Localization Of Cannabinoid Receptors Research Articles

A276 CANNABINOID RECEPTOR 1 IS LOCALIZED IN ENTERIC NEURONS AND EPITHELIUM IN FETAL AND EARLY POSTNATAL MOUSE INTESTINE

Abstract Background The endocannabinoid system (ECS) is necessary for gastrointestinal (GI) homeostasis and serves as a target for exogenous medicinal and recreational cannabinoid compounds. While components of the ECS, including receptors, ligands, and biosynthetic/degradative enzymes, have been increasingly characterized in the adult GI tract of both humans and animal models, similar advances in understanding the role of the ECS in GI developmental programming have not yet been made. Purpose We tested the hypothesis that the ECS plays a role in the developmental programming of the GI tract. Method The developmental timeline of ECS component expression was investigated in the GI tract from fetal and early postnatal mice. Samples from timed-pregnant outbred mice were collected from embryonic days 14 (E14), E16, E18 and postnatal days 1 (P1), P7 and P28, representing a timeline of early colonization of the GI tract by enteric neurons (E14) to 1 week after weaning (P28). Localization of cannabinoid receptor 1 (CB1) expression was examined in fetal and postnatal small intestine by immunohistochemistry. Fetal intestine was processed for RNA extraction and relative expression of ECS components determined by RT-qPCR. Result(s) CB1 expression at E14 was observed in the outer wall of the gut and in the developing mucosa. By E18, expression of CB1 became more localized to the enteric plexuses and to mucosal epithelial cells. In postnatal ileum, at least >50% of myenteric ganglia were found to express CB1 from P1 to P28. CB1 expression continued to be present in the mucosal epithelium of ileum from P1 to P28, with a subset of CB1 expression in enterochromaffin cells, as identified by 5-HT immunolabeling. Significant decreases in expression of mRNA encoding CB1 (p<0.0001) and the ECS biosynthetic enzymes NAPE-PLD (p<0.05) and DAGLa (p<0.05) were found between E14 and E18. Conclusion(s) Components of the ECS are present in the fetal and early postnatal GI tract. Further research is needed to better characterize the roles and mechanisms of action of the ECS in GI development. The potential impact of exogenous cannabis exposure in early life, either by in utero exposure or via the use of medicinal cannabinoids in childhood, highlights the importance of identifying the contributions of the ECS to normal development. Disclosure of Interest None Declared

103 Characterization of the receptors of the endocannabinoid system in equine sperm: Possible role of anandamide in sperm function

Conventional IVF in horses remains challenging. In particular, stallion sperm fails to penetrate the zona pellucida, possibly due to incomplete invitro sperm capacitation. Therefore, there is a need to elucidate, in horses, molecules with a proven role during capacitation in other mammals. Our laboratory has described the relevance of the endocannabinoid system in capacitation of bovine and murine sperm. We reported that anandamide (AEA), an endocannabinoid present in follicular and oviducal fluids, induced capacitation-associated events. The aims of this work were to characterise the localization of cannabinoid receptors in equine sperm and to evaluate the effects of AEA on levels of tyrosine-phosphorylated proteins (pY) and substrates phosphorylated by protein kinase A (pPKA). Both cannabinoid receptors (CB1, CB2, TRPV1) and pPKA and pY were localised in sperm by indirect immunofluorescence. Sperm (15×106mL−1) were incubated, at 38.5°C in air, in modified Tyrode’s-albumin-lactate-pyruvate (TALP) with 25mM NaHCO3, 5mM dextrose and 1mgmL−1 polyvinyl alcohol (PVA; TALP-Bic-PVA) or TALP-Bic-PVA supplemented with AEA (0.1, 1, 10, 100nM, and 1µM) for 4h. After incubation, Western blot was used to determine levels of pY and pPKA in 4.5×106 sperm. Cryopreserved sperm samples from three stallions were evaluated. The normality of data distributions and homoscedasticity were verified with the Shapiro-Wilk and Levene tests, respectively. Data were analysed by one-way ANOVA and Bonferroni post hoc test, with P<0.05 considered significant. Based on immunofluorescence, CB1 was mainly localised in the post-acrosomal region and flagellum (93.4%±5.5, mean±s.d.), CB2 in the post-acrosomal region and middle piece (89.9%±28.3), and TRPV1 in the post-acrosomal region and flagellum (89.3%±9). Sperm positive for pPKA had fluorescence in the middle piece and principal piece of the flagellum. Incubation with 1nM AEA for 4h induced a 61% increase in pPKA levels compared with TALP-Bic-PVA medium alone, with no induction of pY levels in any treatment. In conclusion, cannabinoid receptors were present in equine sperm, and incubation with AEA induced an increase in PKA activity, an essential event associated with sperm capacitation. To our knowledge, this was the first report describing the presence of receptors of the endocannabinoid system in equine sperm and the potential role of AEA in the acquisition of sperm fertilizing ability.

Expression of Cannabinoid Receptors in Human Osteoarthritic Cartilage: Implications for Future Therapies.

Introduction: Cannabinoids have shown to reduce joint damage in animal models of arthritis and reduce matrix metalloproteinase expression in primary human osteoarthritic (OA) chondrocytes. The actions of cannabinoids are mediated by a number of receptors, including cannabinoid receptors 1 and 2 (CB1 and CB2), G-protein-coupled receptors 55 and 18 (GPR55 and GPR18), transient receptor potential vanilloid-1 (TRPV1), and peroxisome proliferator-activated receptors alpha and gamma (PPARα and PPARγ). However, to date very few studies have investigated the expression and localization of these receptors in human chondrocytes, and expression during degeneration, and thus their potential in clinical applications is unknown.Methods: Human articular cartilage from patients with symptomatic OA was graded histologically and the expression and localization of cannabinoid receptors within OA cartilage and underlying bone were determined immunohistochemically. Expression levels across regions of cartilage and changes with degeneration were investigated.Results: Expression of all the cannabinoid receptors investigated was observed with no change with grade of degeneration seen in the expression of CB1, CB2, GPR55, PPARα, and PPARγ. Conversely, the number of chondrocytes within the deep zone of cartilage displaying immunopositivity for GPR18 and TRPV1 was significantly decreased in degenerate cartilage. Receptor expression was higher in chondrocytes than in osteocytes in the underlying bone.Conclusions: Chondrocytes from OA joints were shown to express a wide range of cannabinoid receptors even in degenerate tissues, demonstrating that these cells could respond to cannabinoids. Cannabinoids designed to bind to receptors inhibiting the catabolic and pain pathways within the arthritic joint, while avoiding psychoactive effects, could provide potential arthritis therapies.

O11. Evaluation of the endocannabinoid system in preeclampsia

Introduction The endocannabinoid system plays a key role in female reproduction, including implantation, decidualization and placentation. A growing number of studies indicate that placental and peripheral blood anandamide levels correlate closely with both spontaneous miscarriage and ectopic pregnancy. Anandamide has also been implicated in blood pressure regulation. Objectives In this study, we aimed to analyze placental expression and localization of cannabinoid receptor 1 (CB1), CB2 and fatty acid amid hydrolase (FAAH), as well as circulating anandamide levels in normal pregnancy and preeclampsia. Methods We determined CB1, CB2 and FAAH expressions by Western blotting and immunohistochemistry in placental samples collected directly after Cesarean section in 18 preeclamptic patients and 18 normotensive, healthy pregnant women. Serum anandamide concentrations were measured by high performance liquid chromatography–mass spectrometry (HPLC–MS) technique in 43 preeclamptic patients and 71 healthy pregnant women. Serum total soluble fms-like tyrosine kinase-1 (sFlt-1) and biologically active placental growth factor (PlGF) levels were assessed by electrochemiluminescence immunoassay. Results CB1 expression semi-quantified by Western blotting was significantly higher in preeclamptic placenta, and these findings were confirmed by immunohistochemistry. CB1 immunoreactivity was markedly stronger in syncytiotrophoblasts, the mesenchymal core, decidua, villous capillary endothelial and smooth muscle cells, as well as in the amnion in preeclamptic samples compared to normal pregnancies. However, we did not find significant differences between preeclamptic and normal placenta in terms of CB2 and FAAH expressions and immunoreactivity. Serum levels of anandamide were significantly lower in preeclamptic patients than in healthy pregnant women. Preeclamptic patients had significantly higher sFlt-1 levels and significantly lower PlGF concentrations as compared to healthy pregnant women. Serum anandamide concentrations did not correlate with serum levels of sFlt-1 and PlGF in our healthy pregnant and preeclamptic groups. Conclusion We observed markedly higher expression of CB1 protein in preeclamptic placental tissue. Increased CB1 expression might cause abnormal decidualization and impair trophoblast invasion, thus being involved in the pathogenesis of preeclampsia. Nevertheless, we did not find significant differences between preeclamptic and normal placental tissue regarding CB2 and FAAH expressions. Furthermore, serum anandamide concentrations were decreased in women with preeclampsia. While the detailed pathogenesis of preeclampsia is still unclear, the endocannabinoid system could play a role in the development of the disease.

Localization of Cannabinoid Receptor 1 (CB1) in Submandibular and Sublingual Salivary Glands of Mice throughout Postnatal Development

En vista de que los usuarios humanos de la marihuana a menudo presentan sintomas de sequedad oral, en el presente estudio se intento examinar la localizacion de CB1, que se identifico originalmente en el cerebro, en las glandulas salivales submandibulares y sublinguales durante el desarrollo postnatal en ratones machos. En la glandula submandibular, la inmunoreactividad CB1 fue positiva en la mayoria de las celulas acinares de apariencia granular en su citoplasma apical, mientras que fue negativa en los conductos en la etapa de recien nacidos. La inmunorreactividad disminuyo en las celulas acinares en P1W y no se detecto inmunoreactividad en las celulas acinares en P3W. La inmunoreactividad fue positiva en las celulas ductales en P3W y se mantuvo positiva hasta la etapa P8W. La inmunorreaccion se observo en el plasmalema apical de las celulas ductales intercaladas, mientras que fue distinta en el plasmalema basal de las celulas ductales contorneadas granulares. La inmunotincion mejorada en el plasmalema lateral de las celulas ductales granulares fue distingible solo en P6W. En la glandula sublingual, se detecto inmunoreactividad CB1 en las celulas acinares y se observaron celulas ductales solamente en P4W. Ademas, se demostro que la inmunoreactividad CB1 se produce en las neuronas ganglionares salivales, lo que sugiere la accion CB1 inhibitoria en la secrecion de saliva a traves de la transmision parasimpatica nerviosa.

Expression and localization of cannabinoid receptor 1 in rats' brain treated with acute and repeated morphine.

Morphine induces adaptive changes in gene expression throughout the reward circuitry of brain. Recent research has proven the functional interactions between opioid and endogenous cannabinoid system in the central nervous system (CNS). The cannabinoid receptor 1 (CB1-R) is one of the receptors that mediate the actions of cannabinoids and endocannabinoids in the CNS. Here, we investigated the expression of CB1-R in mRNA and protein levels in the brains of rats treated with acute and repeated morphine. Three groups of rats received intraperitoneal injections (ip injections) of saline, acute morphine (10 mg/kg) and repeated morphine (10 mg/kg, twice daily for 12 consecutive days), and the mRNA levels and protein expressions of CB1-R were examined. RT-PCR and western blot analyses supported that both mRNA and protein levels of CB1-R in cortex, cerebellum and hippocampus were increased by repeated morphine treatment. However, the mRNA level in cerebellum was down-regulated only after acute morphine treatment and would returned to basal levels later. We used immunohistochemistry techniques to determine the functional expression of CB1-R in morphine treated rat's brain. Enzyme-Linked Immunosorbent Assay (ELISA) revealed the significant increase of cytokine (IL-1beta, IL-6) levels in the repeated morphine treatment rats' cortex and hippocampus regions, which are both addiction-related brain areas. In addition, the results from RT-PCR and western blot assay indicated that the expression of CB1-R was directly increased by morphine treatment in vitro. All the results indicated that the CB1-R expression could be changed by morphine exposure and it might be involved in neural immune function, which provided a potential target for neurogenic disease treatment.

Oxidative stress and cannabinoid receptor expression in type-2 diabetic rat pancreas following treatment with Δ⁹-THC.

The objectives of study were (a) to determine alteration of feeding, glucose level and oxidative stress and (b) to investigate expression and localization of cannabinoid receptors in type-2 diabetic rat pancreas treated with Δ(9)-tetrahydrocannabinol (Δ(9)-THC). Rats were randomly divided into four groups: control, Δ(9)-THC, diabetes and diabetes + Δ(9)-THC groups. Diabetic rats were treated with a single dose of nicotinamide (85 mg/kg) 15 min before injection of streptozotocin (65 mg/kg). Δ(9)-THC was administered intraperitoneally at 3 mg/kg/day for 7 days. Body weights and blood glucose level of rats in all groups were measured on days 0, 7, 14 and 21. On day 15 after the Δ(9)-THC injections, pancreatic tissues were removed. Blood glucose levels and body weights of diabetic rats treated with Δ(9)-THC did not show statistically significant changes when compared with the diabetic animals on days 7, 14 and 21. Treatment with Δ(9)-THC significantly increased pancreas glutathione levels, enzyme activities of superoxide dismutase and catalase in diabetes compared with non-treatment diabetes group. The cannabinoid 1 receptor was found in islets, whereas the cannabinoid 2 receptor was found in pancreatic ducts. Their localization in cells was both nuclear and cytoplasmic. We can suggest that Δ(9) -THC may be an important agent for the treatment of oxidative damages induced by diabetes. However, it must be supported with anti-hyperglycaemic agents. Furthermore, the present study for the first time emphasizes that Δ(9)-THC may improve pancreatic cells via cannabinoid receptors in diabetes. The aim of present study was to elucidate the effects of Δ(9)-THC, a natural cannabinoid receptor agonist, on the expression and localization of cannabinoid receptors, and oxidative stress statue in type-2 diabetic rat pancreas. Results demonstrate that the cannabinoid receptors are presented in both Langerhans islets and duct regions. The curative effects of Δ(9)-THC can be occurred via activation of cannabinoid receptors in diabetic rat pancreas. Moreover, it may provide a protective effect against oxidative damage induced by diabetes. Thus, it is suggested that Δ(9)-THC can be a candidate for therapeutic alternatives of diabetes symptoms.

Human urothelial cell lines as potential models for studying cannabinoid and excitatory receptor interactions in the urinary bladder

To characterize human urothelial cell lines' cannabinoid receptor expression and evaluate their possible use for studying signalling interactions with purinergic and muscarinic receptor activation. PCR was used to detect cannabinoid (CB), muscarinic and purinergic receptor transcripts in HCV29 and UROtsa cells, whilst immunofluorescence evaluated protein expression and localization of cannabinoid receptors. The effect of CB1 agonist (ACEA) on carbachol- and ATP-induced changes in intracellular calcium ([Ca(2+)]i) levels was measured using fluorimetry. The ability of ACEA to reduce intracellular cAMP was investigated in HCV29 cells. CB1 and GPR55 receptor transcripts were detected in HCV29 and UROtsa cells, respectively. Immunofluorescence showed positive staining for CB1 in the HCV29 cells. Both cell lines expressed transcript levels for muscarinic receptors, but carbachol did not raise [Ca(2+)]i levels indicating a lack or low expression of G(q)-coupled muscarinic receptors. Transcripts for purinergic receptors were detected; ATP significantly increased [Ca(2+)]i in HCV29 and UROtsa cells by 395 ± 61 and 705 ± 100 nM (mean ± SEM, n = 6), respectively. ACEA did not alter ATP-induced [Ca(2+)]i or cAMP levels in HCV29 cells. Whilst HCV29 cells expressed CB1 and UROtsa cells expressed GPR55 receptors, these were not functionally coupled to the existing purinergic-driven increase in Ca2+ as such they do not represent a good model to study signalling interactions.

Expression and localization of cannabinoid receptors in human immature oocytes and unfertilized metaphase-II oocytes

Endocannabinoid anandamide and cannabinoid receptors have been described in some organs of the female reproductive system, but little is known about the expression of these receptors in human oocytes. The aim of the study was to describe the expression of cannabinoid receptors in human oocytes and to investigate their differential distribution at various stages of meiotic resumption in human oocytes. A total of 750 human oocytes from 214 patients were analysed by Western blot, immunocytochemistry and PCR. For this study, oocytes that were not suitable for intracytoplasmic sperm injection (ICSI) (germinal-vesicle and metaphase-I stages), as well as metaphase-II oocytes that had not developed into an embryo after ICSI were used. Western blot analysis revealed the presence of CB1 and CB2 receptor proteins in human oocytes. CB1 and CB2 receptor immunostaining patterns changed during the various stages of meiotic resumption. Localization of CB1 receptor was peripheral at germinal-vesicle stage, homogeneous over the entire oocyte at metaphase I and peripheral at mature metaphase II. CB2 receptor localization was peripheral at germinal-vesicle and metaphase-I stages but homogeneous over the entire cell at metaphase II. This finding suggests a possible role for endocannabinoids, acting via receptors, in the maturation of female gametes and fertilization. The number of couples with sterility problems attending fertility programmes is rising but treatment is not always successful. Important problems associated with failure to conceive remain unresolved because many physiological aspects of human reproduction are still unknown. Endocannabinoids are endogenous chemical compounds that mimic the action of the main psychoactive component of marijuana, delta-9-tetrahydrocannabinol. An endogenous cannabinoid named anandamide has been found in human follicular fluid. Thus, in order to develop knowledge in this field, in the present study we have described the presence of the cannabinoid receptors CB1 and CB2 (the proteins required to mediate the action of the cannabinoids) in the early stages of meiotic resumption of oocytes (the stages before ovulation) and we could postulate that the endocannabinoids could act in the regulation of maturation of oocytes. Our study, together with other studies, indicates that the endocannabinoid system may play a role in human reproduction.

The Effect of Anandamide on Uterine Nitric Oxide Synthase Activity Depends on the Presence of the Blastocyst

Nitric oxide production, catalyzed by nitric oxide synthase (NOS), should be strictly regulated to allow embryo implantation. Thus, our first aim was to study NOS activity during peri-implantation in the rat uterus. Day 6 inter-implantation sites showed lower NOS activity (0.19±0.01 pmoles L-citrulline mg prot−1 h−1) compared to days 4 (0.34±0.03) and 5 (0.35±0.02) of pregnancy and to day 6 implantation sites (0.33±0.01). This regulation was not observed in pseudopregnancy. Both dormant and active blastocysts maintained NOS activity at similar levels. Anandamide (AEA), an endocannabinoid, binds to cannabinoid receptors type 1 (CB1) and type 2 (CB2), and high concentrations are toxic for implantation and embryo development. Previously, we observed that AEA synthesis presents an inverted pattern compared to NOS activity described here. We adopted a pharmacological approach using AEA, URB-597 (a selective inhibitor of fatty acid amide hydrolase, the enzyme that degrades AEA) and receptor selective antagonists to investigate the effect of AEA on uterine NOS activity in vitro in rat models of implantation. While AEA (0.70±0.02 vs 0.40±0.04) and URB-597 (1.08±0.09 vs 0.83±0.06) inhibited NOS activity in the absence of a blastocyst (pseudopregnancy) through CB2 receptors, AEA did not modulate NOS on day 5 pregnant uterus. Once implantation begins, URB-597 decreased NOS activity on day 6 implantation sites via CB1 receptors (0.25±0.04 vs 0.40±0.05). While a CB1 antagonist augmented NOS activity on day 6 inter-implantation sites (0.17±0.02 vs 0.27±0.02), a CB2 antagonist decreased it (0.17±0.02 vs 0.12±0.01). Finally, we described the expression and localization of cannabinoid receptors during implantation. In conclusion, AEA levels close to and at implantation sites seems to modulate NOS activity and thus nitric oxide production, fundamental for implantation, via cannabinoid receptors. This modulation depends on the presence of the blastocyst. These data establish cannabinoid receptors as an interesting target for the treatment of implantation deficiencies.

Endocannabinoid Signaling Is Required for Development and Critical Period Plasticity of the Whisker Map in Somatosensory Cortex

Type 1 cannabinoid (CB1) receptors mediate widespread synaptic plasticity, but how this contributes to systems-level plasticity and development in vivo is unclear. We tested whether CB1 signaling is required for development and plasticity of the whisker map in rat somatosensory cortex. Treatment with the CB1 antagonist AM251 during an early critical period for layer (L) 2/3 development (beginning postnatal day [P] 12-16) disrupted whisker map development, leading to inappropriate whisker tuning in L2/3 column edges and a blurred map. Early AM251 treatment also prevented experience-dependent plasticity in L2/3, including deprivation-induced synapse weakening and weakening of deprived whisker responses. CB1 blockade after P25 did not disrupt map development or plasticity. AM251 had no acute effect on sensory-evoked spiking and only modestly affected field potentials, suggesting that plasticity effects were not secondary to gross activity changes. These findings implicate CB1-dependent plasticity in systems-level development and early postnatal plasticity of the whisker map.

Cannabinoid CB 1 receptors are expressed in the mouse urinary bladder and their activation modulates afferent bladder activity

Pharmacological studies have indirectly shown the possible presence of cannabinoid receptors in the urinary bladder and their potential role in reducing bladder inflammatory pain. However, the localization of cannabinoid receptors in the urinary bladder remains unknown and there are no published data on the effects of cannabinoids on the sensory system of the bladder. The present study was performed to evaluate the expression of the cannabinoid CB 1 receptors in the mouse urinary bladder and to assess their co-localization with the purinergic P2X 3 receptor, a major player in the transduction of sensory events in the bladder. Also, the effect of intravesical administration of a cannabinoid agonist on the electrical activity of bladder afferent fibers was studied. The expression of mRNA coding for CB 1 receptor was assessed by reverse transcriptase–polymerase chain reaction (RT-PCR). Immunofluorescence experiments were performed to study CB 1 and P2X 3 protein expression in the bladder. The electrical activity of bladder afferent fibers was recorded using an ex vivo bladder-nerve preparation. Mechanical stimulation of the bladder was performed by a controlled slow inflation with an external pump. A bolus of a cannabinoid agonist (AZ12646915) was administered intravesically prior to a second inflation. Afferent activity was measured before and after administration of the cannabinoid compound or its vehicle. The effects of CB 1 receptor antagonist (AM251) on the AZ12646915 response were also analyzed. Cannabinoid receptor CB 1 mRNA was detected in the urinary bladder of the mouse. The protein was found in the urothelium, as well as in nerve fibers. CB 1 and P2X 3 receptors were found to be co-expressed in urothelial cells and in some nerve fibers. In addition, intravesical administration of a cannabinoid receptor agonist reduced the mechanically-evoked activity of bladder afferents in the pelvic nerve. This effect was abolished by the previous administration of the CB 1 antagonist AM251. These data demonstrate the presence of cannabinoid CB 1 receptor mRNA and the protein in the mouse urinary bladder. CB 1 and P2X 3 protein co-localization supports the hypothesis of an interaction between the cannabinoid and the purinergic systems in the transduction of sensory information in the urinary bladder. Finally, the reduction of nerve activity induced by cannabinoid-receptor activation implicates CB 1 receptors in the peripheral modulation of bladder afferent information.

Localization of cannabinoid receptor in the human developing and adult basal ganglia. Higher levels in the striatonigral neurons

In the infant and adult human basal ganglia, the finding of mRNA exclusively in the striatal medium-sized neurons together with the detection of [ 3H]CP55,940 binding sites in the caudate-putamen, accumbens, substantia nigra pars reticulata and globus pallidus suggests cannabinoid receptor localization on the striatal intrinsic enkephalinergic and substance P-projecting neurons and on their nigral and pallidal terminals. However, the consistent finding of higher binding in the substantia nigra pars reticulata and medial part of the globus pallidus over its lateral segment suggests cannabinoid receptor enrichment on the striatal substance P neurons which express selectively the dopamine D 1 receptor.

Characterization and localization of cannabinoid receptors in rat brain: a quantitative in vitro autoradiographic study

A potent, synthetic cannabinoid was radiolabeled and used to characterize and precisely localize cannabinoid receptors in slide-mounted sections of rat brain and pituitary. Assay conditions for 3H-CP55,940 binding in Tris-HCl buffer with 5% BSA were optimized, association and dissociation rate constants determined, and the equilibrium dissociation constant (Kd) calculated (21 nM by liquid scintillation counting, 5.2 nM by quantitative autoradiography). The results of competition studies, using several synthetic cannabinoids, add to prior data showing enantioselectivity of binding and correlation of in vitro potencies with potencies in biological assays of cannabinoid actions. Inhibition of binding by guanine nucleotides was selective and profound: Nonhydrolyzable analogs of GTP and GDP inhibited binding by greater than 90%, and GMP and the nonhydrolyzable ATP analog showed no inhibition. Autoradiography showed great heterogeneity of binding in patterns of labeling that closely conform to cytoarchitectural and functional domains. Very dense 3H-CP55,940 binding is localized to the basal ganglia (lateral caudate-putamen, globus pallidus, entopeduncular nucleus, substantia nigra pars reticulata), cerebellar molecular layer, innermost layers of the olfactory bulb, and portions of the hippocampal formation (CA3 and dentate gyrus molecular layer). Moderately dense binding is found throughout the remaining forebrain. Sparse binding characterizes the brain stem and spinal cord. Densitometry confirmed the quantitative heterogeneity of cannabinoid receptors (10 nM 3H-CP55,940 binding ranged in density from 6.3 pmol/mg protein in the substantia nigra pars reticulata to 0.15 pmol/mg protein in the anterior lobe of the pituitary). The results suggest that the presently characterized cannabinoid receptor mediates physiological and behavioral effects of natural and synthetic cannabinoids, because it is strongly coupled to guanine nucleotide regulatory proteins and is discretely localized to cortical, basal ganglia, and cerebellar structures involved with cognition and movement.

Cannabinoid receptor localization in brain.

[3H]CP 55,940, a radiolabeled synthetic cannabinoid, which is 10-100 times more potent in vivo than delta 9-tetrahydrocannabinol, was used to characterize and localize a specific cannabinoid receptor in brain sections. The potencies of a series of natural and synthetic cannabinoids as competitors of [3H]CP 55,940 binding correlated closely with their relative potencies in several biological assays, suggesting that the receptor characterized in our in vitro assay is the same receptor that mediates behavioral and pharmacological effects of cannabinoids, including human subjective experience. Autoradiography of cannabinoid receptors in brain sections from several mammalian species, including human, reveals a unique and conserved distribution; binding is most dense in outflow nuclei of the basal ganglia--the substantia nigra pars reticulata and globus pallidus--and in the hippocampus and cerebellum. Generally high densities in forebrain and cerebellum implicate roles for cannabinoids in cognition and movement. Sparse densities in lower brainstem areas controlling cardiovascular and respiratory functions may explain why high doses of delta 9-tetrahydrocannabinol are not lethal.