Design and validation of recombinant protein standards for quantitative Western blot analysis of cannabinoid CB1 receptor density in cell membranes: an alternative to radioligand binding methods.

BACKGROUND: Replacement of radioligand binding assays with antibody-antigen interaction-based approaches for quantitative analysis of G protein-coupled receptor (GPCR) levels requires the use of purified protein standards containing the antigen. GPCRs in general and cannabinoid CB1 receptor in particular show a progressive tendency to aggregate and precipitate in aqueous solution outside of their biological context due to the low solubility that the hydrophobic nature imprinted by their seven transmembrane domains. This renders full-length recombinant GPCRs useless for analytical purposes, a problem that can be overcome by engineering soluble recombinant fragments of the receptor containing the antigen. RESULTS: Here we generated highly soluble and stable recombinant protein constructs GST-CB1414-472 and GST-CB1414-442 containing much of the human CB1 receptor C-terminal tail for use as standard and negative control, respectively, in quantitative Western blot analysis of CB1 receptor expression on crude synaptosomes of the adult rat brain cortex. To this end we used three different antibodies, all raised against a peptide comprising the C-terminal residues 443-473 of the mouse CB1 receptor that corresponds to residues 442-472 in the human homolog. Estimated values of CB1 receptor density obtained by quantitative Western blot were of the same order of magnitude but slightly higher than values obtained by the radioligand saturation binding assay. CONCLUSIONS: Collectively, here we provide a suitable Western blot-based design as a simple, cost-effective and radioactivity-free alternative for the quantitative analysis of CB1 receptor expression, and potentially of any GPCR, in a variety of biological samples. The discrepancies between the results obtained by quantitative Western blot and radioligand saturation binding techniques are discussed in the context of their particular theoretical bases and methodological constraints.

Cannabis and cannabinoids as an alternative remedy in metabolic syndrome

Abstract Metabolic syndrome (MetS), an epidemic defined as a group of interconnected physiological, biochemistry, clinical, and metabolic factors, directly increases the risk of cardiovascular disease, atherosclerosis, type 2 diabetes, and death. MetS therapy includes diet, physical exercise, and a poly-pharmacological intervention. Cannabis is mainly recognized for its recreational uses and has several medical applications for neurological diseases, due to its hypnotic, anxiolytic, antinociceptive, anti-inflammatory, and anticonvulsant activities. Although several clinical observations in Cannabis smokers suggest metabolic effects, its utility in metabolic disorders is unclear. This review aims to determine under what conditions Cannabis might be useful in the treatment of MetS. Cannabis contains 120 phytocannabinoids, of which Δ9-THC mediates its psychoactive effects. Cannabinoids exert biological effects through interactions with the endocannabinoid system, which modulates several physiologic and metabolic pathways through cannabinoid receptors (CB1/CB2). Signaling through both receptors inhibits neurotransmitter release. In general, endocannabinoid system stimulation in Cannabis smokers and Δ9-THC signaling through CB1 have been implicated in MetS development, obesity, and type 2 diabetes. In contrast, CB1 antagonists and non-psychotropic phytocannabinoids like cannabidiol reduce these effects through interactions with both cannabinoid and non-cannabinoid receptors. These pharmacological approaches represent a source of new therapeutic agents for MetS. However, more studies are necessary to support the therapeutic potential of Cannabis and cannabinoids in metabolic abnormalities

The expression level of cannabinoid receptors type 1 and 2 in the different types of astrocytomas.

Astrocytomas, the most prevalent primary brain tumors, can be divided by histology and malignancy levels into four following types: pilocytic astrocytoma (grade I), diffuse fibrillary astrocytoma (grade II), anaplastic astrocytoma (grade III), and glioblastoma multiforme (grade IV). For high grade astrocytomas (grade III and grade IV), blood vessels formation is considered as the most important property. The distribution of cannabinoid receptors type 1 (CB1) and cannabinoid receptor type 2 (CB2) in blood vessels and tumor tissue of astrocytoma is still controversial. Asrocytoma tissues were collected from 45 patients under the condition of tumor-related neurosurgical operation. The expression of CB1 and CB2 receptors was assessed using immunofluorescence, quantitative real-time RT-PCR and western blotting. The results indicated an increased expression of CB1 receptors in tumor tissue. There was a significant difference in the mount of CB2 receptors in blood vessels. More was observed in the grade III and glioblastoma (grade IV) than astrocytoma of grade II and control. This study suggested that, the expression increase of cannabinoid receptors is an index for astrocytoma malignancy and can be targeted as a therapeutic approach for the inhibition of astrocytoma growth among patients.

Localization of cannabinoid and cannabinoid related receptors in the cat gastrointestinal tract.

A growing body of literature indicates that activation of cannabinoid receptors may exert beneficial effects on gastrointestinal inflammation and visceral hypersensitivity. The present study aimed to immunohistochemically investigate the distribution of the canonical cannabinoid receptors CB1 (CB1R) and CB2 (CB2R) and the putative cannabinoid receptors G protein-coupled receptor 55 (GPR55), nuclear peroxisome proliferator-activated receptor alpha (PPARα), transient receptor potential ankyrin 1 (TRPA1), and serotonin receptor 5-HT1a 5-HT1aR) in tissue samples of the gastrointestinal tract of the cat. CB1R-immunoreactivity (CB1R-IR) was observed in gastric epithelial cells, intestinal enteroendocrine cells (EECs) and goblet cells, lamina propria mast cells (MCs), and enteric neurons. CB2R-IR was expressed by EECs, enterocytes, and macrophages. GPR55-IR was expressed by EECs, macrophages, immunocytes, and MP neurons. PPARα-IR was expressed by immunocytes, smooth muscle cells, and enteroglial cells. TRPA1-IR was expressed by enteric neurons and intestinal goblet cells. 5-HT1a receptor-IR was expressed by gastrointestinal epithelial cells and gastric smooth muscle cells. Cannabinoid receptors showed a wide distribution in the feline gastrointestinal tract layers. Although not yet confirmed/supported by functional evidences, the present research might represent an anatomical substrate potentially useful to support, in feline species, the therapeutic use of cannabinoids during gastrointestinal inflammatory diseases.

Cellular localization and regulation of receptors and enzymes of the endocannabinoid system in intestinal and systemic inflammation.

Surveys suggest that Cannabis provides benefit for people with inflammatory bowel disease. However, mechanisms underlying beneficial effects are not clear. We performed in situ hybridization RNAscope® combined with immunohistochemistry to show cell-specific distribution and regulation of cannabinoid receptor 1 and 2 (CB1, CB2), G protein-coupled receptor 55 (GPR55), and monoacylglycerol lipase (MGL) mRNA in immune cells using murine models of intestinal and systemic inflammation. In healthy animals, the presence in enteric ganglia is high for CB1 mRNA, but low for CB2 and GPR55 mRNAs. MGL mRNA is predominant throughout the intestinal wall including myenteric neurons, epithelium, circular and longitudinal muscular layers, and the lamina propria. Within the immune system, B220+ cells exhibit high gene expression for CB2 while the expression of CB2 in F4/80+ and CD3+ cells is less prominent. In contrast, GPR55 mRNA is highly present in F4/80+ and CD3+ cells. qRT-PCR of total colonic segments shows that the expression of GPR55 and MGL genes drops during intestinal inflammation. Also at cellular levels, GPR55 and MGL gene expression is reduced in F4/80+, but not CD3+ cells. As to systemic inflammation, reduced gene expression of MGL is observed in ileum by qRT-PCR, while at cellular levels, altered gene expression is also seen for CB1 and GPR55 in CD3+ but not F4/80+ cells. In summary, our study reveals changes in gene expression of members of the endocannabinoid system in situ attesting particularly GPR55 and MGL a distinct cellular role in the regulation of the immune response to intestinal and systemic inflammation.

Endocannabinoids as endometrial inflammatory markers in lactating Holstein cows.

The objective of this study was to consider endocannabinoid system as inflammatory markers in bovine endometrium to better understand the role of this system in regulating many of the functions that are related to inflammatory condition. At day 26 post-partum, fourteen cows were divided into two groups depending on the inflammatory condition: 1- subclinical endometritis (n = 7, with purulent or mucopurulent uterine discharge detectable in the vagina) and 2- healthy (n = 7, No (muco)) purulent discharge. Blood samples were collected at 26 and 30 days relative to calving to determine plasma tumour necrosis factor (TNF) and lipopolysaccharide-binding protein (LBP) concentrations; moreover, uterine biopsy was carried out on day 26 post-partum to measure mRNA abundance of TNF, interleukin-1B (IL1B), interleukin-6 (IL-6), C-X-C motif chemokine ligand 8 (CXCL8), endocannabinoid receptor (CNR2), N-acyl phosphatidylethanolamine phospholipase D (NAPEPLD), fatty acid amide hydrolase (FAAH), N-acylethanolamine acid amidase (NAAA) and monoglyceride lipase (MGLL) by real-time PCR. Results showed mean plasma concentrations of TNF and LBP were lower in healthy cows compared to subclinical endometritis cows (p < .05). Relative mRNA expression for NAAA and FAAH was decreased (p < .05), and relative mRNA expression for CNR2 and NAPEPLD increased in cows with subclinical endometritis compared to healthy cows. In conclusion, relative mRNA expression of TNF, IL1B and CXCL8 and plasma concentration of LBP increased during inflammatory condition along with decreased endocannabinoids hydrolyzing enzyme (NAAA and FAAH), increased enzymes that synthesize endocannabinoids (NAPEPLD) and relative gene expression of the endocannabinoid receptor; together, these contribute to increased endocannabinoids levels during inflammation. Overall, we provide evidence that endocannabinoid system is altered in endometrium tissue during inflammation through increased mRNA expression of CNR2 and synthesis enzyme and decreased mRNA expression of hydrolyzing enzymes interfere with pro-cytokine production and signalling, which may interfere with the onset and progression of inflammation.

Cannabinoids and haemostasis.

Elements of the endocannabinoid system (cannabinoid receptors CB1, CB2, CBPT and CBED, endocannabinoids, enzymes involved in the synthesis and metabolism of endocannabinoids) are located on the structures involved in the process of hemostasis. An increasing level of endocannabinoids was also observed in some pathological conditions, which may occur in disorders of hemostasis. At the same time, disconcertingly, there is an increased number of reports about incidents of cardiovascular events in smokers of marijuana. Experimental and clinical studies demonstrated multidirectional, often contradictory, effects of cannabinoids on hemostasis, including effects of the compounds on platelets, vascular endothelium, fibrinolysis and plasma coagulation systems. The mechanisms of action of cannabinoids on homeostasis depend on the cannabinoid receptors CB1, CB2, CBPT and CBED, receptors of other systems stimulated by endocannabinoids, as well as metabolites of endocannabinoids and nitrogen oxide. The range of biological functions of endo- and plant cannabinoids, expanded to include the process of hemostasis, may constitute a condition for their recognition as a new factor responsible for thromboembolism in smokers of marijuana, in pathological disorders with increased levels of endocannabinoids and in individuals with polymorphisms of FAAH C385A and A385A. On the other hand, there are compelling reasons for anti­hemostatic action of cannabinoids.

A novel near-infrared fluorescence imaging probe that preferentially binds to cannabinoid receptors CB2R over CB1R.

The type 2 cannabinoid receptors (CB2R) have gained much attention recently due to their important regulatory role in a host of pathophysiological processes. However, the exact biological function of CB2R and how this function might change depending on disease progression remains unclear and could be better studied with highly sensitive and selective imaging tools for identifying the receptors. Here we report the first near infrared fluorescence imaging probe (NIR760-XLP6) that binds preferentially to CB2R over the type 1 cannabinoid receptors (CB1R). The selectivity of the probe was demonstrated by fluorescence microscopy using DBT-CB2 and DBT-CB1 cells. Furthermore, in mouse tumor models, NIR760-XLP6 showed significantly higher uptake in DBT-CB2 than that in DBT-CB1 tumors. These findings indicate that NIR760-XLP6 is a promising imaging tool for the study of CB2R regulation.

Delta(9) -tetrahydrocannabinol and cannabidiol as potential curative agents for cancer: A critical examination of the preclinical literature.

An Internet search with search words "cannabis cures cancer" produce a wealth of sites claiming that cannabis has this effect. These sites are freely accessible to the general public and thus contribute to public opinion. But do delta(9) -tetrahydrocannabinol (Δ(9) -THC) and cannabidiol (CBD) cure cancer? In the absence of clinical data other than a safety study and case reports, preclinical data should be evaluated in terms of its predictive value. Using a strict approach where only concentrations and/or models relevant to the clinical situation are considered, the current preclinical data do not yet provide robust evidence that systemically administered Δ(9) -THC will be useful for the curative treatment of cancer. There is more support for an intratumoral route of administration of higher doses of Δ(9) -THC. CBD produces effects in relevant concentrations and models, although more data are needed concerning its use in conjunction with other treatment strategies.

Distribution of lipids in human brain.

The enormous abundance of lipid molecules in the central nervous system (CNS) suggests that their role is not limited to be structural and energetic components of cells. Over the last decades, some lipids in the CNS have been identified as intracellular signalers, while others are known to act as neuromodulators of neurotransmission through binding to specific receptors. Neurotransmitters of lipidic nature, currently known as neurolipids, are synthesized during the metabolism of phospholipid precursors present in cell membranes. Therefore, the anatomical identification of each of the different lipid species in human CNS by imaging mass spectrometry (IMS), in association with other biochemical techniques with spatial resolution, can increase our knowledge on the precise metabolic routes that synthesize these neurolipids and their localization. The present study shows the lipid distribution obtained by MALDI-TOF IMS in human frontal cortex, hippocampus, and striatal area, together with functional autoradiography of cannabinoid and LPA receptors. The combined application of these methods to postmortem human brain samples may be envisioned as critical to further understand neurological diseases, in general, and particularly, the neurodegeneration that accompanies Alzheimer's disease.

Non-invasive imaging of the type 2 cannabinoid receptor, focus on positron emission tomography.

The type 2 cannabinoid receptor (CB(2)R) is a relatively new target for drug development, as the receptor was only discovered in 1993. The CB(2)R is mainly expressed in tissues and organs related to the immune system. However, in pathological conditions, mostly inflammatory, a strong upregulation has been observed. Because of its expression in activated microglia, the type 2 cannabinoid receptor might play an important role in pathologies with a neuroinflammatory component. Positron emission tomography provides a sensitive non-invasive imaging technique to study and quantify receptor expression. In this review, the importance of CB(2)R imaging, the current status and the future possibilities for the development of CB(2)R PET radioligands are discussed.

Localization and function of cannabinoid receptors in the corpus cavernosum: basis for modulation of nitric oxide synthase nerve activity.

BACKGROUND: Anandamide, a proposed endogenous cannabinoid (CB) agonist, has been shown to enhance neurogenic responses in vitro of the rat corpus cavernosal tissue (CC). However, no information is available on the distribution of CB-receptors or effects by anandamide in CC from primates or humans. OBJECTIVE: To characterize the distribution of CB-receptor isoforms in the human and primate CC and to investigate the effects of anandamide on isolated CC preparations. DESIGN, SETTING, AND PARTICIPANTS: CC tissue was excised from the crura penis of six rhesus monkeys and five patients. Expression and distribution of CB1 and CB2 receptors were characterized with Western blot analyses and immunohistochemical investigations. The effects of anandamide on isolated CC preparations were analyzed during pharmacologic and nerve-mediated activation of primate tissue in aerated organ baths. MEASUREMENTS: The expression and localization of CB1 and CB2 receptors in the primate CC and effects of anandamide on nerve-mediated relaxations and pharmacologically evoked contractions. RESULTS AND LIMITATIONS: Western blot experiments revealed CB1 and CB2 receptors at expected band weights. Within and between strands of CC smooth muscle, CB1 and CB2 immunoreactivity (IR) was found in nerve fibers that also expressed IR for nitric oxide synthase (NOS) or transient receptor potential V1 (TRPV1). Neither CB1-IR nor CB2-IR nerves were colocalized with calcitonin-gene-related peptide (CGRP)-containing or tyrosine hydroxylase-containing nerves. No differences were observed between primate and human CC sections. Anandamide (10(-9) to 10(-4) M) had no contractile effects on CC smooth muscle, no relaxant effects on precontracted preparations, and no effect on phenylephrine-induced contractions. However, anandamide (10muM) inhibited electrically evoked smooth-muscle relaxations (34-48%; p

Localisation and function of the endocannabinoid system in the human ovary.

BACKGROUND: Although anandamide (AEA) had been measured in human follicular fluid and is suggested to play a role in ovarian follicle and oocyte maturity, its exact source and role in the human ovary remains unclear. METHODS AND FINDINGS: Immunohistochemical examination of normal human ovaries indicated that the endocannabinoid system was present and widely expressed in the ovarian medulla and cortex with more intense cannabinoid receptor 2 (CB2) than CB1 immunoreactivity in the granulosa cells of primordial, primary, secondary, tertiary follicles, corpus luteum and corpus albicans. The enzymes, fatty acid amide hydrolase (FAAH) and N-acyclphosphatidylethanolamine-phospholipase D (NAPE-PLD), were only found in growing secondary and tertiary follicles and corpora lutea and albicantes. The follicular fluid (FF) AEA concentrations of 260 FF samples, taken from 37 infertile women undergoing controlled ovarian hyperstimulation for in vitro fertilisation and intracytoplasmic sperm injection with embryo transfer, were correlated with ovarian follicle size (P = 0.03). Significantly higher FF AEA concentrations were also observed in mature follicles (1.43+/-0.04 nM; mean+/-SEM) compared to immature follicles (1.26+/-0.06 nM), P = 0.0142 and from follicles containing morphologically assessed mature oocytes (1.56+/-0.11 nM) compared to that containing immature oocytes (0.99+/-0.09 nM), P = 0.0011. ROC analysis indicated that a FF AEA level of 1.09 nM could discriminate between mature and immature oocytes with 72.2% sensitivity and 77.14% specificity, whilst plasma AEA levels and FF AEA levels on oocyte retrieval day were not significantly different (P = 0.23). CONCLUSIONS: These data suggest that AEA is produced in the ovary, is under hormonal control and plays a role in folliculogenesis, preovulatory follicle maturation, oocyte maturity and ovulation.

Identification of the vasodilatory endothelial cannabinoid receptor in the human pulmonary artery.

BACKGROUND: The endocannabinoid anandamide is implicated in the pathogenesis of hypotension in haemorrhagic, endotoxic, and cardiogenic shock. It has been demonstrated in animal, but not in human, vessels that the vasodilatory effects of anandamide and abnormal cannabidiol are partially mediated by an as yet unidentified endothelial cannabinoid receptor. Our study was performed to examine the influence of abnormal cannabidiol on the human pulmonary artery. METHODS: Isolated human pulmonary arteries were obtained from patients without clinical evidence of pulmonary hypertension during resection of lung carcinoma. Vasodilatory effects of abnormal cannabidiol were examined on endothelium-intact vessels preconstricted with serotonin or potassium chloride. RESULTS: Anandamide and abnormal cannabidiol relaxed serotonin-preconstricted vessels concentration-dependently. The effect of abnormal cannabidiol was reduced by endothelium denudation, pertussis toxin and two antagonists of the novel endothelial receptor, cannabidiol and O-1918, but not by the nitric oxide synthase inhibitor L-NAME given together with the cyclooxygenase inhibitor indomethacin. It was also diminished by blockade of calcium-activated potassium channels by the nonselective blocker tetraethylammonium or by combination of selective blockers of small (apamin) and intermediate and large (charybdotoxin) conductance calcium-activated potassium channels. The potency of abnormal cannabidiol to relax vessels was lower in potassium chloride than in serotonin-preconstriced preparations. CONCLUSIONS: Abnormal cannabidiol relaxes human pulmonary arteries in an endothelium-independent and endothelium-dependent manner. The latter component is probably mediated via the putative endothelial cannabinoid receptor, activation of which may release endothelium-derived hyperpolarizing factor, which in turn acts via calcium-activated potassium channels. Abnormal cannabidiol is behaviourally inactive; it may have a therapeutic implication in vascular diseases, especially in the treatment of pulmonary hypertension.

[The endocannabinoid system and prospects her stimulation in clinical medicine].

The history of use by the man of plants of a sort Cannabis totals more than 4000 years. The people have begun to use Cannabis in stone century. On Taiwan archaeologist the rests of utensils made with application of stalks Cannabis more of 10000 years back were found. Cannabis (Cannabis sativa, Cannabis sowing, named also "Indian") - cultural plant, which has set of applications. From it received fibres for hemp of ropes and make a fabric similar on linen. Its stalks went on manufacture glossy of a paper and building of plates. Her sabadilla were used for graziery a bird; oil from sabadilla Cannabis offered as fuel instead of diesel. The greatest popularity Cannabis has received as raw material for reception of products (marijuana, hashish etc.), causing at the man psychotropic--first of all psychomymetic--effects, that at their regular application can result in formation of dependence. Besides the attempts were undertaken to use preparations Cannabis in the medical purposes: at migraine, spasmes, vomiting, pains of a different origin etc.

In silico patent searching reveals a new cannabinoid receptor.

Increasing evidence suggests that some cannabinoids mediate their effects independently of the known cannabinoid CB(1) and CB(2) receptors. Two recently published patents indicate that several cannabinoid receptor ligands also bind to the orphan G-protein-coupled receptor GPR55. This receptor is reported to be expressed in several tissues and might function in lipid or vascular biology. Thus, GPR55 might represent a new cannabinoid receptor.

Differential expression of cannabinoid receptors in the human colon: cannabinoids promote epithelial wound healing.

BACKGROUND & AIMS: Two G-protein-coupled cannabinoid receptors, termed CB1 and CB2, have been identified and several mammalian enteric nervous systems express CB1 receptors and produce endocannabinoids. An immunomodulatory role for the endocannabinoid system in gastrointestinal inflammatory disorders has been proposed and this study sought to determine the location of both cannabinoid receptors in human colon and to investigate epithelial receptor function. METHODS: The location of CB1 and CB2 receptors in human colonic tissue was determined by immunohistochemistry. Primary colonic epithelial cells were treated with both synthetic and endogenous cannabinoids in vitro, and biochemical coupling of the receptors to known signaling events was determined by immunoblotting. Human colonic epithelial cell lines were used in cannabinoid-binding studies and as a model for in vitro wound-healing experiments. RESULTS: CB1-receptor immunoreactivity was evident in normal colonic epithelium, smooth muscle, and the submucosal myenteric plexus. CB1- and CB2-receptor expression was present on plasma cells in the lamina propria, whereas only CB2 was present on macrophages. CB2 immunoreactivity was seen in the epithelium of colonic tissue characteristic of inflammatory bowel disease. Cannabinoids enhanced epithelial wound closure either alone or in combination with lysophosphatidic acid through a CB1-lysophosphatidic acid 1 heteromeric receptor complex. CONCLUSIONS: CB1 receptors are expressed in normal human colon and colonic epithelium is responsive biochemically and functionally to cannabinoids. Increased epithelial CB2-receptor expression in human inflammatory bowel disease tissue implies an immunomodulatory role that may impact on mucosal immunity.

Modulation of morphine-induced Fos-immunoreactivity by the cannabinoid receptor antagonist SR 141716.

A growing body of evidence suggests the existence of a functional interaction between opioid and cannabinoid systems. The present study further investigated this functional interaction by examining the combined effects of morphine and the cannabinoid receptor antagonist SR 141716 on Fos-immunoreactivity (Fos-IR), a marker for neural activation. Male albino Wistar rats were treated with SR 141716 (3 mg/kg, intraperitoneally), morphine HCl (10 mg/kg, subcutaneously), vehicle, or SR 141716 and morphine combined (n = 6 per group). Rats were injected with morphine or its vehicle 30-min after administration of SR 141716 or its vehicle and perfused 3 h later. Locomotor activity and body temperature were both increased in the morphine-treated group and SR 141716 significantly inhibited these effects. Morphine increased Fos-IR in several brain regions including the caudate-putamen (CPu), cortex (cingulate, insular and piriform), nucleus accumbens (NAS) shell, lateral septum (LS), bed nucleus of the stria terminalis (BNST), median preoptic nucleus (MnPO), medial preoptic nucleus (MPO), hypothalamus (paraventricular, dorsomedial and ventromedial), paraventricular thalamic nucleus (PV), amygdala (central and basolateral nuclei), dorsolateral periaqueductal gray, ventral tegmental area (VTA), and Edinger-Westphal nucleus. SR 141716 alone increased Fos-IR in the cortex (cingulate, insular and piriform), NAS (shell), LS, BNST, hypothalamus (paraventricular, dorsomedial and ventromedial), PV, amygdala (central, basolateral and medial nuclei), VTA, and Edinger-Westphal nucleus. SR 141716 attenuated morphine-induced Fos-IR in several regions including the CPu, cortex, NAS (shell), LS, MnPO, MPO, paraventricular and dorsomedial hypothalamus, PV, basolateral amygdala, VTA, and Edinger-Westphal nucleus (EW). These results provide further support for functional interplay between the cannabinoid and opioid systems. Possible behavioural and physiological implications of the interactive effects of SR 141716 on morphine-induced Fos-IR are discussed.