuli.

The endogenous cannabinoid system modulates nicotine reward and dependence.

A growing body of evidence suggests that the endogenous cannabinoid system modulates the addictive properties of nicotine, the main component of tobacco that produces rewarding effects. In our study, complementary transgenic and pharmacological approaches were used to test the hypothesis that the endocannabinoid system modulates nicotine reward and dependence. An acute injection of nicotine elicited normal analgesic and hypothermic effects in cannabinoid receptor (CB)(1) knockout (KO) mice and mice treated with the CB(1) antagonist rimonabant. However, disruption of CB(1) receptor signaling blocked nicotine reward, as assessed in the conditioned place preference (CPP) paradigm. In contrast, genetic deletion, or pharmacological inhibition of fatty acid amide hydrolase (FAAH), the enzyme responsible for catabolism of the endocannabinoid anandamide, enhanced the expression of nicotine CPP. Although the expression of spontaneous nicotine withdrawal (14 days, 24 mg/kg/day nicotine) was unaffected in CB(1) KO mice, acute administration of rimonabant (3 mg/kg) ameliorated somatic withdrawal signs in wild-type mice. Increasing endogenous levels of anandamide through genetic or pharmacological approaches exacerbated the physical somatic signs of spontaneous nicotine withdrawal in a milder withdrawal model (7 days, 24 mg/kg/day nicotine). Moreover, FAAH-compromised mice displayed increased conditioned place aversion in a mecamylamine-precipitated model of nicotine withdrawal. These findings indicate that endocannabinoids play a role in the rewarding properties of nicotine as well as nicotine dependence liability. Specifically, increasing endogenous cannabinoid levels magnifies, although disrupting CB(1) receptor signaling, attenuates nicotine reward and withdrawal. Taken together, these results support the hypothesis that cannabinoid receptor antagonists may offer therapeutic advantages to treat tobacco dependence.

An endocannabinoid mechanism in relapse to drug seeking: a review of animal studies and clinical perspectives.

Detoxification from drug abuse is strongly threatened by the occurrence of renewed episodes of drug intake. In human addicts, relapse to drug seeking may take place even after a considerably long period from the last drug consumption. Over the last decade, the endocannabinoid system has received remarkable attention due to its unique features, including its rewarding properties closely resembling those of the most commonly abused substances and its multiple therapeutic implications. Although limited at present, evidence is now emerging on a possible participation of the endogenous cannabinoid system in the regulation of relapsing phenomena. Both stimulation and blockade of the central cannabinoid CB-sub1 receptor have proved to play an important role in drug- as well as in cue-induced reinstatement of drug seeking behavior. Indeed, while CB-sub1 receptor stimulation may elicit relapse not only to cannabinoid seeking but also to cocaine, heroin, alcohol and methamphetamine, this effect is significantly attenuated, when not fully prevented, by pretreatment with the CB-sub1 receptor antagonist rimonabant. However, corroborating data on the involvement of the cannabinoid system in stress-induced reinstatement are still rather scarce. The present review attempts to collect data obtained from different laboratories using diverse experimental approaches, to provide a comprehensive picture of the recent evidence of a relationship between the cannabinoid system and the neurobiological mechanisms leading to relapse. For each class of abused drugs, the conspicuous progress made in delineating the role of the endocannabinoid system in relapse to drug seeking has been examined by placing particular emphasis on the findings obtained from behavioral studies. After summarizing findings and implications emerging from the reviewed studies, we conclude by briefly discussing what information is still missing and how missing information might be obtained.

The endocannabinoid system and neurogenesis in health and disease.

The endocannabinoid system exerts an important neuromodulatory function in different brain areas and is also known to be involved in the regulation of neural cell fate. Thus, CB(1) cannabinoid receptors are neuroprotective in different models of brain injury, and their expression is altered in various neurodegenerative diseases. Recent findings have demonstrated the presence of a functional endocannabinoid system in neural progenitor cells that participates in the regulation of cell proliferation and differentiation. In this Research Update, the authors address the experimental evidence regarding the regulatory role of cannabinoids in neurogenesis and analyze them in the context of those pathological disorders in which cannabinoid function and altered neuronal or glial generation is most relevant, for example, stroke and multiple sclerosis.

Coevolution between cannabinoid receptors and endocannabinoid ligands.

Genes for receptors and ligands must coevolve to maintain coordinated gene expression and binding affinities. Researchers have debated whether anandamide or 2-arachidonyl glycerol (2-AG) is a more "intrinsic" ligand of cannabinoid receptors. We addressed this debate with a coevolutionary analysis, by examining genes for CB1, CB2, and ten genes that encode ligand metabolic enzymes: abhydrolase domain containing 4 protein, cyclooxygenase 2, diacylglycerol lipase paralogs (DAGLalpha, DAGLbeta), fatty acid amide hydrolase paralogs (FAAH1, FAAH2), monoglyceride lipase, N-acylethanolamine acid amidase, NAPE-selective phospholipase D, and protein tyrosine phosphatase non-receptor type 22. Gene trees (cladograms) of CB1, CB2, and ligand enzymes were obtained by searching for orthologs (tBLASTn) in the genomes of nine phylogenetically diverse species, aligning ortholog sequences with ClustalX, and applying Bayesian analysis (MrBayes). Mirrored cladograms provided evidence of coevolution (i.e., parallel cladogenesis). Next we constructed phylograms of CB1, CB2, and the ten enzymes. Phylogram branch lengths were proportional to three sets of maximum likelihood metrics: all-nucleotide-substitutions and NS/SS ratios (using PAUP()), and Ka/Ks ratios (using FUGE). Spurious correlations in all-nucleotide-substitutions trees (due to phylogenetic bias) and in Ka/Ks ratio trees (due to simplistic modeling) were parsed. Branch lengths from equivalent branches in paired trees were correlated by linear regression. Regression analyses, mirrored cladograms, and phylogenetic profiles produced the same results: close associations between cannabinoid receptors and DAGL enzymes. Therefore we propose that cannabinoid receptors initially coevolved with a fatty acid ester ligand (akin to 2-AG) in ancestral metazoans, and affinity for fatty acid ethanolamide ligands (e.g., AEA) evolved thereafter.

Genetic polymorphisms of the endocannabinoid system.

Disturbances in the endocannabinoid system has been linked to diseases and conditions such as Parkinson's, schizophrenia, pain, energy metabolism, immune modulation, and bone density. Since the early 1990s, a number of genetic polymorphisms in the genes and proteins of the endocannabinoid system have been characterized. Currently identified genetic polymorphisms of the endocannabinoid system are reviewed here with particular consideration given to polymorphisms linked to drug and alcohol abuse, schizophrenia, other mental disorders, and energy metabolism.

Evolutionary origins of the endocannabinoid system.

Endocannabinoid system evolution was estimated by searching for functional orthologs in the genomes of twelve phylogenetically diverse organisms: Homo sapiens, Mus musculus, Takifugu rubripes, Ciona intestinalis, Caenorhabditis elegans, Drosophila melanogaster, Saccharomyces cerevisiae, Arabidopsis thaliana, Plasmodium falciparum, Tetrahymena thermophila, Archaeoglobus fulgidus, and Mycobacterium tuberculosis. Sequences similar to human endocannabinoid exon sequences were derived from filtered BLAST searches, and subjected to phylogenetic testing with ClustalX and tree building programs. Monophyletic clades that agreed with broader phylogenetic evidence (i.e., gene trees displaying topographical congruence with species trees) were considered orthologs. The capacity of orthologs to function as endocannabinoid proteins was predicted with pattern profilers (Pfam, Prosite, TMHMM, and pSORT), and by examining queried sequences for amino acid motifs known to serve critical roles in endocannabinoid protein function (obtained from a database of site-directed mutagenesis studies). This novel transfer of functional information onto gene trees enabled us to better predict the functional origins of the endocannabinoid system. Within this limited number of twelve organisms, the endocannabinoid genes exhibited heterogeneous evolutionary trajectories, with functional orthologs limited to mammals (TRPV1 and GPR55), or vertebrates (CB2 and DAGLbeta), or chordates (MAGL and COX2), or animals (DAGLalpha and CB1-like receptors), or opisthokonta (animals and fungi, NAPE-PLD), or eukaryotes (FAAH). Our methods identified fewer orthologs than did automated annotation systems, such as HomoloGene. Phylogenetic profiles, nonorthologous gene displacement, functional convergence, and coevolution are discussed.

A collaboration investigating endocannabinoid signalling in brain and bone.

Investigations into the cellular and molecular mechanisms underlying the psychoactive effects of cannabis preparations have led to the discovery of the endocannabinoid system. Interest in the central nervous system effects was initially the main focus of the research, but it soon became evident that the endocannabinoid system affects virtually every organ. The research field has therefore experienced a tremendous growth over the last decade and is now truly interdisciplinary. This short review provides a personal account of an interdisciplinary collaboration between Itai Bab from the Hebrew University of Jerusalem and the author. It describes the discovery of the endocannabinoid system in bone and the analysis of its functions. I am summarising the role of CB1 signalling as a modulator of sympathetic inhibition of bone formation. Thus, activation of CB1 receptors on sympathetic nerve terminals in bone, presumably from endocannabinoids released from apposing osteoblasts, reduces the inhibition of bone formation of sympathetic norepinephrine. CB2 receptors on osteoblasts and osteoclasts also modulate the proliferation and functions of these cells. Thus, activation of CB2 stimulates bone formation and represses bone resorption, whereas the genetic disruption of CB2 results in an osteoporosis-like phenotype. This signalling mechanism is clinically relevant, as shown by the association of polymorphisms in the CB2 receptor gene, CNR2, with bone density and osteoporosis. Finally, the review provides a summary of the recently discovered role of endocannabinoid signalling in one elongation. This review will also discuss the benefits of interdisciplinary and international collaborations.

The genetic basis of the endocannabinoid system and drug addiction in humans.

The cannabinoid receptor (CNR1) and the fatty acid amide hydrolase (FAAH) genes are located on chromosomes 6 and 1 in the 6q15 and 1p33 cytogenetic bands, respectively. CNR1 encodes a seven-transmembrane domain protein of 472 amino acids, whereas FAAH encodes one transmembrane domain of 579 amino acids. Several mutations found in these genes lead to altered mRNA stability and transcription rate or a reduction of the activity of the encoded protein. Increasing evidence shows that these functional mutations are related to dependence upon cocaine, alcohol, marijuana, heroin, nicotine and other drugs. One of the most compelling associations is with the C385A single nucleotide polymorphism (SNP), which is found in the FAAH gene. For all of the genetic polymorphisms reviewed here, it is difficult to form overall conclusions due to the high diversity of population samples being studied, ethnicity, the use of volunteers, heterogeneity of the recruitment criteria and the drug addiction phenotype studied. Care should be taken when generalizing the results from different studies. However, many works have repeatedly associated polymorphisms in the CNR1 and FAAH genes with drug-related behaviours; this suggests that these genes should be examined in further genetic studies focusing on drug addiction and other psychiatric disorders.

Contributions of endocannabinoid signaling to psychiatric disorders in humans: genetic and biochemical evidence.

The endocannabinoid signaling system is a widespread, neuromodulatory system in brain and is also widely utilized in the periphery to modulate metabolic functions and the immune system. Preclinical data demonstrate that endocannabinoid signaling is an important stress buffer and modulates emotional and cognitive functions. These data suggest the hypothesis that endocannabinoid signaling could be dysfunctional in a number of mental disorders. Genetic polymorphisms in the human genes for two important proteins of the endocannabinoid signaling system, the CB1 cannabinoid receptor (CB1R) and fatty acid amide hydrolase (FAAH), have been explored in the context of normal and pathological conditions. In the case of the gene for FAAH, the mechanistic relationships among the common genetic polymorphism, the expression of the FAAH protein, and its likely impact on endocannabinoid signaling are understood. However, multiple polymorphisms in the gene for the CB1R occur and are associated with human phenotypic differences without an understanding of the functional relationships among the gene, mRNA, protein, and protein function. The endocannabinoid ligands are found in the circulation, and several studies have identified changes in their concentrations under various conditions. These data are reviewed for the purpose of generating hypotheses and to encourage further studies in this very interesting and important area.