Brain Cannabinoid Receptors Research Articles

A light and electron microscopic study of the CB1 cannabinoid receptor in monkey basal forebrain.

The distribution of the CB1 cannabinoid receptor was studied in the monkey basal forebrain by immunocytochemistry and electron microscopy, using an antibody to the CB1 brain cannabinoid receptor. Large numbers of labelled neurons were observed in the medial septum, nucleus of the diagonal band, and the nucleus basalis of Meynert. The labelled neurons had dimensions similar to those of cholinergic neurons and were larger than those of GABAergic neurons. Double immunolabelling with an antibody to the synthetic enzyme for acetylcholine, choline acetyl transferase (ChAT) showed that CB1-positive neurons were also positive for ChAT, whilst electron microscopy confirmed that CB1-labelled neurons contained lipofuscin granules and dense clusters of rough endoplasmic reticulum, characteristic of cholinergic neurons. The dense labelling of cholinergic neurons for CB1 is interesting from the standpoint of neuroprotection. The CB1 receptor has been shown to couple in an inhibitory manner to voltage dependent calcium channels, and the dense labelling of CB1 in cholinergic neurons would therefore suggest that CB1 receptors could be important in limiting calcium influx through voltage dependent calcium channels in these neurons. This could serve to limit intracellular calcium concentrations, and consequent calcium mediated injury, in these neurons.

Role of the Endogenous Cannabinoid System in the Regulation of Motor Activity

One of the prominent pharmacological features of drugs acting at the brain cannabinoid receptor (CB1) is the induction of alterations in motor behavior. Catalepsy, immobility, ataxia, or the impairment of complex behavioral acts are observed after acute administration of either natural and synthetic cannabinoid receptor agonists or the endogenous CB1ligand anandamide. The dense presence of CB1receptors in the cerebellum and in the basal ganglia, especially at the outflow nuclei (substantia nigra and the internal segment of the globus pallidus), supports the existence of an endogenous cannabinoid system regulating motor activity. In the basal ganglia, the functionality of the anandamide–CB1system is poorly understood. Dual effects are often observed after the administration of CB1ligands in animal models of pharmacological manipulation of basal ganglia transmitter systems, indicating that the activity of the anandamide–CB1system depends on the ongoing activation of the different elements of the basal ganglia. This finding is in agreement with the proposed activity-dependent release of anandamide from a plasmalemma precursor. Additionally, a potential state-dependent bidirectional coupling of the CB1receptor to the adenylate cyclase transduction system has also been described. From this perspective, the endogenous cannabinoid system can be proposed as a local regulator of neurotransmission processes within the basal ganglia. This system may serve as a counterregulatory homeostatic mechanism preserving the functional role of basal ganglia circuits in coding the serial order of events that constitute movement.

Trick or treat from food endocannabinoids?

Beltramo and Piomelli reply — The two most abundant N-acylethanolamines (NAEs) found in chocolate (N-oleyl-ethanolamine and N-linoleylethanolamine) do not activate brain cannabinoid receptors but effectively inhibit anandamide degradation1,2. This observation suggested to us that these compounds might contribute to the hedonic properties of chocolate by causing non-metabolized anandamide to accumulate at its sites of action2.

Selective effects of the endogenous cannabinoid arachidonylethanolamide (anandamide) on regional cerebral blood flow in the rat.

Recent biochemical data suggest that arachidonylethanolamide (AEA; anandamide) may be an endogenous ligand for brain cannabinoid receptors. The functional neuronal consequences of AEA binding to cannabinoid receptors are only poorly understood. Using regional cerebral blood flow (rCBF) as an indirect marker of neuronal activity, acute AEA administration dose-dependently depressed rCBF in unanesthetized rats. Although 3.0 mg/kg was ineffective in altering rCBF, 10 mg/kg led to a decrease in rCBF in seven brain areas including the amygdala, cingulate, frontal, prepyriform, sensorimotor, and claustrocortex. An additional 16 areas responded in a similar manner to AEA, but only after 30 mg/kg, including the CA1 and CA3 regions of the hippocampus, the rostral core portion of the nucleus accumbens, and rostral caudate nucleus. Most of these rCBF effects dissipated between 15 and 20 min after drug administration, with only 4 regions, the basomedial and lateral amygdala, CA3 hippocampus and claustrocortex still depressed 60 min after an acute drug injection. No significant changes in heart rate, blood pressure, or blood gases were seen at the time of rCBF measurement, suggesting that the observed drug effects were neuronally mediated. Taken together with existing behavioral data, these data support the hypothesis that an endogenous cannabinoid neural system exists in mammalian brain and may help to explain the unique behavioral profile seen after cannabinoid administration.

The endogenous cannabinoid anandamide potentiates interleukin-6 production by astrocytes infected with Theiler's murine encephalomyelitis virus by a receptor-mediated pathway

Theiler's murine encephalomyelitis virus (TMEV) infection of a susceptible strain of mice results in virus persistence in the brain and chronic primary immune-mediated demyelination, which resembles multiple sclerosis. Recent attention has focused on the anti-inflammatory and immunosuppressive properties of interleukin-6, a pleiotropic cytokine involved in the regulation of immunological responses, acute phase protein production and hematopoiesis. Anandamide (arachidonoyl ethanolamine) is a natural brain constituent that binds a specific brain cannabinoid receptor. In this study we investigated whether anandamide can modify interleukin-6 production by primary cultures of murine brain cortical astrocytes infected with TMEV. Astrocytes from susceptible (SJL/J) and resistant (BALB/c) strains of mice infected with TMEV (10 5 PFU/well) increased IL-6 release over a period of 24 h. Anandamide caused an enhancement of the release of IL-6 by TMEV-infected astrocytes in a concentration-dependent manner (1–25 μM). Treatment of TMEV-infected astrocytes with 10 μM arachidonyl trifluoromethyl ketone, a potent inhibitor of the amidase that degrades anandamide, was found to potentiate the effects of anandamide on IL-6 release. A novel and selective cannabinoid receptor antagonist, SR 141617A, blocked the enhancing effects of anandamide on IL-6 release by TMEV-infected astrocytes, suggesting a cannabinoid receptor-mediated pathway. The physiological implications of these results are unknown, but may be related to the hypothesis of the protective effects of cannabinoids on neurological disorders like multiple sclerosis.

CANNABIS

The recent discovery of arachidonylethanolamide (anandamide), an endogenous ligand for cannabinoid receptors, and the synthesis of SR141716A, a cannabinoid antagonist selective for brain cannabinoid (CB1) receptors, have provided new tools to explore the mechanisms underlying cannabis abuse and dependence. Drug discrimination is the animal model with the most predictive validity and specificity for investigation of the psychoactive effects of cannabinoids related to their abuse potential, because, unlike many other drugs of abuse, delta9-tetrahydrocannabinol (delta9-THC), the major psychoactive ingredient of marijuana, is not self-administered by animals. Results of delta9-THC discrimination studies have revealed that the subjective effects of cannabis intoxication are pharmacologically selective for centrally active cannabinoid compounds, and that cannabis action at CB1 receptors is involved in medication of these effects. Less clear is the role of endogenous cannabinoid system(s) in cannabis intoxication. Anandamide, named for a Sanskrit word for internal bliss, unreliably substitutes for delta9-THC. Further, substitution, when it is observed, occurs only at doses that also significantly decrease response rates. In contrast, delta9-THC and other structurally diverse cannabinoids fully substitute for delta9-THC at doses that do not substantially affect response rates. Attempts to train animals to discriminate anandamide (or SR141716A) have so far been unsuccessful. Preliminary evidence from drug discrimination studies with more metabolically stable anandamide analogs have suggested that these differences in the discriminative stimulus effects of delta9-THC and anandamide-like cannabinoids are not entirely due to pharmacokinetic factors, but the exact role of internal bliss in cannabis intoxication and dependence is still not completely understood.

Evaluation of cannabimimetic effects of structural analogs of anandamide in rats

Arachidonylethanolamide (anandamide), an endogenous ligand for the cannabinoid receptor, binds competitively to brain cannabinoid receptors and shares many, but not all, of the in vivo effects of Δ 9-tetrahydrocannabinol. In this study, the cannabinoid effects of anandamide analogs in which the anandamide molecule was altered were assessed in a drug discrimination model. Structural manipulations of the anandamide molecule included saturation of the arachidonyl moiety with fluorination (O-586), substitution for either the ethanolamide moiety (O-612 and O-595) or C2′ hydroxyl (O-585), and addition of a methyl group at various positions (O-610, O-680, and O-689). Despite the low binding affinities of the non-methylated compounds ( K i values >2000 nM), all of the analogs had previously shown cannabinoid activity in mice. In the present study, these analogs were tested in a more pharmacologically specific Δ 9-tetrahydrocannabinol discrimination procedure in rats. This animal model is predictive of the subjective effects of marijuana intoxication in humans. Whereas Δ 9-tetrahydrocannabinol and an aminoakylindole fully substituted for the training dose of 3 mg/kg Δ 9-tetrahydrocannabinol, anandamide and its non-methylated analogs were not cannabimimetic in this procedure. Methylation appeared to increase binding affinity ( K i values <150 nM) and efficacy; however, the greatest substitution produced by the methylated analogs occurred only at doses that decreased overall rates of responding, suggesting that these analogs are not fully Δ 9-tetrahydrocannabinol-like. The rapid metabolism of anandamide and some of its analogs undoubtedly contribute to the differences between the pharmacological profiles of the anandamides and classical cannabinoids. These results support the prediction that the subjective effects of anandamide analogs that have been developed thus far would not be cannabimimetic except at high doses.

Appetite suppression and weight loss after the cannabinoid antagonist SR 141716

The effect of the cannabinoid CB 1 receptor antagonist, SR 141716, on food intake and body weight was assessed in adult, non-obese Wistar rats. The daily administration of SR 141716 (2.5 and 10 mg/kg; i.p.) reduced dose-dependently both food intake and body weight. Tolerance to the anorectic effect developed within 5 days; in contrast, body weight in SR 141716-treated rats remained markedly below that of vehicle-treated rats throughout the entire treatment period (14 days). The results suggest that brain cannabinoid receptors are involved in the regulation of appetite and body weight.

Autoradiographic analysis of cannabinoid receptor binding and cannabinoid agonist-stimulated [ 35S]GTP γS binding in morphine-dependent mice

The present study was designed to test the possible existence of changes in brain cannabinoid receptors in morphine-dependent mice. To this end, we compared cannabinoid receptor binding and WIN 55,212-2-stimulated [ 35S]guanylyl-5′- O-( γ-thio)-triphosphate ([ 35S]GTP γS) binding in several brain regions of mice chronically exposed to morphine or saline. The existence of opiate dependence in morphine-injected mice was assessed by analyzing the well-known jumping behavior induced by the blockade of opioid receptors with naloxone, whereas these animals were unresponsive to the blockade of cannabinoid receptors with SR141716. The different structures analyzed exhibited similar cannabinoid receptor binding levels in morphine-dependent and control mice, with the only exception of the globus pallidus, which exhibited a very small, but statistically significant, increase. In addition, the activation of cannabinoid receptors with WIN 55,212-2 increased [ 35S]GTP γS binding in most of the structures examined. The increase was of similar magnitude in morphine-dependent and control mice, except in the substantia nigra, where morphine-dependent mice exhibited lesser [ 35S]GTP γS binding levels in basal conditions, although a significantly higher WIN 55,212-2-stimulated binding. Other structures, such as the central gray substance, where there was a poor agonist-induced stimulation in control mice, exhibited, however, higher levels of WIN 55,212-2-stimulated [ 35S]GTP γS binding in morphine-dependent mice, whereas these animals tended to exhibit a higher [ 35S]GTP γS binding levels in basal conditions, although a lesser and not statistically significant WIN 55,212-2-stimulated binding, in the deep layers of the cerebral cortex. Thus, the data support the potential existence of a specific effect of morphine in the coupling of cannabinoid receptors to GTP-binding proteins, rather than on receptor binding, although this was observed only in the substantia nigra and central gray substance.

Complex pharmacology of natural cannabivoids: Evidence for partial agonist activity of Δ 9-tetrahydrocannabinol and antagonist activity of cannabidiol on rat brain cannabinoid receptors

Δ 9-tetrahydrocannabinol (Δ 9-THC), cannabinol and cannabidiol are three important natural cannabinoids from the Marijuana plant ( Cannabis sativa). Using [ 35S]GTP-γ-S binding on rat cerebellar homogenate as an index of cannabinoid receptor activation we show that: Δ 9-THC does not induce the maximal effect obtained by classical cannabinoid receptor agonists such as CP55940. Moreover at high concentration Δ 9-THC exhibits antagonist properties. Cannabinol is a weak agonist on rat cerebellar cannabinoid receptors and cannabidiol behaves as an antagonist acting in the micromolar range.

Early Cannabinoid Exposure as a Source of Vulnerability to Opiate Addiction: A Model in Laboratory Rodents

Recent findings have identified an endogenous brain system mediating the actions of cannabis sativa preparations. This system includes the brain cannabinoid receptor (CB-1) and its endogenous ligands anandamide and 2-arachidonoyl-glycerol. The endogenous cannabinoid system is not only present in the adult brain, but is also active at early stages of brain development. Studies developed at our laboratory have revealed that maternal exposure to psychoactive cannabinoid results in neuro-developmental alterations. A model is proposed in which early Δ9-tetrahydrocannabinol (THC) exposure during critical developmental periods results in permanent alterations in brain function by either the stimulation of CB-1 receptors present during the development, or by the alterations in maternal glucocorticoid secretion. Those alterations will be revealed in adulthood after challenges either with drugs (i.e. opiates) or with environmental stressors (i.e. novelty). They will include a modified pattern of neuro-chemical, endocrine, and behavioral responses that might lead ultimately to inadaptation and vulnerability to opiate abuse.

Immunohistochemical localization of the neural cannabinoid receptor in rat brain.

The cannabinoid receptor family consists of two inhibitory G-protein-coupled receptors, CB1 and CB2. CB1 is distributed primarily in neural tissue, whereas CB2 is distributed predominantly in immune cells. The distribution of cannabinoid receptors in neural tissue has been demonstrated by using ligand binding autoradiography with CP55,940, a high-affinity cannabinoid receptor ligand, and in situ hybridization. However, the localization of CB1 within individual cells in the brain remains to be defined. In the present study, domain-specific polyclonal antibody to amino acids 83-98 of CB1 was used to define the expression of the neural cannabinoid receptor at the histochemical level. The use of CB1-specific antiserum is advantageous in view of recent reports that CB2 also is expressed in the brain and binds CP55,940. Thus, utilization of anti-CB1 antiserum would allow for the specific detection of CB1 protein expression. The regional staining pattern for CB1 in rat brain was consistent with that reported for CB1 using ligand binding autoradiography and in situ hybridization. Intense immunoreactivity was present in the hippocampal formation, the basal ganglia, and the molecular layer of the cerebellum. Moderate immunohistochemical staining was observed in the olfactory bulb, piriform cortex, cerebral cortex, and the granular layer of the cerebellum. In addition, immunoreactive staining was concentrated on afferent projections and dendritic processes of neuronal cells and was present within cell bodies and on cell surfaces. These data indicate that the anti-CB1 antibody is a sensitive probe for the unequivocal histological discrimination of CB1 protein expression.

Immunohistochemical localization of the neural cannabinoid receptor in rat brain

Immunohistochemical localization of the neural cannabinoid receptor in rat brain

Interaction of brain cannabinoid receptors with guanine nucleotide binding protein: A radioligand binding study

The binding of a classical cannabinoid agonist, [ 3H]R-(+)-(2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrol[1,2,3-de]-l,4-benzoxazin-6-yl)(1-napthalenyl)methanone monomethanesulfonate ([ 3H] WIN55212-2), and a selective cannabinoid receptor (CB 1) antagonist, N-(piperidin-1-yl)-5-(4-chlorophenyl)- l-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride ([ 3H]SR141716A), to rat cannabinoid receptors was evaluated using rat cerebellar membranes. Guanine nucleotides inhibited [ 3H]WIN55212-2 binding by approximately 50% at 10 (JtM and enhanced [5H]SR141716A binding very slightly. In the same tissue, the binding of guanosine 5′-O-[γ-[ 35S]thio]triphosphate ([ 35S]GTP-γ-S) was characterized and the influence of cannabinomimetics evaluated on this binding. Cannabinoid receptor agonists enhanced [ 35 S]GTP-γ-S binding, whereas SR141716A was devoid of action by itself but antagonized the action of cannabinoid receptor agonists. The good correlation obtained between the half maximum efficient concentration ( ec 50 ) values in [ 35S]GTP-γ-S binding and the ic 50 values [ 3H]WIN55212-2 binding shows that [ 35S]GTP-γ-S binding could be a good functional assay for brain cannabinoid receptors.

Modulation of rat brain cannabinoid receptors after chronic morphine treatment.

Intraperitoneal injection of delta9-THC (7.5 mg/kg) in rats made tolerant to morphine by s.c. implantation of morphine pellets had a much greater analgesic effect than in placebo pellet plus delta9-THC treatment. To investigate whether this was due to some change in cannabinoid receptor levels and/or expression induced by chronic morphine, we designed this autoradiographic binding study coupled with in situ hybridization on sagittal sections of the treated rat brains. Binding showed a significant increase in CB1 receptor density (15%) specifically in the caudate-putamen, in parallel with a significant enhancement of CB1 mRNA in the same area (20%). We suggest that morphine chronic treatment leads to a functional modulation between the opioid and cannabinoid systems at least for analgesia in a specific area, in this case the striatum.

Binding of the non-classical cannabinoid CP 55,940, and the diarylpyrazole AM251 to rodent brain cannabinoid receptors

The binding of [ 123I]AM251 (a radioiodinated analog of the cannabinoid CB1 receptor antagonist SR141716A) was compared to that of [ 3H]CP 55,940 in mouse and rat brain preparations. Scatchard analysis of the binding of [ 123I]AM251 and [ 3H]CP 55,940 to membranes prepared from mouse cerebellum, striatum and hippocampus yielded similar Bmax values (15–41 pmol g wet wt tissue). Kd values were lower for [ 123I]AM251 (0.23–0.62 nM) than for [ 3H]CP 55,940 (1.3–4 nM). CP 55,940 and SR141716A increased dissociation of [ 123I]AM251 from binding sites in mouse cerebellar homogenates to a similar extent. The structurally dissimilar cannabinoid receptor ligands THC, methanandamide, WIN 55, 212-2, CP 55,940 and SR141716A were each able to fully compete with binding of both [ 123I]AM251 and [ 3H]CP 55,940 in mouse cerebellum. In vitro autoradiography demonstrated that the distribution of binding sites for [ 123I]AM251 in rat brain was very similar to published distributions of binding sites for [ 3H]CP 55,940. Together, these observations suggest that AM251 binds to the same site (the cannabinoid CB1 receptor) in rodent brains as CP 55,940. However, the binding site domains which interact with AM251 and CP 55,940 may not be identical, since IC 50 values for cannabinoid receptor ligands depended on whether [ 123I]AM251 or [ 3H]CP 55,940 was used as radioligand.

Expression of a brain-type cannabinoid receptor (CB 1) in alveolar Type II cells in the lung: regulation by hydrocortisone

Using the polymerase chain reaction with degenerate primers to identify novel G-protein-coupled receptors of the rat alveolar Type II cell, we identified sequences expressed by the Type II cell identical to the sequence of the rat brain cannabinoid receptor (CB 1). The use of Northern blot analysis to examine expression of CB 1 mRNA in rat tissues revealed differences between the brain and lung. While rat brain expressed a 6.0 kb mRNA as previously described, rat lung expressed mRNA of 4.5 and 6.0 kb. Isolated lung alveolar Type II cells also expressed mRNA of 4.5 and 6.0 kb as determined by Northern analysis. However, only freshly isolated Type II cells contained cannabinoid receptor mRNA. Reverse transcriptase-polymerase chain reaction (RT-PCR) failed to detect CB 1 mRNA in Type II cells maintained in culture for 1 or 2 days. We next determined developmental changes in lung CB 1 mRNA expression using semi-quantitative RT-PCR. CB 1 expression was detected as early as gestational day 16 in rat lung and mRNA levels increased to fetal day 20 before birth, before declining to adult levels. Fetal rat lung explants were utilized to further examine the ontogeny and hormonal effects on CB 1 mRNA expression. Hydrocortisone induced a dose-dependent expression in 15-day and 18-day explants, similar to previous results for surfactant-associated proteins. Our results demonstrate expression of CB 1 mRNA in rat alveolar Type II cells and rat lung. This expression is ontogenically and hormonally regulated, with maximal expression noted just prior to birth in rat lung. Since CB 1 mRNA is only expressed in freshly isolated Type II cells, CB 1 may be useful as a Type II cell marker.

Structural requirements for binding of anandamide-type compounds to the brain cannabinoid receptor.

In order to establish the structural requirements for binding to the brain cannabinoid receptor (CB1), we have synthesized numerous fatty acid amides, ethanolamides, and some related simple derivatives and have determined their Ki values. A few alpha-methyl- or alpha, alpha-dimethylarachidonoylalkylamides were also examined. In the 20:4, n-6 series, the unsubstituted amide is inactive; N-monoalkylation, at least up to a branched pentyl group, leads to significant binding. N,N-Dialkylation, with or without hydroxylation on one of the alkyl groups, leads to elimination of activity. Hydroxylation of the N-monoalkyl group at the omega carbon atom retains activity. In the 20x, n-6 series, x has to be either 3 or 4; the presence of only two double bonds leads to inactivation. In the n-3 series, the limited data reported suggest that the derived ethanolamides are either inactive or less active than comparable compounds in the n-6 series. Alkylation or dialkylation of the alpha carbon adjacent to the carbonyl group retains the level of binding in the case of anandamide (compounds 48, 49); however, alpha-monomethylation or alpha,alpha-dimethylation of N-propyl derivatives (50-53) potentiates binding and leads to the most active compounds seen in the present work (Ki values of 6.9 +/- 0.7 to 8.4 +/- 1.1 nM). We have confirmed that the presence of a chiral center on the N-alkyl substituent may lead to enantiomers which differ in their levels of binding (compounds 54, 57 and 55, 56).

Binding of aminoalkylindoles to noncannabinoid binding sites in NG108-15 cells.

1. Aminoalkylindoles, typified by WIN 55212-2, bind to G protein-coupled cannabinoid receptors in brain. Although cannabinoids inhibit adenylyl cyclase in NG108-15 neuroblastoma x glioma hybrid cells, cannabinoid receptor binding in these cells has not been described previously. This study compares pharmacological characteristics of [3H]WIN 55212-2 binding sites in rat cerebellar membranes and in NG108-15 membranes. 2. Although the KD of specified [3H]WIN 55212-2 binding was similar in brain and NG108-15 membranes, the Bmax was 10 times lower in NG108-15 than in cerebellar membranes. In both brain and NG108-15 membranes, aminoalkylindole analogues were relatively potent in displacing [3H]WIN 55212-2 binding. However, IC50 values for more traditional cannabinoids were significantly higher in NG108-15 membranes than in brain, e.g., the Ki values for CP55,940 were 1.2 nM in brain and > 5000nM in NG108-15 membranes. Moreover, sodium and GTP-gamma-S decreased [3H]WIN 55212-2 binding in brain but not in NG108-15 membranes. 3. These data suggest that WIN 55212-2 does not label traditional cannabinoid receptors in NG108-15 cells and that these novel aminoalkylindole binding sites are not coupled to G proteins.

Endogenous lipids that activate cannabinoid receptors. Formation and inactivation.

The discovery of cannabinoid receptors in brain and in peripheral tissues has prompted the search for endogenous substances with cannabis-like properties. This search has led to the isolation of a lipid derivative, N-arachidonoylethanolamine (anandamide), which mimics the psychoactive effects of plant-derived and synthetic cannabinoids (Devane et al., 1992). Like Δ9-tetrahydrocannabinol, the active principle of Cannabis, anandamide binds with high affinity to cannabinoid CB1-type receptors, inhibits the twitch response in mouse vas deferens, and modulates the activities of adenylyl cyclase and voltage-dependent Ca2+ channels in neuroblastoma cells. Moreover, when administered in vivo, anandamide produces a series of behavioral responses characteristic of cannabinoid drugs (for review, see Mechoulam et al., 1994).KeywordsPhosphatidic AcidPhosphatidic AcidBioactive LipidEndogenous LipidBrain AstrocyteThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.