Precipitated cannabinoid withdrawal is reversed by Delta(9)-tetrahydrocannabinol or clonidine.

The availability of the cannabinoid antagonist, SR 141716A, to precipitate withdrawal following repeated cannabinoid administration provides a model to investigate the mechanisms underlying cannabinoid dependence as well as potential treatments to alleviate withdrawal symptoms. The goal of the present study was to determine whether SR 141716A-precipitated withdrawal symptoms in Delta(9)-tetrahydrocannabinol (Delta(9)-THC)-tolerant mice could be alleviated by either readministration of Delta(9)-THC or clonidine, an alpha(2)-receptor agonist. SR 141716A elicited paw tremors in Delta(9)-THC-tolerant mice, but produced a significant increase in head shakes independently of repeated Delta(9)-THC treatment. Readministration of Delta(9)-THC, following SR 141716A-precipitated withdrawal, reversed paw tremors (ED(50)=9.9 mg/kg), but failed to reduce head shaking behavior. Clonidine reversed SR 141716A-precipitated paw tremors (ED(50)=0.18 mg/kg) and blocked head shakes at all doses tested. The reversal effects did not appear to be the result of motor impairment because neither decreases in spontaneous locomotor activity nor motor incoordination, as assessed in the inverted screen test, could account for the effects. These findings suggest that SR 141716A precipitates paw tremors in mice by competing with Delta(9)-THC at the CB(1) receptor, though it also produced head shaking in nondependent animals. Finally, the observation that clonidine alleviated SR 141716A-precipitated paw tremors suggests its potential as a treatment for cannabinoid dependence.     

The pharmacological activity of inhalation exposure to marijuana smoke in mice

Although the majority of cannabinoid users smoke marijuana, the preponderance of laboratory animal research is based on administration of Delta9-tetrahydrocannabinol (Delta9-THC) or other cannabinoid agents via injection. The aim of the present study was to evaluate the impact of inhaling marijuana, or ethanol-extracted placebo smoke in the mouse model of cannabinoid activity by assessing inhibition of spontaneous activity, antinociception, catalepsy, and body temperature. In order to determine dosimetry, blood levels of Delta9-THC were obtained following either marijuana exposure or intravenous injection of Delta(9)-THC. Inhalation exposure to marijuana produced dose-related increases in antinociception and catalepsy, with estimated ED50 doses of Delta9-THC of 2.4 and 3.8 mg/kg, respectively. However, hypothermia and locomotor depression occurred in both the placebo- and marijuana-exposed mice. The CB1 receptor antagonist, SR 141716A antagonized the antinociceptive effects of marijuana (AD50 = 0.6 mg/kg), but only slightly decreased marijuana-induced catalepsy, and failed to alter either the hypothermic or locomotor depressive effects. In contrast, SR 141716A antagonized the antinociceptive, cataleptic, and hypothermic effects of intravenously administered Delta9-THC in mice that were exposed to air alone, though all subjects exhibited locomotor depression, possibly related to the restraint. In accordance with reports of others, these data suggest that exposure to smoke alone has pharmacological consequences. Our findings also indicate that marijuana-induced antinociception is mediated through a CB1-receptor mechanism of action and are consistent with the notion that Delta9-THC is mainly responsible for this effect.     

Reversal of cannabinoids (delta9-THC) by the benzoflavone moiety from methanol extract of Passiflora incarnata Linneaus in mice: a possible therapy for cannabinoid addiction

The newly reported benzoflavone moiety from the plant Passiflora incarnata Linneaus has been evaluated in light of traditional reports on the use of P. incarnata in breaking down cannabis addiction. In the modern or allopathic system of therapeutics, there has been no suitable remedy to combat the severe withdrawal effects of various cannabis products, including marihuana, marijuana, bhang, hashish, ganja, etc., the world-wide consumption of which has attained alarming proportions especially among the younger generation. Mice were given a 10-mg-kg(-1) twice-daily dose of delta9-tetrahydrocannabinol (delta9-THC) by mouth for six days to make them dependent upon cannabinoids. Concurrently, other groups of mice were administered delta9-THC along with a 10- or 20-mg-kg(-1) twice-daily dose of the benzoflavone moiety from P. incarnata orally for 6 days. Upon measuring locomotor activity during the treatment regimen, it was noticed that the mice receiving the P. incarnata extract and delta9-THC together developed significantly less tolerance and dependence, relative to the mice receiving delta9-THC alone. Upon administration of SR-141716A, a selective cannabinoid-receptor antagonist (10 mg kg(-1), p.o.) to all the groups of mice on the 7th day, an artificial withdrawal was produced due to an abrupt decline of delta9-THC levels in mouse brain. However, the typical withdrawal effects like paw tremors and head shakes were significantly less in the mice given delta9-THC+P. incarnata benzoflavone moiety for 6 days. Upon administration of 20 mg kg(-1) of the P. incarnata benzoflavone moiety to mice showing severe symptoms of withdrawal due to administration of SR-141716A, there was a marked attenuation of withdrawal effects, thereby suggesting the usefulness of the benzoflavone moiety in delta9-THC withdrawal. Thus, the benzoflavone moiety of P. incarnata, when administered concurrently with delta9-THC, prevented the development of tolerance and dependence of cannabinoids in mice. Even an acute administration of the benzoflavone moiety (20 mg kg(-1), p.o.) significantly blocked the expression of withdrawal effects in delta9-THC-dependent mice.     

Physiochemical and pharmacological characterization of a Delta(9)-THC aerosol generated by a metered dose inhaler

The goal of the present study was to formulate a Delta(9)-tetrahydrocannabinol (Delta(9)-THC) metered-dose inhaler (MDI) that can be used to provide a systemic dose of Delta(9)-THC via inhalation. Following physiochemical characterization and accelerated stability testing of the aerosol, mice were exposed to the aerosol and evaluated for pharmacological effects indicative of cannabinoid activity, including hypomotilìty, antinociception, catalepsy, and hypothermia. The fine particle dose of Delta(9)-THC was 0.22 +/- 0.03 mg (mean +/- S.D.) or 25% of the emitted dose and was not affected by accelerated stability testing. A 10-min exposure to aerosolized Delta(9)-THC elicited hypomotility, antinociception, catalepsy, and hypothermia. Additionally, Delta(9)-THC concentrations in blood and brain at the antinociceptive ED(50) dose were similar for both inhalation and intravenous routes of administration. Finally, pretreatment with the CB(1) receptor antagonist SR 141716A (10 mg/kg, i.p.) significantly antagonized all of the Delta(9)-THC-induced effects. These results indicate that an MDI is a viable method to deliver a systemic dose of Delta(9)-THC that elicits a full spectrum of cannabinoid pharmacological effects in mice that is mediated via a CB(1) receptor mechanism of action. Further development of a Delta(9)-THC MDI could provide an appropriate delivery device for the therapeutic use of cannabinoids, thereby reducing the need for medicinal marijuana.     

New perspectives in the studies on endocannabinoid and cannabis: abnormal behaviors associate with CB1 cannabinoid receptor and development of therapeutic application

Delta9-tetrahydrocannabinol (Delta9-THC), the major psychoactive component of marijuana, induces catalepsy-like immobilization and impairment of spatial memory in rats. Delta9-THC also induces aggressive behavior in isolated housing stress. These abnormal behaviors could be counteracted by SR141716A, a CB1 cannabinoid receptor antagonist. Also Delta9-THC inhibited release of glutamate in the dorsal hippocampus, but this inhibition could be antagonized by SR141716A in an in vivo microdialysis study. Moreover, NMDA and AMPA-type glutamate receptor enhancers improved the Delta9-THC-induced impairment of spatial memory. On the other hand, Delta9-THC markedly inhibited the neurodegeneration in experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis and reduced the elevated glutamate level of cerebrospinal fluid induced by EAE. These therapeutic effects on EAE were reversed by SR141716A. Taken together, our results demonstrate that the inhibition of glutamate release via activation of the CB1-cannabinoid receptor is one mechanism involved in Delta9-THC-induced impairment of spatial memory, and the therapeutic effect of Delta9-THC on EAE, and a Delta9-THC analog might provide an effective treatment for psychosis and neurodegenerative diseases.     

Lack of evidence for appetitive effects of Delta 9-tetrahydrocannabinol in the intracranial self-stimulation and conditioned place preference procedures in rodents

Data on the ability of Delta 9-tetrahydrocannabinol (THC) to modify reward processes in experimental animals are inconsistent. This study examined the effects of Delta 9-THC on brain reward function using the rate-frequency curve shift paradigm of intracranial self-stimulation (ICSS) and the conditioned place preference (CPP) paradigm. In ICSS tests, rats were implanted with electrodes into the medial forebrain bundle. After brain stimulation reward thresholds stabilized, rats received intraperitoneal injections of Delta 9-THC (0, 0.5, 1 and 2 mg/kg) or the CB1 receptor antagonist SR141716A (0, 0.02 mg/kg) and Delta 9-THC (0, 2 mg/kg). The two highest doses of Delta 9-THC significantly increased the threshold ICSS frequency. SR141716A reversed the action of Delta 9-THC (2 mg/kg), without affecting reward thresholds by itself. In the CPP test, mice received intraperitoneal injections of Delta 9-THC (0, 1 or 3 mg/kg). Delta 9-THC showed neither statistically significant preference nor aversion in either of the doses tested. These findings indicate that Delta 9-THC, in contrast to other drugs of abuse, does not facilitate ICSS or support CPP under the present experimental conditions, but rather has a dose-dependent inhibitory influence on ICSS.     

Immunomodulation by cannabinoids is absent in mice deficient for the cannabinoid CB(2) receptor

The reemergence on the debate of the use of marijuana for medicinal purposes has been the impetus for developing an acceptable delivery form of aerosolized cannabinoids. The goals of the present study were to: (1) develop and characterize the physical properties of an aerosolized form of Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the major psychoactive constituent present in marijuana; and (2) assess the pharmacological effects of cannabinoid inhalation in mice. A Small Particle Aerosol Generator (SPAG) nebulizer, used to generate the aerosol, had an output of approximately 0.154 mg/l of aerosolized Delta(9)-THC with a 2.0 microm mass median aerodynamic diameter and a 2.2 geometric standard deviation (GSD). Virtually all the particles were less than 5.0 microm in diameter suggesting that they were sufficiently small to penetrate deeply into the lungs. Inhalation exposure to aerosolized Delta(9)-THC in mice elicited antinociceptive effects that were dependent on concentration and exposure time with an estimated Delta(9)-THC dose of 1.8 mg/kg. On the other hand, inhalation exposure to Delta(9)-THC failed to produce two other indices indicative of cannabinoid activity, hypothermia and decreases in spontaneous locomotor activity. The antinociceptive effects occurred within 5 min of exposure and lasted approximately 40 min in duration. The cannabinoid receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide HCl (SR 141716A), but not naloxone, blocked these antinociceptive effects (AD(50)=0.09 mg/kg) indicating a cannabinoid receptor mechanism of action. Similarly, inhalation exposure to a water soluble cannabinoid analog, 3-(5'-cyano-1', 1'dimethylheptyl)-1-(4-N-morpholinobutyrloxy)-Delta(8)-te trahydrocann abinol (O-1057), produced antinociception that was blocked by SR 141716A. These results demonstrate that the development of an aerosolized form of cannabinoids for human medicinal use is feasible.     

Reversal of SR 141716A-induced head-twitch and ear-scratch responses in mice by delta 9-THC and other cannabinoids.

Recently, we have shown that cannabinoids of diverse structure block the ability of the selective 5-HT(2A/C) agonist DOI to produce the head-twitch response (HTR) and the ear-scratch response (ESR) in mice. The cannabinoid CB(1) antagonist/inverse agonist SR 141716A also induces these behaviors in mice. The purposes of the present study were: (1) to investigate whether Delta(9)-tetrahydrocannabinol (Delta(9)-THC) and other cannabinoids HU-210 and WIN 55, 212-2 can prevent SR 141716A-induced HTR and ESR and (2) to evaluate any correlation between the ID(50) potency order of the cited cannabinoids in blocking SR 141716A-induced HTR and ESR and their ED(50) order of potency in reducing spontaneous locomotor activity and rearing behavior. For the SR 141716A reversal study, different groups of mice were injected intraperitoneally with either vehicle or varying doses of the following cannabinoids: Delta(9)-THC (2.5-20 mg/kg), Delta(8)-THC (5-20 mg/kg), HU-210 (0.05-0.5 mg/kg), CP 55, 940 (0.5-2.5 mg/kg) and WIN 55, 212-2 (2.5-10 mg/kg). Thirty minutes later, each mouse received SR 141716A (2.5 mg/kg ip) and the frequencies of the induced behaviors (mean +/- S.E.M.) were recorded for the next 30 min. The effects of the cited doses of cannabinoids were also examined on spontaneous locomotor activity and rearing frequency for a 20-min duration 10 min after cannabinoid injection. The tested cannabinoids reduced the frequencies of HTR and ESR in SR 141716A-injected mice. These agents also attenuated the cited naturally occurring repertoire of motor parameters in mice. Although large potency differences were observed among the cited cannabinoids, each tested cannabinoid was relatively equipotent in preventing locomotor parameters and SR 141716A-induced behaviors. The ID(50) potency order of cannabinoids in blocking SR 141716A-induced HTR and ESR were similar (HU-210>CP 55, 940>WIN 55, 212-2 > or = Delta 9)-THC=Delta(8)-THC), and are comparable with: (1) their ED(50) potency order in attenuating both spontaneous locomotor activity and rearing behavior (HU-210>CP 55, 940>WIN 55, 212-2>Delta(9)-THC=Delta(8)-THC) and (2) their published ED(50) potency order for producing the tetrad of behaviors in mice as well as their rank order of binding affinities for cannabinoid CB(1) receptors. The present data show that cannabinoids of diverse structure prevent SR 141716A-induced HTR and ESR, and inhibition of these behaviors by cannabinoids could be used as a new index of cannabimimetic activity.     

Effects of Delta 9-tetrahydrocannabinol, (R)-methanandamide, SR 141716,and d-amphetamine before and during daily Delta 9-tetrahydrocannabinol dosing

We examined the effects of Delta 9-tetrahydrocannabinol (Delta 9-THC), (R)-(+)-arachidonyl-1'-hydroxy-2'-propylamide ((R)-methanandamide, AM 356), SR 141716, and d-amphetamine on fixed-ratio (FR) responding maintained by food in rats before and during daily dosing with Delta 9-THC. Rats responded under a FR 10 schedule of food reinforcement. Cumulative dose-response curves for the various drugs were determined before and during daily Delta 9-THC administration. All four drugs dose-dependently decreased responding both before and during daily dosing with Delta 9-THC (18 mg/kg/day). The dose-response curves for both Delta 9-THC and (R)-methanandamide were shifted to the right with daily dosing with Delta 9-THC, indicating tolerance to the effects of Delta 9-THC and cross-tolerance to the effects of (R)-methanandamide. The doses of d-amphetamine examined produced similar effects both before and during daily dosing with Delta 9-THC. The effects of SR 141716 were not consistently altered by daily Delta 9-THC administration. These results indicate that tolerance develops to the effects of Delta 9-THC, when Delta 9-THC is administered repeatedly. These results also indicate that cross-tolerance to (R)-methanandamide develops with repeated Delta 9-THC administration.     

Dependence of mesolimbic dopamine transmission on delta9-tetrahydrocannabinol.

Rats were administered daily for 8 days with increasing doses (2-12 mg/kg/day) of delta9-tetrahydrocannabinol (delta9-THC) and than challenged with different doses of SR141716A, an antagonist of cannabinoid receptors. SR141716A dose dependently reduced dialysate dopamine (DA) in the nucleus accumbens shell and precipitated a physical withdrawal syndrome. No such effects were obtained after administration of SR141716A to saline controls.     

Intravenous self-administration of the cannabinoid CB1 receptor agonist WIN 55,212-2 in rats

RATIONALE: Delta9-tetrahydrocannabinol (Delta9-THC), the main psychoactive ingredient of marijuana, as well as synthetic cannabinoid (CB1) receptor agonists, has led to negative or equivocal results when tested with the intravenous self-administration procedure, the best validated behavioural model for evaluating abuse liability of drugs in experimental animals. We recently reported, however, that the synthetic CB1 receptor agonist WIN 55,212-2 is intravenously self-administered by drug-naive mice and that its self-administration is blocked by the cannabinoid CB1 receptor antagonist SR 141716A. OBJECTIVE: To assess a reliable model of cannabinoid intravenous self-administration in rats. Long Evans male rats were allowed the opportunity to self-administer WIN 55,212-2 at doses ranging from 6.25 to 50 microg/kg per injection, under a fixed-ratio 1 (FR1) schedule of reinforcement and nose-pokes as the operant responses. The effect of either a change in the unit drug dose available or a pretreatment with the specific CB1 receptor antagonist SR 141716A were then investigated (maintenance phase). Finally, the extinction of the self-administration behaviour was evaluated. RESULTS: Response rate depended on the drug dose available, with maximum rates occurring at 12.5 microg/kg per injection. Response rate increased following pretreatment with the specific CB1 receptor antagonist, SR 141716A. Moreover, operant behaviour rapidly extinguished following both the substitution of saline or vehicle for cannabinoid and the disconnection of the drug delivery pumps. CONCLUSION: Rats will intravenously self-administer the synthetic CB1 receptor agonist WIN 55,212-2 under specific experimental conditions, thus allowing further investigation of the neurobiological mechanisms underlying cannabinoid-taking behaviour.     

Effects of the cannabinoid CB(1) receptor antagonist, SR141716A, after Delta(9)-tetrahydrocannabinol withdrawal

Rats were trained to lever press according to variable interval 10 s schedules during daily experimental sessions composed of six 3 min food reinforcement periods and were treated twice daily for 6 days with either vehicle or escalating regimens of Delta(9)-tetrahydrocannabinol. On days 7 and 8, the rats were challenged with vehicle and cumulative doses of SR141716A (N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4, -dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxyamide hydrochloride), a cannabinoid CB(1) receptor antagonist, up to 3 and 9 mg/kg, respectively. Response rates increased during Delta(9)-tetrahydrocannabinol withdrawal and towards those of the vehicle treatment group suggesting a waning of the direct effects of Delta(9)-tetrahydrocannabinol. SR141716A reduced response rates but only in rats pre-treated with Delta(9)-tetrahydrocannabinol. These data suggest that dependence upon Delta(9)-tetrahydrocannabinol was induced and SR141716A precipitated withdrawal.     

Cannabinoids prevent the acute hyperthermia and partially protect against the 5-HT depleting effects of MDMA ("Ecstasy") in rats

Cannabinoid-MDMA interactions were examined in male Wistar rats. MDMA (4 x 5 mg/kg or 2 x 10 mg/kg over 4 h on each of 2 days) was administered with or without Delta 9-tetrahydrocannabinol (THC) (4 x 2.5 mg/kg), the synthetic cannabinoid receptor agonist CP 55,940 (2 x 0.1 or 0.2 mg/kg) or the cannabinoid receptor antagonist SR 141716 (2 x 5 mg/kg). Co-administered Delta 9-THC and CP 55,940 but not SR 141716 prevented MDMA-induced hyperthermia, causing a powerful hypothermia. Co-administered Delta 9-THC, CP 55,940 and SR 141716 all tended to decrease MDMA-induced hyperactivity. Co-administered Delta 9-THC provided protection against the long-term increases in anxiety seen in the emergence test, but not the social interaction test, 6 weeks after MDMA treatment. Co-administered Delta 9-THC and CP 55,940, but not SR 141716, partly prevented the long-term 5-HT and 5-HIAA depletion caused by MDMA in various brain regions. SR 141716 administered with CP 55,940 and MDMA prevented the hypothermic response to the CP 55,940/MDMA combination but did not alter the CP 55,940 attenuation of MDMA-induced 5-HT depletion. These results suggest a partial protective effect of co-administered cannabinoid receptor agonists on MDMA-induced 5-HT depletion and long-term anxiety. This action appears to operate independently of cannabinoid CB1 receptors.     

Central mediation of the cannabinoid cue: activity of a selective CB1 antagonist, SR 141716A

Active cannabimimetic drugs are known to bind to two receptor subtypes: one, called CB1, is mainly localised in the central nervous system while the other (CB2) is expressed preferentially in the immune system. SR 141716A has been demonstrated to have a nanomolar affinity for CB1 receptor subtypes and a micromolar affinity for CB2 receptors. Moreover, it is an effective antagonist at these receptors both in vitro (antagonism of cannabinoid activity in vas deferens) and in vivo (suppression of the hypothermia elicited by WIN 55,212-2). The present experiments were thus undertaken to investigate the role of CB1 receptors in cannabinoid discrimination. Rats were trained to discriminate WIN 55,212-2 (0.3mg/kg s.c.) from saline in a standard operant (FR10) food rewarded discrimination procedure. Acquisition of the discrimination required 16 days on average and the ED(50) of WIN 55,212-2 was 0.032mg/kg s.c. CP55,940 and delta-9-tetrahydrocannabinol (Delta(9)-THC) generalised to the WIN 55,212-2 stimulus with the respective ED(50)s of 0.007mg/kg (s.c.) and 0.64mg/kg (p.o.). Pretreatment with SR 141716A antagonised the cue elicited by WIN 55,212-2 (ED(50) = 1.6mg/kg) as well as the generalisation to CP 55,940 (ED(50) = 0.08mg/kg) and to Delta(9)-THC (ED(50) = 0.15mg/kg). SR 140098 is a CB1 antagonist as potent as SR 141716A in vitro. This compound is unlikely to pass into the brain since it failed to displace [(3)H]-CP55, 940 from rat brain membranes ex vivo, and to reverse WIN 55,212-2-induced hypothermia. SR 140098, in contrast to SR 141716A, did not antagonise the WIN 55,212-2 stimulus. Taken together, the present results demonstrate that the brain CB1 receptor subtype mediates the cannabinoid cue.     

Role of different brain structures in the behavioural expression of WIN 55,212-2 withdrawal in mice

We have evaluated several responses induced by the cannabinoid agonist WIN 55,212-2 related to its addictive properties, including rewarding effects and the development of physical dependence in mice. Moreover, we have studied the specific involvement of several brain regions with high density of CB1 cannabinoid receptors, such as striatum, hippocampus, amygdala and cerebellum, in the behavioural expression of SR 141716A-precipitated WIN 55,212-2 withdrawal. The systemic administration of the CB1 receptor antagonist SR 141716A (10 mg kg(-1), s.c.) precipitated behavioural signs of withdrawal in mice chronically treated with WIN 55,212-2 (1 and 2 mg kg(-1), intraperitoneal (i.p.)), revealing the development of physical dependence. The microinjection of SR 141716A (1.5 and 3 micrograms) into the cerebellum induced severe manifestations of abstinence in mice dependent on WIN 55,212-2 (1 mg kg(-1), i.p.). Out of 10 signs evaluated, seven were statistically significant: wet dog shakes, body tremor, paw tremor, piloerection, mastication, genital licks and sniffing. When the cannabinoid antagonist was administered into the hippocampus and the amygdala, a moderate but significant withdrawal syndrome was also observed. However, no signs of abstinence were induced when SR 141716A was microinjected into the striatum. WIN 55,212-2 produced rewarding effects in the place-conditioning paradigm in mice pre-exposed to a priming injection of the drug. These results show a reliable behavioural model to reveal rewarding effects and physical dependence induced by the repeated administration of WIN 55,212-2 in mice. The cerebellum and to a lesser extent the hippocampus and the amygdala participate in the behavioural expression of cannabinoid withdrawal.     

Marijuana smoke and Delta(9)-tetrahydrocannabinol promote necrotic cell death but inhibit Fas-mediated apoptosis

Marijuana smoke shares many components in common with tobacco smoke except for the presence of Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the psychotropic compound found only in Cannibis sativa. Delta(9)-THC has been shown to potentiate smoke-induced oxidative stress and necrotic cell death. In the present study, our objective was to determine the effects of Delta(9)-THC on the balance between Fas-induced apoptosis and necrosis in A549 lung tumor cells. We found that Fas-induced activation of caspase-3 was inhibited by whole smoke from both tobacco and marijuana cigarettes. Gas-phase smoke, which generates high levels of intracellular reactive oxygen species, had no effect on caspase-3 activity. However, particulate-phase smoke (tar) was a potent inhibitor of Fas-induced caspase-3 activity, with marijuana tar being more potent than either tobacco or placebo marijuana tar (lacking Delta(9)-THC). Delta(9)-THC also inhibited Fas-induced caspase-3 activity in A549 cells. In contrast, no inhibition was observed when Delta(9)-THC was incubated with activated caspase-3 enzyme, suggesting that Delta(9)-THC acts on the cell pathway(s) leading to caspase-3 activation and not directly on enzyme function. Flow cytometry was used to measure the percentage of cells undergoing apoptosis (staining for annexin V) versus necrosis (staining for propidium iodide) and confirmed that both marijuana tar extract and synthetic Delta(9)-THC inhibit Fas-induced apoptosis while promoting necrosis. These observations suggest that the Delta(9)-THC contained in marijuana smoke disrupts elements of the apoptotic pathway, thereby shifting the balance between apoptotic and necrotic cell death. This shift may affect both the carcinogenic and immunologic consequences of marijuana smoke exposure.     

Discriminative stimulus effects of the cannabinoid antagonist, SR 141716A, in delta -sup-9-tetrahydrocannabinol-treated rhesus monkeys

This study examined whether the cannabinoid antagonist, SR 141716A, could be established as a discriminative stimulus in rhesus monkeys treated with delta -sup-9-tetrahydrocannabinol (delta -sup-9-THC). Stimulus control was established with SR 141716A (1.0 mg/kg) in 3 delta -sup-9-THC-treated monkeys (1.12 mg/kg/day) in 113-124 sessions. The SR 141716A discriminative stimulus was dose related, attenuated by an acute injection of delta -sup-9-THC, and not mimicked by cocaine or ketamine. SR 141716A-appropriate responding occasioned by temporary discontinuation of delta -sup-9-THC treatment was attenuated by delta -sup-9-THC and not ketamine. The SR 141716A discriminative stimulus in delta -sup-9-THC-treated monkeys appears to be mediated by cannabinoid receptors and could be related to delta -sup-9-THC withdrawal.     

Discriminative stimulus effects of the cannabinoid CB1 antagonist SR 141716A in rhesus monkeys pretreated with Δ9-tetrahydrocannabinol

Rationale Drug discrimination can be used to examine tolerance and dependence in agonist-treated animals by establishing an appropriate antagonist as a discriminative stimulus.Objective Establish intravenous SR 141716A as a discriminative stimulus in four rhesus monkeys pretreated with a relatively small dose of Δ⁹-tetrahydrocannabinol (Δ⁹-THC).Methods Rhesus monkeys received i.v. Δ⁹-THC (0.32 mg/kg) and discriminated i.v. SR 141716A (1 mg/kg) from vehicle while responding under a fixed ratio (FR) 5 schedule of stimulus-shock termination.Results The discriminative stimulus effects of SR 141716A were dose-dependent (ED₅₀=0.33 mg/kg) and were mimicked by the CB₁ antagonist AM 251 (ED₅₀=0.98 mg/kg), but not by a benzodiazepine (midazolam) or an N-methyl-D-aspartate antagonist (ketamine). An additional dose (0.32 mg/kg in addition to 0.32 mg/kg administered before the session) of Δ⁹-THC shifted the SR 141716A dose–effect curve 3-fold rightward. Omitting Δ⁹-THC before test sessions resulted in responding on the SR 141716A lever that was attenuated by subsequent administration of Δ⁹-THC (ED₅₀=0.13 mg/kg), CP 55940 (ED₅₀=0.013 mg/kg), and WIN 55212-2 (ED₅₀=0.35 mg/kg); midazolam and ketamine did not attenuate responding on the SR 141716A lever. SR 141716A (1 mg/kg) shifted the Δ⁹-THC and CP 55940 dose–effect curves 3.4-fold rightward; the WIN 55212-2 dose–effect curve was not significantly modified by a dose of 1 mg/kg of SR 141716A.Conclusions SR 141716A can be established as a discriminative stimulus in animals pretreated with Δ⁹-THC, and this assay is selective for cannabinoid activity. Differential antagonism of cannabinoids by SR 141716A might indicate that the mechanism of action of WIN 55212-2 is not identical to other cannabinoids. This study demonstrates that, under the appropriate conditions, drug discrimination has utility for examining cannabinoid dependence and withdrawal.     

Behavioral and molecular changes elicited by acute administration of SR141716 to Delta9-tetrahydrocannabinol-tolerant rats: an experimental model of cannabinoid abstinence

Whether chronic cannabinoid consumption produces a dependent state comparable to that occurring with other drugs (e.g. the appearance of withdrawal signs when consumption is interrupted), and whether chronic cannabinoid consumption increases the risk of consuming other drugs of greater addictive power, are probably the two questions relating to cannabinoid addiction that provoke the most controversy. The present study was designed to further explore these two questions in laboratory animals. Firstly, we examined the effects of an acute challenge with SR141716 (an antagonist for the cannabinoid CB(1) receptor) in Delta(9)-tetrahydrocannabinol (Delta(9)-THC)-tolerant rats. This antagonist has been reported to precipitate a cannabinoid withdrawal syndrome. Thus, the administration of SR141716 to Delta(9)-THC-tolerant rats reduced inactivity in the open-field test and enhanced responses as tremor, turning and retropulsion-these responses that were only slightly enhanced in control rats. The administration of SR141716 increased the plasma prolactin and the corticosterone concentration in controls, but these increases were much lesser in Delta(9)-THC-tolerant rats. In addition, CRF-mRNA levels in the paraventricular hypothalamic nucleus, while reduced in SR141716-treated controls, were significantly increased in Delta(9)-THC-tolerant rats. The analysis of endocannabinoids also revealed that the administration of SR141716, which was mostly inactive in control rats, was able to reverse the changes in anandamide or 2-arachidonoylglycerol concentrations found in Delta(9)-THC-tolerant rats, in the striatum, limbic forebrain, diencephalon, cerebellum and brainstem, but not in the midbrain and hippocampus. As a second objective, we evaluated whether Delta(9)-THC-tolerant rats were more vulnerable to morphine in a self-administration paradigm. The Delta(9)-THC-tolerant and control rats self-administered morphine to a similar extent, in concordance with the similar values of dopaminergic activity in limbic and motor regions. In summary, our data indicate that Delta(9)-THC-tolerant rats were not more vulnerable to the reinforcing properties of morphine. However, they responded to the blockade of CB(1) receptors by exhibiting slightly but possibly relevant differences in behavioral, endocrine and molecular parameters compared to the response in non-tolerant rats. This is indicative of the existence of a withdrawal syndrome in cannabinoid-tolerant rats that is mild compared with abstinence in opioid-dependent rats.     

Tolerance to the disruptive effects of Delta(9)-THC on learning in rats

Tolerance to the effects of the cannabinoid agonist Delta(9)-tetrahydrocannabinol (Delta(9)-THC) was characterized in rats responding under a multiple schedule of repeated acquisition and performance. During the acquisition component, subjects acquired a different three-response sequence each session, whereas in the performance component the sequence was the same each session. Responding was maintained under a second-order fixed-ratio 2 (FR2) schedule of food reinforcement. Acute doses of Delta(9)-THC (1-10 mg/kg) decreased rate and accuracy in both components, whereas doses of the cannabinoid (CB1) receptor antagonist N-(piperidin-1-yn-)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A; 0.32 and 1 mg/kg) were ineffective. While 5.6 mg/kg of Delta(9)-THC disrupted responding when administered acutely, tolerance to the rate-decreasing and error-increasing effects of this dose developed in both components after daily administration. When 1 mg/kg of SR141716A was substituted for Delta(9)-THC during chronic administration, this previously ineffective dose selectively increased within-session errors in the acquisition component of the multiple schedule. During the postchronic phase, subjects were generally less sensitive to the disruptive effects of Delta(9)-THC. In summary, these data demonstrated that tolerance to Delta(9)-THC developed across two different behavioral tasks and that learning was generally more sensitive than performance to the effects of SR141716A during chronic treatment with Delta(9)-THC.