Impaired action of anxiolytic drugs in mice deficient in cannabinoid CB1 receptors

The role of cannabinoid CB(1) receptors in the action of anxiolytics was examined. Deletion of CB(1) receptors resulted in increased anxiety-like behaviours in light/dark box, elevated plus maze and social interaction tests. Mutant mice presented basal low corticosterone concentrations and low proopiomelanocortin gene expression in the anterior lobe of the pituitary gland compared to wild-type mice. Ten minutes of restraint stress resulted in a twofold increase in corticosterone concentrations in the plasma of mutant mice, compared to wild-type mice. Bromazepam (50 or 100 microg/kg) markedly increased the time spent in light area in wild-type animals, though both doses were without effect in mutant mice. Administration of buspirone (1 or 2 mg/kg) produced anxiolytic effects in wild-type mice. In contrast, only the highest dose of buspirone had anxiolytic results in mutant mice. Our findings reveal that CB(1) receptors are involved in the regulation of emotional responses, and play a pivotal role in the action mechanism of anxiolytics. They suggest that alterations in the functional activity of the CB(1) receptor may be related to the emergence of anxiety disorders, and may affect treatment with anxiolytics.     

Influence of age, postmortem delay and freezing storage period on cannabinoid receptor density and functionality in human brain

It has been suggested that cannabimimetic drugs could be of interest in the treatment of several nervous disorders. Thus, it is important to analyse the distribution and properties of cannabinoid (CB) receptors directly in human brain. As postmortem human tissue is subjected to the effects of several biological variables, we have analyzed by autoradiography the influence of age, postmortem delay and freezing storage period (at -25 degrees C) on two parameters corresponding to cannabinoid CB1 receptors in human frontal cortex: receptor density and degree of activation of G-proteins ([35S]GTPgammaS assays). A significant decrease in the amount of both receptor density and agonist-stimulated G-protein activity was observed with age, revealing a mean reduction of about 10% per decade. In contrast, no significant correlations were found with postmortem delay either for CB1 receptors density or functionality. Finally, both parameters (receptor density and [35S]GTPgammaS response) were significantly reduced with freezing storage period at -25 degrees C in frontal cortical layers. Non-linear analysis of these data yielded values between 12 and 24 months of storage for a 50% reduction. In conclusion, when studying CB1 receptor properties in human brain samples, a careful analysis (and matching) for variables such as age and freezing storage period has to be carried out.     

Reduced sensitivity to reward in CB1 knockout mice

Rationale Previous studies have demonstrated that the activation and blockade of the cannabinoid type 1 receptor (CB1) leads to an enhancement and decrease of the consumption of food and other orally ingested reinforcers, respectively.Objective To gain further knowledge about the role of CB1 in sucrose/saccharin reinforcing efficacy and intake, we tested CB1 knockout (CB1-KO) and littermate wild-type (WT) control mice in several self-administration experimental protocols.Methods Operant (fixed or progressive ratio schedule) and non-operant conditioning procedures were used. In addition, a choice analysis based on the matching law as well as a microstructural analysis of the intra-session pattern of self-administration was performed.Results CB1-KO mice consume less sucrose under operant conditions or when using a two-bottle free choice procedure. Moreover, as revealed by additional behavioural analysis, CB1-KO mice exhibit a decreased sensitivity to the rewarding properties of sucrose. In agreement with this finding, the differences between WT and CB1-KO mice faded away when the palatability of sucrose was devaluated by adding quinine, but not when a non-caloric sweetener, saccharin, was available.Conclusions These results demonstrate a modulatory role of CB1 in the determination of the rewarding properties of sucrose and probably, as suggested by previous studies, other reinforcers.     

Down-regulation of cannabinoid receptor agonist-stimulated [S]GTPγS binding in synaptic plasma membrane from chronic ethanol exposed mouse

In our previous study, we demonstrated that chronic ethanol (EtOH) exposure down-regulated the cannabinoid receptors (CB1) in mouse brain synaptic plasma membrane (SPM) (Basavarajappa et al., Brain Res. 793 (1998) 212–218). In the present study, we investigated the effect of chronic EtOH (4-day inhalation) on the CB1 agonist stimulated guanosine-5′-O-(3-[³⁵S]thio)-triphosphate ([³⁵S]GTPγS) binding in SPM from mouse. Our results indicate that the net CP55,940 stimulated [³⁵S]GTPγS binding was increased with increasing concentrations of CP55,940 and GDP. This net CP55,940 (1.5 μM) stimulated [³⁵S]GTPγS binding was reduced significantly (−25%) in SPM from chronic EtOH group (175±5.25%, control; 150±8.14%, EtOH; P<0.05). This effect occurs without any significant changes on basal [³⁵S]GTPγS binding (152.1±10.7 for control, 147.4±5.0 fmol/mg protein for chronic EtOH group, P>0.05). Non-linear regression analysis of net CP55,940 stimulated [³⁵S]GTPγS binding in SPM showed that the B_max of cannabinoid stimulated binding was significantly reduced in chronic EtOH exposed mouse (B_max=7.58±0.22 for control; 6.42±0.20 pmol/mg protein for EtOH group; P<0.05) without any significant changes in the G-protein affinity (K_d=2.68±0.24 for control; 3.42±0.31 nM for EtOH group; P>0.05). The pharmacological specificity of CP55,940 stimulated [³⁵S]GTPγS binding in SPM was examined with CB1 receptor antagonist, SR141716A and these studies indicated that CP55,940 stimulated [³⁵S]GTPγS binding was blocked by SR141716A with a decrease (P<0.05) in the IC₅₀ values in the SPM from chronic EtOH group. These results suggest that the observed down-regulation of CB1 receptors by chronic EtOH has a profound effect on desensitization of cannabinoid-activated signal transduction and possible involvement of CB1 receptors in EtOH tolerance and dependence.     

Cannabinoids inhibit pre- and postjunctionally sympathetic neurotransmission in rat mesenteric arteries

The effects of cannabinoids on sympathetic neurotransmission in the rat isolated perfused mesenteric arterial bed, were investigated. Electrically evoked sympathetic neurogenic vasocontraction was inhibited by the cannabinoid receptor agonists 11-hydroxy-dimethylheptyl-Delta(8)-tetrahydrocannabinol (HU210), (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]trans-4-(3-hydroxypropyl)-cyclohexanol (CP55,940) and methanandamide, and by (+)-11-hydroxy-Delta(8)-tetrahydrocannabinol (HU211), a (+)-stereoisomer of HU210. The inhibition was unaffected by cannabinoid CB(1) and CB(2) receptor antagonists. Electrically evoked release of endogenous noradrenaline from sympathetic nerves was inhibited by HU210, but not by HU211. Inhibition was blocked by a cannabinoid CB(1), but not a CB(2), receptor antagonist. HU210 attenuated contractions to noradrenaline, and all of the cannabinoids blocked contractions to KCl. Capsaicin pre-treatment had no significant effect on HU210- and CP55,940-mediated inhibition of sympathetic neurogenic contraction, but partly blocked inhibition mediated by methanandamide. These data show that cannabinoids can inhibit, by distinct pre- and postjunctional actions, sympathetic neurotransmission in the rat mesenteric arterial bed. The pre-junctional action is mediated by a cannabinoid CB(1)-like receptor, but the postjunctional action does not appear to involve either cannabinoid CB(1) or CB(2) receptors.     

Modulation of gastric emptying and gastrointestinal transit in rats through intestinal cannabinoid CB(1) receptors

We studied the delay in gastric emptying and gastrointestinal transit induced by the cannabinoid receptor agonists (+)-WIN 55,212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate) and CP 55,940 ((-)-cis-3[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol), as prevented by the selective cannabinoid CB(1)-receptor antagonist SR141716 ((N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide)) in rats after systemic or central drug administration. Oral SR141716 showed comparable potency (ID(50) range 1.0-3.9 mg/kg) in antagonizing gastric emptying and gastrointestinal transit delay by (+)-WIN 55,212-2 or CP 55,940. Gastric emptying and gastrointestinal transit delay after intracerebroventricular (i.c.v.) (+)-WIN 55,212-2 was prevented by oral or i.c.v. SR141716, but i.c.v. SR141716 did not significantly reduce the effect of i.p. (+)-WIN 55,212-2. Pertussis toxin prevented the delaying action of i.c.v. (+)-WIN 55,212-2 on both gastric emptying and gastrointestinal transit, but had no effect on (+)-WIN 55,212-2 i.p. These findings are consistent with a primary role of peripheral cannabinoid CB(1) receptor mechanisms in gastrointestinal transit delay by specific agonists.     

Description of a Bivalent Cannabinoid Ligand with Hypophagic Properties

A series of bivalent cannabinoid ligands is proposed. The synthesis of double amides based on the rimonabant structure separated by an alkyl chain and the evaluation of their affinities for cannabinoid receptors are reported. The data of 4d confirmed that a bivalent structure is a suitable scaffold for CB₁ cannabinoid receptor binding. The compound 4d was selected for in vitro and in vivo pharmacological evaluations. Moreover, intraperitoneal administration of 4d to food‐deprived rats resulted in a dose‐dependent inhibition of feeding that was maintained up to 240 min.     

Physiological evidence of a postsynaptic inhibition of the tail flick reflex by a cannabinoid receptor agonist

Current evidence indicates that cannabinoids are antinociceptive and this effect is in part mediated by spinal mechanisms. Anatomical studies have localized cannabinoid CB₁ receptors to pre- and postsynaptic sites within the spinal cord. However, behavioural tests have not clearly indicated the relative importance of each of these sites. In this study, the tail flick test was used as a model of acute pain in the rat to determine the site of action of WIN 55,212-2 ((R)-(+)-[2,3-dihydro-5-methyl-3[(4-morpholinyl)methyl]pyrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate), a synthetic cannabinoid receptor agonist. WIN 55,212-2 (3 mg/kg, i.p.) increased the latency of tail withdrawal from a noxious radiant heat source, indicating it is antinociceptive in this model. Using the same paradigm, WIN 55,212-2 was then tested against the synaptically-induced nociceptive hypersensitivity in response to noxious thermal stimulation of the tail (hot water tail immersion). WIN 55,212-2 blocked this hypersensitivity, confirming a spinal site of action of the cannabinoid receptor agonist. Further, WIN 55,212-2 blocked the nociceptive hypersensitivity induced by intrathecal administration of substance P. As substance P acts on postsynaptic tachykinin NK1 receptors in the dorsal horn of the spinal cord, the data are interpreted to suggest that WIN 55,212-2 expressed its anti-hypersensitivity effects at least partially via a postsynaptic site of action; the data do not rule out a presynaptic site of action. This study suggests that cannabinoids may induce analgesia via a postsynaptic site of action in the spinal cord, as well as the possibility that they may interact with substance P signaling.     

Cannabinoid-induced conditioned place preference in the spontaneously hypertensive rat–an animal model of attention deficit hyperactivity disorder

Rationale  Cannabis preparations are the most widely consumed illicit drugs, and their use typically begins in adolescence. The prevalence of cannabis abuse is higher in patients with attention deficit/hyperactivity disorder (ADHD) than in the general population, yet, knowledge about the motivational properties of cannabinoids in animal models of ADHD are lacking. Objective  To compare the motivational effects of the synthetic cannabinoid agonist WIN55,212-2 (WIN) in adolescent and adult spontaneously hypertensive rats (SHR), a validated animal model of ADHD, and Wistar rats, representing a “normal” genetically heterogeneous population. We also asked whether the effects of WIN depended (1) on the activation of the cerebral subtype of cannabinoid receptors, namely, the CB₁ cannabinoid receptor and (2) on putative changes by WIN in blood pressure. Methods  WIN was tested under an unbiased conditioned place preference (CPP) paradigm. Blood pressure after WIN administration was also monitored in additional groups of rats. Results  In the Wistar rats, WIN produced place aversion only in the adult but not adolescent rats. In contrast, WIN produced CPP in both adolescent and adult SHR rats. The behavioral effects of WIN were CB₁-mediated and not related to blood pressure. Conclusion  The contrasting effects of WIN in Wistar and SHR, and the higher resistance of adolescent rats to the aversive and rewarding effects of WIN in these two strains suggests that both adolescence and the ADHD-like profile exhibited by the SHR strain constitute factors that influence the motivational properties of cannabinoids.     

Investigation of endocannabinoid modulation of conditioned responding evoked by a nicotine CS and the Pavlovian stimulus effects of CP 55,940 in adult male rats

Rationale  The cannabinoid CB₁ receptor antagonist/inverse agonist rimonabant (SR 141716) has been shown to block reinforcing and rewarding effects of nicotine. Research has not investigated whether the cannabinoid system is involved in the interoceptive stimulus effects of nicotine functioning as a conditional stimulus (CS). Objective  We examined the effects of rimonabant and the CB_1/2 receptor agonist, CP 55,940, on responding evoked by a nicotine CS in rats. Additionally, we determined whether CP 55,940 functioned as a CS or a Pavlovian positive drug feature Materials and methods  Pavlovian discrimination training involved intermixed nicotine (0.2 mg base/kg) and saline sessions with intermittent access to water only on nicotine. Antagonism tests with rimonabant (0.1-3 mg/kg) and substitution tests with CP 55,940 (0.003–0.1 mg/kg) followed. An effective dose of CP 55,940 was tested against the nicotine generalization curve. A separate group received CS training with CP 55,940 (0.01 mg/kg). Two other groups were trained using CP 55,940 (0.01 or 0.03 mg/kg) as a positive drug feature in which a brief light CS signaled access to water only on CP 55,940 sessions Results  Rimonabant blocked nicotine-evoked responding. CP 55,940 partially substituted for nicotine and enhanced responding to lower nicotine doses. Overall, CP 55,940 did not acquire control of conditioned responding in either Pavlovian drug discrimination task Conclusions  The cannabinoid system was involved in the CS effects of nicotine. This finding is counter to the operant drug discrimination research with nicotine as a discriminative stimulus, warranting further research into this possible dissociation.