Neurobiological mechanisms of cannabinoid addiction.

The endocannabinoid system is implicated in the regulation of a variety of physiological processes, among which conditioning, motivation, habit forming, memory, learning, and cognition play pivotal roles in drug reinforcement and reward. In this article we will give a synopsis of last developments in research on cannabinoid actions on brain reward circuits coming from behavioral, neurochemical and electrophysiological studies. Central cannabinoid-induced effects as measured by animal models of addiction, in vivo cerebral microdialysis, in vitro and in vivo electrophysiological recording techniques, will be reviewed. Brain sites that have been implicated in the mediation of addictive cannabinoid properties include primarily the ventral tegmental area, the nucleus accumbens, and the medial prefrontal cortex, although the amygdala, the substantia nigra, the globus pallidus, and the hippocampus have also been shown to be critical structures mediating motivational and reinforcing effects of cannabinoids. Putative neurobiological mechanisms underlying these effects will be delineated.

Cannabinoid self-administration in rats: sex differences and the influence of ovarian function.

BACKGROUND AND PURPOSE: We recently demonstrated the existence of strain differences in self-administration of the cannabinoid CB1 receptor agonist WIN55,212-2 (WIN) by Long Evans (LE) and Lister Hooded (LH) but not Sprague-Dawley (SD) male rats. This follow-up study is aimed at verifying whether sex and ovarian hormones might also be critical factors in the initiation, retention and extinction of WIN self-administration. EXPERIMENTAL APPROACH: LE, LH and SD male and female rats, the latter either intact or bilaterally ovariectomized (OVX), were trained to self-administer WIN (12.5 microg kg(-1) per infusion) under a FR1 reinforcement schedule, using lever-pressing. KEY RESULTS: Data showed that contrary to the findings in SD rats, LE and LH rats developed robust cannabinoid intake, with rates of responding for WIN being constantly higher in intact females than in males (+45 and +42% for LE and LH strains, respectively). In comparison with intact females, OVX females of both strains acquired self-administration at lower rates, displaying slower acquisition, lower drug intake (-42 and -52% for LE and LH, respectively) and longer extinction. CONCLUSIONS AND IMPLICATIONS: These findings provide the first evidence of significant sex differences in cannabinoid self-administration, females acquiring stable WIN intake at higher rates and more rapidly than males. Moreover, when compared to intact females, a lower percentage of LE and LH OVX rats acquired and maintained stable drug intake, suggesting that ovarian hormones might represent a critical factor in modulating the reinforcing effect of cannabinoids.

EP 91073 prevents EP 80661-induced penile erection: new evidence for the existence of specific EP peptide receptors mediating penile erection.

The effect of EP 91073, EP 51389, EP 70555 and EP 51216, peptide analogues of the growth hormone releasing peptide hexarelin, on penile erection induced by EP 80661 or EP 60761 injected into the paraventricular nucleus of the hypothalamus, was studied in male rats. Of the above peptides only EP 91073 (0.2-1 microg) was found capable of reducing penile erection induced by EP 80661 or EP 60761, when given into the paraventricular nucleus. Despite its ability to prevent EP peptide-induced penile erection, EP 91073 (1 microg) was unable to prevent penile erection induced by the dopamine receptor agonist apomorphine (50 ng), oxytocin (30 ng) and N-methyl-D-aspartic acid (50 ng), when given into the paraventricular nucleus 10 min prior to the above substances. The EP 91073-induced prevention of penile erection occurred with a reduction in the increase in nitric oxide production that occurs in the paraventricular nucleus concomitant to penile erection induced by EP 80661 and EP 60761, as measured by intracerebral vertical microdialysis. The present results are in line with the hypothesis that EP 80661 and EP 60761 induce penile erection by activating specific receptors in the paraventricular nucleus, located possibly in oxytocinergic neurons mediating penile erection, and show that EP 91073 acts as an antagonist of these EP peptide receptors mediating penile erection.

Effect of excitatory amino acid, dopamine, and oxytocin receptor antagonists on noncontact penile erections and paraventricular nitric oxide production in male rats.

In male rats, noncontact erections occur concomitantly with an increase in NO2- and NO3- in the paraventricular nucleus of the hypothalamus (PVN). In the present study, both responses were reduced by the blockade of PVN excitatory amino acid receptors by dizocilpine, (+)-MK-801(1 and 5 microg), but not by 6-cyano-7-nitro-quinoxaline-2,3-dione (5 microg) or (+)-2-amino-4-phosphono-butanoic acid (5 microg). Also ineffective when injected into the PVN were the dopamine antagonists SCH 23390 (5 microg), S(+)-raclopride (10 microg), and cis-flupenthixol (10 microg), and the oxytocin antagonist d(CH2)5Tyr(Me)2-Om8-vasotocin (1 microg). However, when the last was given into the lateral ventricles, it reduced noncontact erections without modifying NO2- and NO3- increases. These results suggest that excitatory amino acid transmission increases in the PVN during noncontact erections. This may contribute to increased NO production in the PVN, and it may activate oxytocin neurons mediating this sexual response.

Different effects of omega-conotoxin on penile erection, yawning and paraventricular nitric oxide in male rats.

A dose of apomorphine or oxytocin that induces penile erection and yawning increases nitric oxide production in the paraventricular nucleus of the hypothalamus, as determined by the increase in NO2- and NO3- concentration induced by these substances in the paraventricular dialysate obtained from male rats. All the above responses were prevented by a dose of omega-conotoxin-GVIA as low as 5 ng. This potent inhibitor of N-type Ca2+ channels was injected into the paraventricular nucleus 15 min before apomorphine (50 ng) or oxytocin (10 ng). In contrast, omega-conotoxin was ineffective when the above responses were induced by N-methyl-D-aspartic acid (50 ng). The peptide toxin (5 ng) was also ineffective on the penile erection and yawning induced by the nitric oxide donors sodium nitroprusside (50 microg) or hydroxylamine (50 microg), injected into the paraventricular nucleus. The present results suggest that omega-conotoxin-sensitive Ca2+ channels are involved in the activation of nitric oxide synthase, penile erection and yawning induced by apomorphine and oxytocin, but not by N-methyl-D-aspartic acid, at the paraventricular level.

EP 60761 and EP 50885, two hexarelin analogues, induce penile erection in rats.

The effect of hexarelin and four related peptide analogues, EP 40904, EP 40737, EP 50885 and EP 60761, injected into the paraventricular nucleus of the hypothalamus of male rats in doses between 2 and 2000 ng on spontaneous penile erection was studied. Of these peptides, EP 60761 and EP 50885, but not hexarelin, EP 40904 or EP 40737, increased dose-dependently the number of spontaneous penile erections. EP 60761 was active already at the dose of 20 ng, which induced the sexual response in 70% of the treated rats. The maximal response was induced by 200 ng of the peptide. EP 50885 was less potent than EP 60761, with 1000 ng being the minimal effective dose and 2000 ng as the dose required to induce the maximal response. At the doses used, both peptides also increased slightly the number of spontaneous yawning episodes. EP 60761- and EP 50885-induced penile erection was prevented by the oxytocin receptor antagonist [d(CH(2))(5)Tyr(Me)(2)-Orn(8)]vasotocin (0.1-1 microg) given intracerebroventricularly (i.c.v.), but not into the paraventricular nucleus (0.1-1 microg), by the competitive nitric oxide (NO) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) given either into the paraventricular nucleus (10-20 microg) or i.c.v. (75-150 microg), by the N-type Ca(2+) channel blocker omega-conotoxin-GVIA (2-5 ng) or by the opiate morphine (1-10 microg), but not by the dopamine receptor antagonist (Z)-4-[3-[2-(trifluoromethyl)-9H-thioxanthen-9-ylidene]propyl]-1-p ipe razine-ethanol (cis-flupenthixol) (10 microg) or by the N-methyl-D-aspartic acid (NMDA) receptor antagonist (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine ((+)-MK-801) (1 microg), all given into the paraventricular nucleus before either peptide. The present results show that EP 60761 and EP 50885 induced penile erection by increasing central oxytocin transmission, possibly by activating NO synthase in the cell bodies of oxytocinergic neurons located in the paraventricular nucleus that control penile erection.

EP 60761- and EP 50885-induced penile erection: structure-activity studies and comparison with apomorphine, oxytocin and N-methyl-D-aspartic acid.

The effect of 10 peptides structurally related to the growth hormone (GH) releasing peptide hexarelin, injected into the paraventricular nucleus of the hypothalamus (PVN), on penile erection was studied in male rats. Six out of the 10 peptides tested induced penile erection in a dose-dependent manner. Among them, the most potent were EP 80661, EP 60761 and EP 91072, which were active at doses of 20-200 ng. The potency of these peptides in inducing penile erection is comparable to that of apomorphine, oxytocin and N-methyl-D-aspartic acid similarly injected into the PVN. Other peptides found active were EP 50885, EP 90101 and EP 91071, which induced penile erection at doses of 200-2000 ng. In contrast, EP 51322, EP 70555, EP 51216 and EP 91073 were inactive, as were hexarelin, EP 40904 and EP 40737 in a previous study. The majority of EP peptides found active when injected into the PVN induced penile erection, although to a lesser extent, also when given systemically (endovenously). The proerectile effect of EP peptides was prevented by the oxytocin receptor antagonist [d(CH2)5 Tyr(Me)2-Orn8]-vasotocin given into the lateral ventricles but not into the PVN, by the nitric oxide (NO) synthase inhibitor N(G)-nitro-1-arginine methyl ester given either into the lateral ventricles or into the PVN, by the N-type Ca2+ channel blocker omega-conotoxin GVIA and by morphine, but not by the dopamine receptor antagonist cis-flupenthixol or by the N-methyl-D-aspartic acid receptor antagonist dizolcipine, given into the PVN. As the structure-activity relationship of EP peptides for proerectile activity is different from those of other biological actions of these compounds, ie for GH release and eating behaviour, the present results suggest that EP peptides induce penile erection by acting on specific hypothalamic receptor sites that activate paraventricular oxytocinergic neurons projecting to extrahypothalamic brain areas that mediate this sexual function by a mechanism similar to that of dopamine receptor agonists, oxytocin and N-methyl-D-aspartic acid.

Penile erection induced by EP 80661 and other hexarelin peptide analogues: involvement of paraventricular nitric oxide.

The effect of GAB-D-Trp(2-Me)-D-Trp(2-Me)-LysNH(2) (EP 80661), GAB-D-Trp(2-Me)-D-Trp(2-Me)-D-Trp(2-Me)-LysNH(2) (EP 60761), GAB-D-Trp(2-Me)-LysNH(2) (EP 91071) and GAB-D-Trp(2-Me)-D-beta Nal-Phe-LysNH(2) (EP 50885), four hexarelin peptide analogues that induce penile erection when injected into the paraventricular nucleus of the hypothalamus of male rats, on the concentration of NO(2)(-) and NO(3)(-) in the paraventricular dialysate was studied in male rats. EP peptides (1 microg) induced penile erection and increased the concentration of NO(2)(-) and NO(3)(-) in the paraventricular dialysate. In contrast, hexarelin (1 microg) was ineffective on either penile erection or paraventricular NO(2)(-) and NO(3)(-). EP peptide-induced penile erection was prevented by the nitric oxide synthase inhibitor N(G)-nitro-l-arginine methylester given into the paraventricular nucleus (20 microg), which also reduced the concomitant increase of NO(2)(-) and NO(3)(-) concentration in the paraventricular dialysate. In contrast, the oxytocin receptor antagonist [d(CH(2))(5)Tyr(Me)(2)-Orn(8)]vasotocin (1 microg) given into the paraventricular nucleus, was ineffective on penile erection and on the NO(2)(-) and NO(3)(-) increase induced by EP peptides, despite its ability to prevent the sexual response induced by the above peptides when given into the lateral ventricles. The present results show that EP peptides induce penile erection by activating nitric oxide synthase in the paraventricular nucleus of the hypothalamus, possibly in the cell bodies of oxytocinergic neurons that control penile erection.

Activation of gamma-aminobutyric acid(A) receptors in the paraventricular nucleus of the hypothalamus reduces apomorphine-, N-methyl-D-aspartic acid- and oxytocin-induced penile erection and yawning in male rats.

The effect of muscimol and baclofen injected into the paraventricular nucleus of the hypothalamus on penile erection and yawning induced by apomorphine, oxytocin and N-methyl-D-aspartic acid (NMDA) was studied in male rats. Muscimol (20-200 ng), but not baclofen (200 ng), injected into the paraventricular nucleus of the hypothalamus 10 min before apomorphine (50 ng), oxytocin (10 ng) or NMDA (50 ng) reduced penile erection and yawning induced by the above compounds given into the paraventricular nucleus. Bicuculline (250 ng) injected into the paraventricular nucleus 5 min before muscimol (100 ng) prevented the inhibitory effect of muscimol on penile erection and yawning induced by apomorphine, oxytocin and NMDA. The present results show that gamma-aminobutyric acid (GABA) inhibits penile erection and yawning by acting on GABA(A) receptors in the paraventricular nucleus of the hypothalamus.

The oxytocin antagonist d(CH2)5Tyr(Me)2-Orn8-vasotocin reduces non-contact penile erections in male rats.

Male rats show four to six penile erection episodes when put for 80 min in the presence of an inaccessible receptive female. These non-contact penile erections were reduced dose-dependently by d(CH2)5Tyr(Me)2-Orn8-vasotocin, a potent and selective oxytocin receptor antagonist, when given into the lateral ventricles (0.1, 0.5 and 1 microg), but not when given into the paraventricular nucleus of the hypothalamus (0.1 and 1 microg). In contrast, non-contact erections were reduced by N(G)-nitro-L-arginine methyl ester, a competitive inhibitor of nitric oxide synthase, given into the lateral ventricles (50, 100 and 200 microg), or into the paraventricular nucleus (10 and 20 microg). The present results show that central oxytocin is involved in the expression of penile erection induced not only by drugs but also by sexual physiological stimuli.

Cannabinoid-opioid interactions in drug discrimination and self-administration: effect of maternal, postnatal, adolescent and adult exposure to the drugs.

Cannabinoids and opioids are known to strictly interact in many physiological and pathological functions, including addiction. The endogenous opioid system is significantly influenced by maternal or perinatal cannabinoid exposure, major changes concerning operant behaviour in adult animals. Copious data suggests that adolescence is also a particularly sensitive period of life not only for the initiation of abusing illicit drugs, but also for the effects that these drugs exert on the neural circuitries leading to drug dependence. This paper examines the role played by the age of drug exposure in the susceptibility to discriminative and reinforcing effects of both cannabinoids and opioids. We first revisited evidence of alterations in the density and functionality of mu-opioid and CB1 cannabinoid receptors in reward-related brain regions caused by either maternal, postnatal, adolescent or adult exposure to opioids and cannabinoids. Then, we reviewed behavioural evidence of the long-term consequences of exposure to opioids and cannabinoids during gestation, postnatal period, adolescence or adulthood, focusing mostly on drug discrimination and self-administration studies. Overall, evidence confirms a neurobiological convergence of the cannabinoid and opioid systems that is manifest at both receptor and behavioural levels. Although discrepant results have been reported, some data support the gateway hypothesis that adolescent cannabis exposure contributes to greater opioid intake in adulthood. However, it should be kept into consideration that in humans genetic, environmental, and social factors could influence the direct neurobiological effects of early cannabis exposure to the progression to adult drug abuse.

Drug- and cue-induced reinstatement of cannabinoid-seeking behaviour in male and female rats: influence of ovarian hormones.

BACKGROUND AND PURPOSE: Animal and human studies have shown that sex and hormones are key factors in modulating addiction. Previously, we have demonstrated that self-administration of the cannabinoid CB(1) receptor agonist WIN55,212-2 (WIN; 12.5 microg.kg(-1) per infusion) is dependent on sex, intact female rats being more sensitive than males to the reinforcing properties of cannabinoids, and on the oestrous cycle, ovariectomized (OVX) females being less responsive than intact females. EXPERIMENTAL APPROACH: This follow-up study investigated whether sex and ovarian function also affect reinstatement of cannabinoid-seeking in rats after exposure to drug or cue priming. KEY RESULTS: After priming with 0.15 or 0.3 mg.kg(-1) WIN, intact female rats exhibited stronger reinstatement than males and OVX females. Responses of intact female rats were higher than those of male and OVX rats even after priming with a drug-associated visual (Light) or auditory (Tone) cue, or a WIN + Light combination. However, latency to the first response did not differ between intact and OVX female rats, and males showed the longest latency to initiate lever-pressing activity. CONCLUSIONS AND IMPLICATIONS: Our study provides compelling evidence for a pivotal role of sex and the oestrous cycle in modulating cannabinoid-seeking, with ovariectomy diminishing drug and cue-induced reinstatement. However, it is possible that sex differences during self-administration training are responsible for sex differences in reinstatement. Finding that not only drug primings but also acute exposure to drug-associated cues can reinstate responding in rats could have significant implications for the development of pharmacological and behavioural treatments of abstinent female and male marijuana smokers.

Cannabinoid mechanism in reinstatement of heroin-seeking after a long period of abstinence in rats.

Because opioid and cannabinoid systems have been reported to interact in the modulation of addictive behaviour, this study was aimed at investigating the ability of cannabinoid agents to reinstate or prevent heroin-seeking behaviour after a prolonged period of extinction. In rats previously trained to self-administer heroin intravenously, non-contingent non-reinforced priming administrations of heroin and cannabinoids were presented after long-term extinction, and lever pressing following injections was observed. Results showed that: (i) intravenous priming infusions of heroin (0.1 and 0.2 mg/kg) lead to reinstatement of drug-seeking behaviour; (ii) intraperitoneal priming injections of the central cannabinoid receptor agonists R-(+)-(2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrol[1,2,3-de]-1,4-benzoxazinyl) (1-naphthalenyl)methanonemesylate (WIN 55,212-2, 0.15 and 0.3 mg/kg) and (-)-cis-3-[2-hydroxy-4(1,1-dimethyl-heptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol (CP 55,940, 0.05 and 0.1 mg/kg), but not delta9-tetrahydrocannabinol (delta9-THC, 0.1-1.0 mg/kg), effectively restored heroin-seeking behaviour; (iii) intraperitoneal priming injection of the central cannabinoid receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)4-methyl-1H-pyrazole-3-carboxamide (SR 141716A, 0.3 mg/kg) did not reinstate responding, but (iv) completely prevented heroin-induced reinstatement of drug-seeking behaviour. Moreover, heroin-seeking behaviour was still present for a few days following cannabinoid primings, indicating a long-lasting effect of cannabinoids on responding for heroin. These findings indicate that relapse to heroin after an extended drug-free period is triggered by cannabinoid agonists and that SR 141716A prevents drug-seeking behaviour, suggesting that the use of the cannabinoid antagonist could have some therapeutic benefits in heroin-induced relapse.

Morphine injected into the paraventricular nucleus of the hypothalamus prevents noncontact penile erections and impairs copulation: involvement of nitric oxide.

Male rats show four to six penile erection episodes when put in the presence of an inaccessible receptive female for 80 min. These noncontact erections occur concomitantly with an increase in nitric oxide production in the paraventricular nucleus of the hypothalamus. This is shown by the increases in the NO2- and NO3- concentrations in the paraventricular dialysate obtained from these males by in vivo microdialysis. The NO2- concentration increased from 0.75 +/- 0. 10 microm to 2.89 +/- 0.39 microm and that of NO3- from 4.13 +/- 0. 58 microm to 9.5 +/- 1.2 microm. Morphine (0.5, 1 and 5 microg), given unilaterally into the paraventricular nucleus 15 min before the introduction of the receptive female, prevented the NO2- and NO3- increases, and noncontact erections, dose-dependently. In contrast, the kappa opioid receptor agonist U-69 593 (5 microg) was ineffective. The effects of morphine on NO2- and NO3-, and on noncontact erections, were prevented by the opiate receptor antagonist naloxone (10 microg) injected into the paraventricular nucleus 15 min before morphine. The NO2- and NO3- concentrations were also increased in the paraventricular dialysate of male rats during copulation, i.e. when in copula penile erections occurred. As found with noncontact erections, morphine, but not U-69 593, injected into the paraventricular nucleus prevented the NO2- and NO3- increases and impaired copulatory behaviour, and naloxone prevented these responses when given before morphine. Although some diffusion of the opiate to surrounding brain areas cannot be completely ruled out, the present results suggest that morphine acts through mu receptors in the paraventricular nucleus to impair noncontact erections and copulation. These effects of morphine are apparently mediated by a prevention of the increased nitric oxide production that occurs in the paraventricular nucleus of the hypothalamus of male rats during sexual activity.

Differential orexigenic effects of hexarelin and its analogs in the rat hypothalamus: indication for multiple growth hormone secretagogue receptor subtypes.

We have previously reported that hexarelin and some of its analogs, including EP 50885, stimulated GH secretion and feeding after systemic administration in the rat, whereas EP 40904 selectively stimulated food intake and EP 40737 only GH release. The precise mechanism of growth hormone-releasing peptides (GHRPs) actions is still unclear, but the integrity of the arcuate nucleus of the hypothalamus (ARC) appears crucial for their endocrine effects. To better characterize the site(s) and mechanisms(s) of the orexigenic action of GHRPs, we have investigated their effects after infusion into the arcuate, paraventricular, ventromedial and medial preoptic areas of the hypothalamus. Food intake was measured for 60 min following injection of the test compound (2 microg/rat). Hexarelin, EP 40904 and EP 50885 had significant orexigenic effects after injection into the ARC. A specific NPY antagonist significantly inhibited the effect of hexarelin, whereas a GHRH antagonist was ineffective. In the paraventricular nucleus, only EP 50885 stimulated feeding, whereas all peptides were ineffective in the ventromedial nucleus and medial preoptic area. Taken altogether, these results demonstrate that GHRPs are endowed with site-specific orexigenic actions and that endogenous NPY, but not GHRH, mediates these effects. The additional orexigenic action of EP 50885 in the paraventricular nucleus suggests the existence of a GHRP receptor subtype different from the already cloned one.