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Rhabdomyolysis has been reported in patients who abuse synthetic cannabinoids. However, no studies have yet assessed whether these cases reflect the direct cytotoxicity of synthetic cannabinoids on skeletal muscle, a possibility that the present study sought to address. Specifically, this study investigated the cytotoxicity of the synthetic cannabinoid CP-55,940, a compound that acts equally on both types of cannabinoid receptors (CB1 and CB2), in a human embryonic rhabdomyosarcoma (RD) cell line. Exposure of these cells to CP-55,940 resulted in concentration-dependent decreases in cell viability. These effects were attenuated by pre-incubation with AM251 (30 µM), a selective CB1 receptor antagonist, but not by pre-incubation with AM630 (30 µM), a selective CB2 receptor antagonist. Following treatment with CP-55,940, RD cells exhibited apoptosis, as indicated by the accumulation of annexin-V, activation of caspase-3, and a loss of the mitochondrial membrane potential. Additionally, CP-55,940 treatment of RD cells led to increases in intracellular Ca2+ levels. CP-55,940-induced cell death was significantly attenuated in the absence of extracellular Ca2+, and was partially decreased by pre-incubation with verapamil (5 µM) or diltiazem (5 µM), compounds that block the l-type Ca2+ channel. Our results indicate that the cytotoxicity of CP-55,940 towards RD cells (skeletal muscle cells) is mediated by the CB1 receptor, but not by the CB2 receptor. Our results further suggest that calcium influx through the l-type channel may play an important role in the apoptosis induced by these compounds.

Cannabinoids, including phytocannabinoids, synthetic cannabinoids and endogenous cannabinoids, can be neuroprotective, rewarding or aversive. The aversive effects of cannabinoids may hinder their medical and recreational applications. It is unknown which type of cannabinoid (CB) receptors mediates the direct aversive effects of synthetic cannabinoid CP 55,940 which is an analog of Δ9-THC (Δ9-tetrahydrocannabinol), the major psychoactive component of marijuana. In this study, we address this question by taking advantage of systematic type 1 CB receptor (CB1R) knockout mice and conditional reinstatement of this receptor only in astrocytes. We show that CP 55,940 at a concentration of 1 mg/kg induces conditioned place aversion (CPA) and the CPA effect of CP 55,940 is mediated by astroglial CB1Rs. Inhibiting cyclooxygenase-2 (COX-2) eliminates CP 55,940-induced CPA in mice that only express CB1Rs in astrocytes. These findings conclude that CPA effect of CP 55,940 is mediated by astroglial CB1 cannabinoid receptors through COX-2 signaling, suggesting that selective COX-2 inhibition or precise isolation of astroglial CB1Rs activity may be the strategy for treating aversive response of medical and recreational administrations of marijuana.

Pavlovian conditioned approach paradigms are used to characterize the nature of motivational behaviors in response to stimuli as either directed toward the cue (i.e., sign-tracking) or the site of reward delivery (i.e., goal-tracking). Recent evidence has shown that activity of the endocannabinoid system increases dopaminergic activity in the mesocorticolimbic system, and other studies have shown that sign-tracking behaviors are dependent on dopamine. Therefore, we hypothesized that administration of a cannabinoid agonist would increase sign-tracking and decrease goal-tracking behaviors. Forty-seven adult male Sprague Dawley rats were given a low, medium, or high dose of the cannabinoid agonist CP-55,940 (N=12 per group) or saline (N=11) before Pavlovian conditioned approach training. A separate group of rats (N=32) were sacrificed after PCA training for measurement of cannabinoid receptor type 1 (CB1) and fatty acid amide hydrolase (FAAH) using in situ hybridization. Contrary to our initial hypothesis, CP-55,940 dose-dependently decreased sign-tracking and increased goal-tracking behavior. CB1 expression was higher in sign-trackers compared to goal-trackers in the prelimbic cortex, but there were no significant differences in CB1 or FAAH expression in the infralimbic cortex, dCA1, dCA3, dorsal dentate gyrus, or amygdala. These results demonstrate that cannabinoid signaling can specifically influence behavioral biases toward sign- or goal-tracking. Pre-existing differences in CB1 expression patterns, particularly in the prelimbic cortex, could contribute to individual differences in the tendency to attribute incentive salience to reward cues.

Previous work has shown that cannabinoids increase feeding, while cholecystokinin (CCK) has an anorexigenic effect on food intake. Receptors for these hormones are located on cell bodies of vagal afferent nerves in the nodose ganglia. An interaction between CCK and endocannabinoid receptors has been suggested. The purpose of these studies is to explore the effect of pretreatment with a cannabinoid agonist, CP 55,940, on nodose neuron activation by CCK. To determine the effect of CP 55,940 and CCK on neuron activation, rats were anesthetized and nodose ganglia were excised. The neurons were dissociated and placed in culture on coverslips. The cells were treated with media; CP 55,940; CCK; CP 55,940 followed by CCK; or AM 251, a CB1 receptor antagonist, and CP 55,940 followed by CCK. Immunohistochemistry was performed to stain the cells for cFos as a measure of cell activation. Neurons were identified using neurofilament immunoreactivity. The neurons on each slip were counted using fluorescence imaging, and the number of neurons that were cFos positive was counted in order to calculate the percentage of activated neurons per coverslip. Pretreatment with CP 55,940 decreased the percentage of neurons expressing cFos‐immunoreactivity in response to CCK. This observation suggests that cannabinoids inhibit CCK activation of nodose ganglion neurons.

Relative to Δ9-tetrahydrocannabinol (THC), the synthetic cannabinoid CP 55,940 (CP) is significantly more potent and efficacious at cannabinoid receptors, the primary targets for endogenous cannabinoids (eCBs). eCBs belong to a large, interconnected lipidome of bioactive signaling molecules with a myriad of effects in optimal and pathological function. Recreational use of highly potent and efficacious synthetic cannabinoids is common amongst adolescents, potentially impacting brain development. Knowledge of the molecular outcomes of synthetic cannabinoid use will be important to develop more targeted therapies for synthetic cannabinoid intoxication and to prevent long-term disruption to the CNS. Here, we test the hypothesis that CP has age and region-dependent effects on the brain lipidome. Adolescent [post-natal day (PND) 35 and PND 50] and young adult female mice were given either an acute dose of CP or vehicle and brains were collected 2 h later. Eight brain regions were dissected and levels of ∼80 lipids were screened from each region using HPLC/MS/MS. CP had widespread effects on the brain lipidome in all age groups. Interestingly, more changes were observed in the PND 35 mice and more were reductions in a lipid's concentration, including region-dependent lowering of eCB levels. CP levels were highest in the cortex at PND 35, the hippocampus at PND 50, and in the cerebellum in the adult. These data provide novel insights into how high-potency, synthetic cannabinoids drive different, age-dependent, cellular signaling effects in the brain.

Studies on cannabinoids have reported contradictory findings, showing both aversion and rewarding outcomes in conditioned place preference (CPP). Various possibilities have been suggested to explain the aversive properties of cannabinoids, including the pharmacokinetics profile and dose selection. In this study, we have established a CPP method to investigate the effects of modulating astroglial glutamate transporters in cannabinoid dependence using a cannabinoid receptor 1 (CB1R) agonist, CP 55,940 (CP). Previous reports using CPP paradigm demonstrated the involvement of glutamatergic system in seeking behavior of several drugs of abuse such as cocaine, heroin and nicotine. Glutamate homeostasis is maintained by several astroglial glutamate transporters, such as glutamate transporter 1 (GLT-1), cystine/glutamate transporter (xCT) and glutamate aspartate transporter (GLAST). In this study, we investigated the effects of Ampicillin/Sulbactam, β-lactam compounds known to upregulate GLT-1 and xCT, on cannabinoid seeking behavior using CP. We found first that one prime dose of CP induced CP reinstatement; this effect was associated, in part, with significant downregulation of xCT expression in the nucleus accumbens, dorsomedial prefrontal cortex and amygdala. Moreover, GLT-1 expression was downregulated in the amygdala. Importantly, Ampicillin/Sulbactam treatment during the extinction phase attenuated CP-induced reinstatement and restored the expression of GLT-1 and xCT in mesocorticolimbic brain regions. These findings suggest that β-lactams may play a potential therapeutic role in attenuating dependence to cannabinoids, in part, through upregulation of GLT-1 and xCT.

BACKGROUND: Cannabinoids have shown promise for the treatment of intractable pain states and may represent an alternative pharmacotherapy for pain management. A growing body of clinical evidence suggests a role for sex in pain perception and in cannabinoid response. We examined cannabinoid sensitivity and tolerance in male and female mice expressing a desensitization-resistant form (S426A/S430A) of the cannabinoid type 1 (CB(1)) receptor. MATERIALS AND METHODS: Mice were assessed for acute and inflammatory nociceptive behaviors in the formalin test following pretreatment with either vehicle or mixed CB(1)/CB(2) receptor agonists, Δ(9)-THC (1–6 mg/kg) or CP55,940 (0.06–0.2 mg/kg). Tolerance to the effects of 6 mg/kg Δ(9)-THC or 0.1 mg/kg CP55,940 was examined via the formalin test following chronic daily dosing. RESULTS: Female mice showed decreased sensitivity to the effects of Δ(9)-THC and CP55,940 compared to male controls. The S426A/S430A mutation increased the attenuation of nociceptive behaviors for both agonists in both sexes. Female mice displayed delayed tolerance to Δ(9)-THC compared to male mice while the S426A/S430A mutation conferred a delay in tolerance to Δ(9)-THC in both sexes. Male S426A/S430A mutant mice also display resistance to tolerance to CP55,940 compared to wild-type controls. CONCLUSION: This study demonstrates sex and genotype differences in response for two different cannabinoid agonists. The results underscore the importance of including both male and female subjects in pre-clinical studies of pain and cannabinoid pharmacology.

Cannabinoid receptor (CBs) agonists affect the growth of tumor cells via activation of deadly cascades. The spectrum of action of these agents and the precise role of the endocannabinoid system (ECS) on oncogenic processes remain elusive. Herein we compared the effects of synthetic (CP 55-940 and WIN 55,212-2) and endogenous (anandamide or AEA) CBs agonists (10-20 μM) on morphological changes, cell viability, and induction of apoptosis in primary astrocytes and in two glioblastoma cell lines (C6 and U373 cells) in order to characterize their possible differential actions on brain tumor cells. None of the CBs agonist tested induced changes in cell viability or morphology in primary astrocytes. In contrast, CP 55-940 significantly decreased cell viability in C6 and U373 cells at 5 days of treatment, whereas AEA and WIN 55,212-2 moderately decreased cell viability in both cell lines. Treatment of U373 and C6 for 3 and 5 days with AEA or WIN 55,212-2 produced discrete morphological changes in cell bodies, whereas the exposure to CP 55-940 induced soma degradation. CP 55-940 also induced apoptosis in both C6 and U373 cell lines. Our results support a more effective action of CP 55-940 to produce cell death of both cell lines through apoptotic mechanisms. Comparative aspects between cannabinoids with different profiles are necessary for the design of potential treatments against glial tumors.

In humans, impulsivity measured as false alarms in a Go/No-Go task is reportedly decreased by amphetamine and is not affected by oxycodone and delta(9)-tetrahydrocannabinol. To model these findings in animals, three rhesus monkeys were trained to perform a food-reinforced Go/No-Go task. In this task, amphetamine was found to decrease false alarms (i.e. responding during No-Go trials), but only at doses that also decreased hits (i.e. responding during Go trials). Morphine generally decreased hits but not false alarms. The cannabinoid receptor agonist CP 55, 940 decreased both false alarms and hits, but only at doses that also decreased the number of trials completed. Additional studies in animals and humans are necessary to delineate the conditions under which amphetamine and other psychoactive drugs affect impulsivity in Go/No-Go tasks.

Recent behavioral reports suggest that repeated exposure to cannabis and synthetic cannabinoid agonists is linked with mental disorders associated with dysfunction of serotonin 2A (5-HT2A) receptor neurotransmission such as anxiety and depression. Here, we studied the effect of a nonselective cannabinoid agonist, CP55940, on the activity of 5-HT2A receptors in hypothalamic paraventricular nucleus (PVN). We detected that repeated exposure to CP55940 enhanced the prolactin and corticosterone neuroendocrine responses mediated by 5-HT2A receptors and increased the membrane-associated levels of 5-HT2A receptors in PVN. Importantly, we also detected increased anxiety-like behaviors in CP55940 treated rats compared to controls. The data presented here suggest that the mechanisms mediating the cannabinoid-induced upregulation of 5-HT2A receptors would be brain-region specific, as we were unable to detect a CP55940-induced upregulation of 5-HT2A mRNA. Our results might provide insight into the molecular mechanism by which repeated exposure to cannabinoids could be associated with the pathophysiology of neuropsychiatric disorders.

A novel alkynylthiophene series of cannabinoid CB1 receptor antagonists has been described to exhibit distinct intrinsic activities with minimal substructure modifications. The three representatives, BPR0432, BPR0568 and BPR0569, functioning as a neutral antagonist, an inverse agonist and a partial agonist, respectively, in GTP binding assay, were further characterized for their downstream signaling activities in relation to in vivo efficacy in appetite suppression to diets of different macronutrients. Interestingly, these three derivatives all behaved as inverse agonists with the potency of BPR0432 > BPR0568 > BPR0569 in cAMP assay. After administered to non-deprived rats, the potency in appetite suppression was positively related to their strength in intrinsic activity in the first hour of intake. The preferential suppression to high fat and high carbohydrate diets was revealed after 6 h and only appeared in the treatment of BPR0568, presumably due to its metabolic stability in addition to its intrinsic activity. These results indicated the suppression of appetite was controlled in a biphasic manner, and these three structurally close but functionally distinct compounds are invaluable tools in elucidating the mechanism of neuronal response to appetite and palatability.

We have analysed the long-term effects of adolescent (postnatal day 28–43) exposure of male and female rats to nicotine (NIC, 1.4 mg/kg/day) and/or the cannabinoid agonist CP 55,940 (CP, 0.4 mg/kg/day) on the following parameters measured in the adulthood: (1) the memory ability evaluated in the object location task (OL) and in the novel object test (NOT); (2) the anxiety-like behaviour in the elevated plus maze; and (3) nicotinic and CB1 cannabinoid receptors in cingulated cortex and hippocampus. In the OL, all pharmacological treatments induced significant decreases in the DI of females, whereas no significant effects were found among males. In the NOT, NIC-treated females showed a significantly reduced DI, whereas the effect of the cannabinoid agonist (a decrease in the DI) was only significant in males. The anxiety-related behaviour was not changed by any drug. Both, nicotine and cannabinoid treatments induced a long-lasting increase in CB1 receptor activity (CP-stimulated GTPγS binding) in male rats, and the nicotine treatment also induced a decrease in nicotinic receptor density in the prefrontal cortex of females. The results show gender-dependent harmful effects of both drugs and long-lasting changes in CB1 and nicotinic receptors.

Selective agonists of cannabinoid receptors Win 55 212 and CP 55 940, labeled with hydrogen isotopes, were prepared. The content of isotopomers in deuterium-labeled Win 55 212 and CP 55 940, and also the deuterium distribution in fragments of the [2H]Win 55212 molecule were determined by mass spectrometry. [3H]Win 55 212 and [3H]CP 55 940 with molar radioactivities of 55 and 70 Ci mmol−1, respectively, were prepared by the reaction with gaseous tritium. The efficiency of isotope exchange with activated hydrogen species under the conditions of primary and secondary hydrogen spillover is discussed.

There is a bi-directionality in hormone-cannabinoid interactions: cannabinoids affect prominent endocrine axes (such as the hypothalamic-pituitary-gonadal), and gonadal hormones modulate cannabinoid effects. This review will summarize recent research on these interactions, with a specific focus upon their implications for motivated behavior. Sexual behavior will serve as a "case study." I will explore the hypothesis that ovarian hormones, in particular estradiol, may serve to release estrous behavior from endocannabinoid inhibition. Hormonal regulation of the endogenous cannabinoid system also affects processes that underlie drug abuse. This review will briefly discuss sex differences in behavioral responses to cannabinoids and explore potential mechanisms by which gonadal hormones alter cannabinoid reward. An examination of this research informs our perspective on how hormones and endocannabinoids may affect drug-seeking behavior as a whole and the development of addiction.

The cannabinoid CB(1) 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).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 featurePavlovian 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.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.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.

Human cannabinoid receptors 1 (hCB 1 R) and 2 (hCB 2 R) are expressed in the CNS and couple to G i /G o -proteins. The aim of this study was to compare coupling of hCB 1 R and hCB 2 R to Gα i2 β 1 γ 2 in Sf9 insect cells. High-affinity agonist binding at hCB 1 R, but not at hCB 2 R, was resistant to guanine nucleotides. hCB 1 R activated Gα i2 β 1 γ 2 much more rapidly than hCB 2 R in the [ 35 S]guanosine 5′-[γ-thio]triphosphate ([ 35 S]GTPγS) binding assay. Moreover, hCB 1 R exhibited a higher constitutive activity than hCB 2 R as assessed by the relative inhibitory effects of inverse agonists on [ 35 S]GTPγS binding and steady-state high-affinity GTPase activity compared to the stimulatory effects of the hCB 1/2 R agonist CP 55,940 [(-)- cis -3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]- trans -4-(3-hydroxypropyl)cyclohexanol]. Gα i2 β 1 γ 2 coupled to hCB 2 R exhibited higher GDP- and GTPγS-affinities than Gα i2 β 1 γ 2 coupled to hCB 1 R. NaCl effectively reduced constitutive activity of hCB 1 R but not of hCB 2 R. Collectively, hCB 1 R and hCB 2 R couple differentially to Gα i2 β 1 γ 2 . Moreover, hCB 1 R exhibits higher constitutive activity than hCB 2 R. These differences point to distinct functions of hCB 1 R and hCB 2 R in the CNS.

A series of 1-deoxy analogs of CP-47,497 (8 and 13, n = 0 to 7) and 1-deoxy analogs of CP-55,940 (9, n = 0 to 7) have been synthesized and their affinities for the cannabinoid CB1 and CB2 receptors have been determined. Although the majority of these compounds exhibit selectivity for the CB2 receptor none have greater than modest affinity for either receptor. The interactions of these 1-deoxy nontraditional cannabinoids with the CB2 receptor are discussed.

Cannabinoids have been shown to increase the extracellular levels of glutamate in vivo and in vitro, but no studies have evaluated the possible involvement of glial glutamate reuptake system. The present study investigates whether cannabinoids and endocannabinoid, anandamide have an effect on astroglial excitatory amino acid (EAA) transport. The kinetics of glutamate transport was studied in rat cortical astrocytes, using the radiolabeled, non-metabolized amino acid, d -[ 3 H] aspartate in the absence or presence of cannabinoid receptor agonists. The results show that in vehicle controls the uptake of d -aspartate was rapid, sodium-dependent and saturated within the first 5 min, resulting in a K m 7.365 ± 1.16 μmol/L ( n = 5) and the maximum velocity ( V max ) 1207 ± 51 nmol/mg protein/min. Addition of the synthetic cannabinoid analog R (+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolol][1,2,3de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone (WIN 55,212-2; 3 μmol/L) increased the K m (26.25 ± 4.84 μmol/L) without affecting the V max (1122 ± 77 nmol/mg protein/min), suggesting the inhibition was competitive and reversible. Various other cannabinoid agonists also inhibited d -aspartate uptake in a dose-dependent and stereospecific manner. The cannabinoid inhibition of EAA transport was partially blocked by the cannabinoid type-1 (CB1) receptor antagonist N -(piperidin-1-yl-5(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl- 1H -pyrazole-3-carboxamidehydrochloride (SR141716A; 100 nmol/L). The inhibitory effects of WIN 55,212-2, or its endogenous counterpart anandamide were reversed by 98,059, an inhibitor of mitogen-activated kinase (MAPK) kinase (MEK). These results suggest that cannabinoids and endocannabinoids may constitute a novel class of inhibitors of astroglial glutamate transport system.

Patients with cancer frequently report pain that can be difficult to manage. This study examined the antihyperalgesic effects of a cannabinoid receptor agonist, CP 55,940, in a murine model of cancer pain. Implantation of fibrosarcoma cells into and around the calcaneous bone in mice produced mechanical hyperalgesia (decreased paw withdrawal thresholds and increased frequency of paw withdrawals). On day 13 after implantation, mechanical hyperalgesia, nociception, and catalepsy were assessed. Mice were randomly assigned to receive CP 55,940 (0.01-3 mg/kg, i.p.) or vehicle and behavioral measures were redetermined. CP 55,940 dose-dependently attenuated tumor-evoked mechanical hyperalgesia. To examine the effect of catalepsy on the antihyperalgesic effect of CP 55,940, mice with tumor-evoked hyperalgesia were pretreated with the dopamine agonist apomorphine prior to administration of CP 55,940. Apomorphine attenuated the cataleptic effect of CP 55,940 but did not attenuate its antihyperalgesic effect. In a separate group of mice with tumor-evoked hyperalgesia, administration of the dopamine antagonist spiperone produced catalepsy that was approximately 2.5 fold greater than that produced by CP 55,490. Whereas this dose of CP 55,940 completely reversed tumor-evoked mechanical hyperalgesia, spiperone only attenuated mechanical hyperalgesia by approximately 35%. Thus, the cataleptic effects of CP 55,940 did not fully account for its antihyperalgesic effect. The antihyperalgesic effect of CP 55,940 was mediated via the cannabinoid CB1 but not CB2 receptor. Finally, repeated administration of CP 55,940 produced a short-term and a longer-term attenuation of tumor-evoked hyperalgesia. These results suggest that cannabinoids may be a useful alternative or adjunct therapy for treating cancer pain.

Low doses of cannabinoids applied intrathecally attenuate capsaicin-evoked heat and mechanical hyperalgesia via CB1 receptors. Although cannabinoids produce antinociception, in part, by attenuating responses of nociceptive neurons in the spinal cord, few studies have examined the effect of cannabinoids on sensitization of spinal neurons. We therefore investigated whether a cannabinoid receptor agonist, CP 55,940, attenuated excitation and sensitization of spinal nociceptive neurons produced by intraplantar injection of 0.1% capsaicin (10 μl). In rats, wide-dynamic-range (WDR) and high-threshold (HT) neurons were classified according to responses evoked by mechanical stimuli of varying intensity. CP 55,940 (10 μg in 50 μl) or vehicle was applied directly to the spinal cord and responses to mechanical (von Frey monofilament) and heat stimuli were recorded 10 min after drug treatment. CP 55,940 alone did not alter responses to mechanical stimuli; however the enhanced responses to mechanical stimuli after injection of capsaicin into the receptive field were dose dependently attenuated in both HT and WDR neurons. Vehicle-treated neurons increased their response to 300.6 ± 52.1% of baseline after capsaicin, whereas CP 55,940-treated neurons responded at 153.0 ± 27.1% of baseline. The effects of CP 55,940 on sensitization to heat were less pronounced; however, CP 55,940 attenuated the capsaicin-evoked decrease in heat threshold in HT neurons. The attenuation by CP 55,940 of sensitization to mechanical stimuli was blocked by pretreatment of the spinal cord with the CB1 receptor antagonist, SR141716A. These studies demonstrate that cannabinoid application to the spinal cord prevents central sensitization.

Although many studies have examined the acute behavioural effects of cannabinoids in rodents, few have examined the lasting effects of cannabinoids at different developmental ages. This study compared lasting effects of cannabinoid exposure occurring in adolescence to that occurring in early adulthood. Forty, 30-day old (adolescent) and 18, 56-day old (adult) female albino Wistar rats were injected with vehicle or incremental doses of the cannabinoid receptor agonist (-)- cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]- trans-4-(3-hydroxypropyl) cyclohexanol (CP 55,940) once per day for 21 consecutive days (150, 200 and 300 μg/kg i.p. for 3, 8 and 10 days, respectively). Following a 21-day drug-free period, working memory was assessed using an object recognition task. Locomotor activity was also measured in the object recognition apparatus via a ceiling-mounted passive infrared sensor. Three days later, anxiety was assessed using a social interaction test. In the object recognition task, significantly poorer working memory was observed in the adolescent but not adult CP 55,940-treated rats. Adolescent, but not adult CP 55,940-treated rats, also exhibited a significant decrease in social interaction with a novel conspecific. These results suggest that chronic exposure to a cannabinoid receptor agonist well after the immediate postnatal period, but before reaching sexual maturity, can lead to increased anxiety and a lasting impairment of working memory.

Addictive drugs have a number of commonalities in animal behavioral models. They lower intracranial self-stimulation (ICSS) thresholds, support self-administration, and produce conditioned place preference (CPP). However, cannabinoids appear atypical as drugs of abuse, since there are controversial data in the literature concerning their reinforcing properties.The aim of the present study was to examine the effects of cannabinoids on brain reward using the rate-frequency curve shift paradigm of ICSS.Male Sprague-Dawley rats were implanted with electrodes into the medial forebrain bundle (MFB). Rate-frequency functions were determined by logarithmically decreasing the number of cathodal pulses in a stimulation train from a value that sustained maximal responding to one that did not sustain responding. After brain stimulation reward thresholds stabilized rats received intraperitoneal (IP) injections of the potent CB1 receptor agonists WIN 55,212-2 (graded doses 0.1, 0.3, 1 and 3 mg/kg), CP 55,940 (graded doses 10, 30, 56 and 100 microg/kg), or HU-210 (graded doses 10, 30, 100 microg/kg).With the exception of the highest dose of all cannabinoid agonists tested, which significantly increased the threshold frequency required for MFB ICSS, all other doses of the tested drugs did not affect ICSS thresholds. The CB1 receptor antagonist SR141716A reversed the actions of WIN 55,212-2 and CP 55,940, but not HU-210. However, the selective CB1 cannabinoid receptor antagonist AM 251 counteracted the effect of HU-210. Both CB1 receptor antagonists, at the doses used in the present study, did not affect reward thresholds by themselves.The present results indicate that cannabinoid agonists do not exhibit reinforcing properties in the ICSS paradigm, but rather have an inhibitory influence on reward mechanisms. The results suggest that the anhedonic effects of cannabinoids are probably mediated by cannabinoid CB1 receptors.

Animals trained to lick for a sucrose solution of a given incentive value that subsequently encounter an incentive downshift (i.e. 32-4% sucrose) display an exaggerated decrease in the amount consumed, relative to unshifted controls. This change has been classified as a successive negative contrast (SNC) effect. The emotional component to this robust behavioural change is dynamic and changes from post-shift day (PSD) 1 to 2. Anxiolytics block SNC, but the possible link between anxiety and SNC needs further exploration. Both nicotine and a cannabinoid receptor agonist have been reported to change anxiety and both have actions on the reward process, but their effects on SNC have not been investigated.To determine: (1) whether exposure to SNC evokes an anxiogenic response; (2) whether an anxiolytic dose of nicotine has the same effects on SNC as those of chlordiazepoxide; (3) the effects of a low (anxiolytic) and a high (anxiogenic) dose of the cannabinoid receptor agonist CP 55,940 on SNC.Two groups of animals were given access to high (32%) or low (4%) sucrose solutions for 5 min per day for 10 days. On PSD 1 and 2, the shifted group had access to a devalued incentive (from 32 to 4% sucrose) and the unshifted group remained at 4% sucrose. The volumes (ml) of sucrose solution consumed were measured pre-shift and on PSD 1 and 2. In experiment 1, immediately after SNC testing on PSD 1 and 2, the rats were tested in the social interaction and elevated plus-maze tests of anxiety. In experiment 2, the effects of chlordiazepoxide (5 and 7.5 mg/kg) and nicotine (0.1 mg/kg) were examined on PSD 1 and 2. In experiment 3, the effects of CP 55,940 (5 and 40 microg/kg) were examined on PSD 1 and 2.There were no anxiogenic effects of shift in either test of anxiety on either test day. However, on PSD 1, the shifted group had significantly higher locomotor activity and spent a higher percentage of time on the open arms, perhaps reflecting search strategies. Nicotine was without significant effect on SNC on either test day. On PSD 1, chlordiazepoxide (5 mg/kg) and CP 55,940 (5 and 40 microg/kg, IP) blocked SNC. On PSD 2, both doses of chlordiazepoxide and the low, anxiolytic dose of CP 55,940 (5 microg/kg) blocked SNC, the high dose of CP 55,940 was without effect.The pattern of results allows for the separation between effects on anxiety and SNC. The block of contrast on PSD 1 was independent of changes in anxiety, since both anxiolytic and anxiogenic drug doses were effective. It is suggested that this may provide an animal model of disappointment in which the cannabinoid system plays an important role. An anxiolytic action would seem to be a necessary, but not a sufficient, action to block SNC on PSD 2.

Cannabinoids have been shown to influence food intake, and until recently, the neural pathways mediating these effects have remained obscure. It has been previously shown that intracerebroventricular injection of delta-9-tetrahydrocannabinol (Delta(9)-THC) causes increased consumption of palatable foods in rats, and we postulated the involvement of the hindbrain in this cannabinoid-induced food intake. Cannulated rats (both female and male groups) trained to consume sweetened condensed milk received either lateral or fourth ventricle injections of CP 55,940 and were presented with sweetened condensed milk 15 min after injection. Rats were injected over a range of doses between 100 pg and 10 microg per rat. Milk intake was recorded for a total of 3 h. Lateral ventricle injection of CP 55,940 increased milk intake at doses in the microgram range. However, CP 55,940 was effective in increasing food intake at nanogram doses when injected into the fourth ventricle. Finally, male rats appeared to be more sensitive to CP 55,940 than female rats inasmuch as milk consumption was increased at the 1 ng dose in male rats, whereas only the 10 ng dose was effective in females. These results indicate that CP 55,940 may act in the hindbrain to influence feeding behavior in rats.

Rationale: Despite the increasing use of cannabis among adolescents, there is scarce information about the long-term effects of cannabinoid receptor agonists in appropriate animal models. Objectives: We aimed to investigate the behavioural features of adult male and female Wistar rats that had been exposed to a chronic treatment with the cannabinoid receptor agonist CP 55,940 (CP) during the juvenile period. Methods: CP (0.4 mg/kg i.p.) or its corresponding vehicle was administered once daily, from day 35 to day 45. In adulthood, the animals were tested in the holeboard, the open field and the elevated plus-maze, under different stress (illumination) conditions. After a resting period, the serum corticosterone levels (radioimmunoassay) of the animals were measured. The effects of CP on food intake and somatic growth were monitored throughout the experimental period. Results: The CP treatment induced significant sex-dependent effects on holeboard activity, as well as a decrease in the level of emotionality/anxiety in the open field and in the plus-maze. The animals receiving CP also showed diminished food intake and body weights during the treatment period, but both parameters recovered normal values during the period after treatment. No significant effect of the CP treatment on corticosterone levels was found. Conclusions: The results demonstrate that chronic administration of CP during the peri-adolescent period resulted in marked behavioural effects in adulthood. The nature of these effects depended on the sex of the animals and on the specific behavioural test. The possible neurobiological substrates underlying the effects of CP are discussed.

Like other G-protein coupled receptors with hydrophobic ligands, the human cannabinoid receptor 1 (CB1) is thought to bind its ligands within the transmembrane region of the receptor. However, for some of these receptors the extracellular loops (ECs) have also been shown to play a role in ligand recognition and selectivity. We have taken a mutagenesis approach to examine the role of the amino terminus, EC1, and EC3 of CB1 in ligand binding. Eight mutant receptors, each with a dipeptide insertion, were constructed, expressed, and evaluated for binding to the cannabinoid ligands (-)-cis-3[2-hydroxy-4-(1',1'-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP 55,940) and N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR 141716A). Mutants with insertions in the membrane distal region of the amino terminus or EC3 maintained affinity for both ligands. Those with insertions in the membrane proximal region of the amino terminus or EC1 exhibited a loss of affinity for CP 55,940 while retaining wild-type affinity for SR 141716A. Representative mutants were analyzed for agonist-induced inhibition of cyclic AMP accumulation, and the results indicated that G-protein coupling remained intact. Alanine substitution mutants were made to address whether it was the perturbation of the overall structure of the region or the displacement of particular side chains that was responsible for the loss of CP 55,940 binding. We conclude that a structurally intact EC1, but not the comparably short EC3, is essential for high-affinity CP 55,940 binding.

Ligand binding to the cannabinoid receptor of brain membranes has been characterized using [ 3 H] CP 55,940 and the Multiscreen Filtration System. Binding of [ 3 H]CP 55,940 is saturable and reaches equilibrium by 45 min at room temperature. At a concentration of 10 pg of membrane protein/well, the K D for [ 3 H]CP 55,940 is 461 pM and the B max is 860 mfmol/mg of protein. The apparent K D of [ 3 H]CP 55,940 is dependent upon tissue protein concentration, increasing to 2,450 pM at 100 μg of membrane protein. Binding of [ 3 H]CP 55,940 is dependent upon the concentration of bovine serum albumin in the buffer; the highest ratio of specific to nonspecific binding occurs between 0.5 and 1.0 mg/ml. The K i of anandamide, a putative endogenous ligand of the cannabinoid receptor, is 1.3 pM in buffer alone and 143 nM in the presence of 0.15 mM phenylmethylsulfonyl fluoride. When [ 14 C] anandamide is incubated with rat forebrain membranes at room temperature, it is degraded to arachidonic acid; the hydrolysis is inhibited by 0.15 mM phenylmethylsulfonyl fluoride. These results support the hypothesis that anandamide is a high-affinity ligand of the cannabinoid receptor and that it is rapidly degraded by membrane fractions

I.c.v. self-administration of MDMA (0.01–2 μg per infusion), alone and in combination with CP 55,940 (0.4 μg infusion−1), was studied on an operant responding procedure. On the basis of individual preference for one of two levers, developed during training, rats were allowed to self-administer vehicle from the preferred lever and MDMA from the other. Pressings on the MDMA associated-lever, except for the maximal unit dose, progressively increased. The combination of CP 55,940 with MDMA (1 μg infusion−1) reduced the number of drug-associated lever pressings compared to the single drugs. Pre-treatment with SR 141716A (0.5 mg kg−1 i.p.), 15 min before each daily session, significantly increased MDMA self-administration. These findings suggest that MDMA self-administration is under endogenous tonic control by the endocannabinoid system. British Journal of Pharmacology (2002) 136, 1089–1092. doi:10.1038/sj.bjp.0704825

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-210CP 55, 940WIN 55, 212-2or = 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-210CP 55, 940WIN 55, 212-2Delta(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.

Several effects of the abused synthetic cannabinoid JWH-018 were compared to those of Δ 9 -tetrahydrocannabinol (Δ 9 -THC) in rhesus monkeys. JWH-018 (0.1 mg/kg i.v.) was established as a discriminative stimulus and rimonabant was used to examine mechanisms responsible for discrimination as well as operant response rate-decreasing and hypothermic effects. JWH-018 dose-dependently increased drug-lever responding (ED 50 =0.01 mg/kg) and decreased response rate (ED 50 =0.064 mg/kg). Among various cannabinoids, the relative potency for producing discriminative stimulus and rate-decreasing effects was the same: CP-55940=JWH-018>Δ 9 -THC=WIN-55212-2=JWH-073. The benzodiazepine agonist midazolam and the NMDA antagonist ketamine did not exert JWH-018 like discriminative stimulus effects up to doses that disrupted responding. JWH-018 and Δ 9 -THC decreased rectal temperature by 2.2 and 2.8 °C, respectively; the doses decreasing temperature by 2 °C were 0.21 and 1.14 mg/kg, respectively. Antagonism did not differ between JWH-018 and Δ 9 -THC, but did differ among effects. The apparent affinities of rimonabant calculated in the presence of JWH-018 and Δ 9 -THC were not different from each other for antagonism of discriminative stimulus effects (6.58 and 6.59, respectively) or hypothermic effects (7.08 and 7.19, respectively). Apparent affinity estimates are consistent with the same receptors mediating the discriminative stimulus and hypothermic effects of both JWH-018 and Δ 9 -THC. However, there was more limited and less orderly antagonism of rate-decreasing effects, suggesting that an additional receptor mechanism is involved in mediating the effects of cannabinoids on response rate. Overall, these results strongly suggest that JWH-018 and Δ 9 -THC act at the same receptors to produce several of their shared psychopharmacological effects.

Lewis rats were trained to self-stimulate the medial forebrain bundle (MFB) using a rate-frequency paradigm. They were then tested for the effects of the cannabinoid receptor agonist CP 55,940, the selective cannabinoid receptor antagonist SR 141716 and the dopamine D 1 receptor antagonist SCH 23390. CP 55,940 (0, 10, 25 and 50 μg/kg i.p.) had no effect on MFB self-stimulation behaviour as assessed by the M 50 , the stimulation frequency at which half-maximal response rates were obtained. With SR 141716, only a very high dose (20 mg/kg i.p.) caused a significant inhibition of the rewarding efficacy of the stimulation. This was seen as an increase in the M 50 . All other doses of SR 141716 (0, 1, 3, 10 mg/kg i.p.) were ineffective in modulating the M 50 . By comparison, a relatively low dose (0.06 mg/kg i.p.) of SCH 23390 caused a large increase in M 50 . These results indicate a relatively modest influence, if any at all, of exogenous or endogenous cannabinoids on reward-relevant neurotransmission.