Daniela Viganò

Changes in the cannabinoid receptor binding, G protein coupling, and cyclic AMP cascade in the CNS of rats tolerant to and dependent on the synthetic cannabinoid compound CP55,940.

Abstract: Chronic exposure to CP55,940 produced a significant down‐regulation of cannabinoid receptors in the striatum, cortex, hippocampus, and cerebellum of rat brain. At 24 h after SR141716‐precipitated withdrawal, we observed a tendency to return to basal levels in the striatum and cortex, whereas the specific binding remained lower in the hippocampus and cerebellum. When we surveyed cannabinoid receptor‐activated G proteins, in chronic CP55,940‐treated rats the guanosine 5′‐O‐(3‐[35S]thiotriphosphate) ([35S]GTPγS) binding assay revealed a decrease of activated G proteins in the striatum, cortex, and hippocampus, whereas no significant changes were seen in the cerebellum. At 24 h after the SR141716‐precipitated withdrawal, [35S]GTPγS binding increased compared with that of rats chronically exposed to CP55,940, attaining the control level except for cerebellum, where we observed a trend to overcome the control amounts. Concerning the cyclic AMP (cAMP) cascade, which represents the major intracellular signaling pathway activated by cannabinoid receptors, in the cerebral areas from rats chronically exposed to CP55,940 we found alteration in neither cAMP levels nor protein kinase A activity. In the brain regions taken from CP55,940‐withdrawn rats, we only observed a significant up‐regulation in the cerebellum. Our findings suggest that receptor desensitization and down‐regulation are strictly involved in the development of cannabinoid tolerance, whereas alterations in the cAMP cascade in the cerebellum could be relevant in the mediation of the motor component of cannabinoid abstinence.

Relative involvement of cannabinoid CB1 and CB2 receptors in the Δ9-tetrahydrocannabinol-induced inhibition of natural killer activity

We demonstrated that in vivo administration of Delta(9)-tetrahydrocannabinol in mice (15 mg/kg s.c.) significantly inhibited natural killer cell (NK) cytolytic activity without affecting Concanavalin A (ConA)-induced splenocyte proliferation. Moreover, we investigated the effect of in vivo pretreatment with cannabinoid receptor antagonists, namely, the selective cannabinoid CB(1) receptor antagonist SR 141716 [N-piperidin-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-3-pyrazolecarboxamide] and the selective cannabinoid CB(2) receptor antagonist SR 144528 ¿N-[(1S)-endo-1,3, 3-trimethyl bicyclo [2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazo le- 3-carboxamide¿, on Delta(9)-tetrahydrocannabinol-induced inhibition of NK cytolytic activity. Both antagonists partially reversed the Delta(9)-tetrahydrocannabinol inhibition of NK cytolytic activity, although the cannabinoid CB(1) receptor antagonist was more effective than the cannabinoid CB(2) receptor antagonist. The parallel measurement of interferon gamma and interleukin 2 levels revealed that Delta(9)-tetrahydrocannabinol significantly reduced (about 70%) the former cytokine without affecting the latter. Cannabinoid CB(1) and CB(2) receptor antagonists completely reversed the interferon gamma reduction induced by Delta(9)-tetrahydrocannabinol. Our results indicate that both types of cannabinoid receptors are involved in the complex network mediating NK cytolytic activity.

Chronic Δ-9-tetrahydrocannabinol treatment increases cAMP levels and cAMP-dependent protein kinase activity in some rat brain regions

Abstract When Δ9-tetrahydrocannabinol (Δ9-THC,15 mg/kg) was injected intraperitoneally twice a day for 6 days, tolerance to its analgesic effect appeared to be complete. Chronic exposure to Δ9-THC caused a significant reduction in CB1 receptor binding in all brain areas that contain this receptor. Cannabinoid receptor density was markedly reduced in the cerebellum (52%), hippocampus (40%) and globus pallidum (47%) compared to 30% in the cortex and striatum. Chronic exposure enhanced the cAMP pathway, as shown by the significant increase of cAMP levels and PKA activity in the areas with receptor down-regulation (cerebellum, striatum and cortex). We propose that the increase in cAMP cascade is part of the biochemical basis of cannabinoid tolerance.

Long-term treatment with SR141716A, the CB1 receptor antagonist, influences morphine withdrawal syndrome.

The role of the cannabinoid system in morphine withdrawal was examined through long-term CB1 receptor antagonist administration in morphine pellet implanted rats. SR141716A chronic treatment (5mg/kg i.p. twice a day for four days) did not influence the development of tolerance to the morphine analgesic effect but significantly reduced the intensity of naloxone-induced opiate withdrawal in tolerant rats: Specifically there was a significant reduction in the number of digging, teeth chattering and penile licking and the incidence of diarrhoea while other signs such as writhing, head dog shakes and rearing were unaffected. These results suggest that the pharmacological treatment with SR141716A could be of some interest in ameliorating opiate withdrawal syndrome.

In vivo characterization of the specific cannabinoid receptor antagonist, SR141716A: behavioral and cellular responses after acute and chronic treatments.

To characterize the behavioral and biochemical effects of the cannabinoid CB1 antagonist SR141716A, we injected the compound intraperitoneally (ip) at doses from 0.625 mg/kg to 5 mg/kg in rats. SR141716A per se induced a dose‐dependent increase of some behavioral signs such as wet dog and head shakes, forepaw fluttering, grooming, and facial rubbing. When the highest dose of SR141716A (5 mg/kg ip) was injected once a day for four days, tolerance developed to most of the behavioral signs, although with different time courses, except for grooming behavior, which was still significantly different from controls after the fourth injection although reduced by 38% from the first. To characterize the biochemical mechanism underlying these effects, we designed a series of biochemical studies on specific cerebral areas from rats treated with the highest dose of SR141716A (5 mg/kg ip). Thirty minutes after SR141716A injection, cAMP accumulation in the cortex, striatum, hippocampus, mesencephalon, and cerebellum was the same as in controls, whereas protein kinase A (PKA) activity was significantly increased in the hippocampus (65%) and striatum (87%). To explain this difference, we performed a cAMP assay at an early time (10 min) and found a significant increase in the striatum and hippocampus, suggesting that the change in cAMP level is the earliest event in the G protein‐coupled receptor transduction pathway ending in a pharmacological effect after 30 min. When the same assays were done in tolerant animals, no change was seen in either cAMP levels or PKA activity in the brain areas considered. To conclude, we found in vivo that SR141716A acts through activation of the cAMP cascade and our results represent an important point for developing potential therapeutic application for SR141716A. Synapse 35:8–14, 2000.

Loss of Cannabinoid-Stimulated Guanosine 5′-O-(3-[35S]Thiotriphosphate) Binding Without Receptor Down-Regulation in Brain Regions of Anandamide-Tolerant Rats

Abstract: The endogenous cannabinoid anandamide has been reported toproduce well‐defined behavioral tolerance, but studies on the possiblemechanisms underlying this process are few and often contradictory. Thepresent study was designed to survey the cellular events involved inanandamide tolerance, in terms of the effects on receptor number, couplingwith G proteins, and activation of the cyclic AMP (cAMP) cascade. Chronictreatment of rats with anandamide (20 mg/kg i.p. for 15 days) resulted inbehavioral tolerance without any change in cannabinoid receptor binding in thebrain regions studied (striatum, cortex, hippocampus, and cerebellum),suggesting that receptor down‐regulation was not involved in the developmentof anandamide behavioral tolerance. In contrast, prolonged exposure toanandamide significantly reduced agonist‐stimulated guanosine5′‐O‐(3‐[35S]thiotriphosphate) binding in the sameareas, with losses of >50%, suggesting that receptor desensitization may bepart of the molecular mechanism underlying this tolerance. Finally, concerningthe cAMP cascade—the most well‐known intracellular signaling pathwaysactivated by CB1 receptors—in the brain regions from ratstolerant to anandamide, we found no alteration in cAMP levels or in proteinkinase A activity. We propose that anandamide, unlikeΔ9‐tetrahydrocannabinol and other cannabinoids, does notalter the receptor system at multiple levels but that desensitization of theCB1 receptor might account for behavioral tolerance to thedrug.

The psychoactive ingredient of marijuana induces behavioural sensitization.

Here we describe, for the first time, the occurrence of behavioural sensitization after chronic exposure to Δ9‐tetrahydrocannabinol. Rats were treated twice a day, for five days, with increasing doses (5, 10, 20, 40, 40u2003mg/kg i.p.) of Δ9‐tetrahydrocannabinol or its vehicle and after 20u2003days of withdrawal, animals were challenged with 5u2003mg/kg (i.p.) of the drug and their behaviour was assessed. Contrary to the motor inhibition induced in control rats, challenge with Δ9‐tetrahydrocannabinol in pre‐exposed animals elicited a complex behavioural syndrome mainly characterized by oral stereotyped items. Due to the relevance of behavioural sensitization in drug‐seeking behaviour that persists long after discontinuation of drug use, our findings suggest that cannabinoids could trigger neurobiological alteration not dissimilar from those observed with more harmful abused drugs.

Immune function alterations in mice tolerant to Δ9-tetrahydrocannabinol: functional and biochemical parameters

We studied the effect of acute (1 h) or chronic exposure (7 and 14 days) to delta9-tetrahydrocannabinol (delta9-THC) on immune parameters in male Swiss mice. One hour after a dose of 10 mg/kg s.c., the splenocyte proliferative response to ConA and NK activity were not inhibited, but there was a significant decrease in the production of IL-2. After 7 days of treatment, when mice were tolerant to delta9-THC-induced analgesia, these functional parameters were strongly inhibited and there was a persistent reduction in IL-2 and IFNgamma. With 14 days exposure to the drug, splenocyte proliferation was significantly reduced only with 5 microg/ml ConA, and NK activity was still significantly depressed (about 37%). IL-2 had returned to the control value, whereas IFNgamma was still 40% down. Flow cytometry analysis of spleen cell composition indicated no changes after the acute and 7 day treatments, but at 14 days there was a 20% decrease in the number of T lymphocytes, mirrored by a 26% increase of B lymphocytes. In conclusion, in vivo exposure to psychoactive doses of delta9-THC has profound effects on immune function. This implies some important questions in relation to the liberalization of marijuana and its therapeutic uses.

Cellular mechanisms of Δ9-tetrahydrocannabinol behavioural sensitization

We investigated the cellular events linked to the induction of cannabinoid behavioural sensitization. In sensitized rats, autoradiographic binding studies with [3H]CP‐55,940 showed a significant increase in cannabinoid receptor binding, specifically in the cerebellum, with no changes in the other brain areas where basal CB1‐receptor expression is observed. In vitro autoradiography of CP‐55,940‐stimulated [35S]GTPγS binding provided a picture of cannabinoid receptor‐mediated G protein activation. Basal [35S]GTPγS binding was not affected, whereas sensitized rats showed a significant increase of net [35S]GTPγS binding in the caudate putamen and cerebellum. Autoradiographic studies suggested that only these two areas had altered receptor functionality. We therefore focused our intracellular investigations only there, first surveying the responsiveness of the cAMP system to cannabinoids. CP‐55,940 was unable to inhibit forskolin‐induced cAMP accumulation in the cerebellum of sensitized animals, but no difference was observed between groups in the caudate putamen. Finally, we surveyed the levels of CREB and AP‐1 binding activity, in the same two areas and found no difference in sensitized rats. The intracellular picture in sensitized rats suggests that besides the cAMP cascade, other signalling pathways may participate in the development of cannabinoid sensitization.

Intrauterine position has long-term influence on brain μ-opioid receptor density and behaviour in mice

In multiparous rodents, a naturally occurring variation in degree of exposure to sex steroids during the prenatal phase of sexual differentiation derives from the in-utero proximity to opposite sex foetuses. So far, the studies on intrauterine position (IUP) phenomenon have mostly focused on traits relating to reproduction and behaviour, while its influence on neurochemical substrates and pharmacological response has been largely unexplored. We investigated possible variations in the function and the profile of expression of the mu-opioid receptor system in three groups of adult mice from known IUP: 2M mice (located between two males), 0M (between two females), and 1M (between a male and a female). Autoradiographic study revealed in female mice that proximity to at least a male in utero (1M and 2M position) resulted associated at adulthood with an increased density of midbrain mu-opioid receptors. Behavioural observations were conducted following injection with the specific mu-opioid agonist Fentanyl (at 0, 0.01 or 0.05 mg/kg IP). A drug-conditioned place preference test confirmed that 1M and 2M subjects were also more sensitive to the rewarding effects of the drug, since mice spent significantly more time in the drug-paired compartment than 0M subjects. In a hot-plate test, 2M subjects showed levels of drug-induced analgesia that were much higher than other IUP groups. No reliable differences were observed between the IUP groups for locomotor activity upon drug treatment. Overall, these data indicate for the first time that the organisation of the mu-opioid receptor system in the brain, as well as a differential vulnerability to abuse of opiate drugs can be modulated by epigenetic variables such as the prenatal in utero contiguity to male foetuses.