DA D2R activation stimulates AEA levels, thus, drugs of abuse that increase brain DA levels activate DA D2Rs, and in turn, increase AEA levels. We have tested the effects of abused drugs in producing CB1R mediated generalization in THC-discrimination tests in rats. Cocaine, and amphetamine injected alone did not produce effects significantly different from vehicle, but potentiated the THC-like effects of THC. Nicotine and the D2/3 DAR agonist quinpirole alone did not generalize to the THC cue, but both drugs did so in animals pretreated with the inhibitor of FAAH that metabolizes AEA. Nicotine and quinpirole also potentiated the effects of THC. We have suggested that AEA is released by drugs through activation of D2R mediated mechanism. So, administered alone these drugs do not stimulate AEA levels sufficiently to provide CB1-mediated THC-like effects, but potentiate ineffective small doses of THC. However, when the same drugs are administered in combination with URB-597, AEA levels are magnified by blockade of its metabolism, and its concentration could thus activate CB1R producing THC discriminative effects. Recently it has also been shown that effects of AEA and blockers of its metabolism (e.g. URB-597) might be mediated not only by the endocannabinoid system, but also by PPAR-alpha. Indeed, blockade of AEA metabolism by URB-597 might increase levels of oleoylamide (OEA) or palmytoilamide (PEA), as well as AEA. While AEA has both CB1R and PPAR-alpha activities, OEA and PEA are selective ligands for PPAR, with negligible activity at CB1R. We showed that drugs acting specifically at brain PPAR-alpha can block the addictive actions of nicotine in rats and monkeys. Psychostimulant sensitization might play a role in the path to abuse and addiction. Even a single exposure to psychostimulants produce sensitization by increasing strength of excitatory synapses in midbrain DAergic regions. This synaptic plasticity is related to alterations in the cannabinoid system. We hypothesized that development of psychostimulant sensitization might involve stimulation of brain endocannabinoid levels that can bind to and activate CB1Rs. We started this project studying cocaine sensitization in mice, measured as increased stimulation of behavioral activities before and after sensitizing doses of cocaine. We hypothesized that development of cocaine sensitization requires release of endocannabinoids, and is reversed by CB1R blockade. Thus, low doses of cocaine that do not induce behavioral sensitization might become effective when animals are pretreated with enhancers of endocannabinoid levels. DA transmission, believed to mediate behavioral and reinforcing effects of cocaine, will also be measured before and after cocaine sensitization. Our results have confirmed that a single exposure to cocaine induces behavioral sensitization in mice. Rimonabant, CB1 antagonist, injected before cocaine, reduced the behavioral sensitization. Cocaine- sensitization was paralleled by a larger stimulation of DA levels, in the NAC core, but not in the NAC shell. Blockade of endocannabinoid metabolism, by pretreatment with URB-597, enhanced the extracellular levels of endocannabinoids released by cocaine, and this enhancement was related to the sensitization by doses of cocaine otherwise not effective. We have also found that the same enhancement of AEA levels by cocaine will produce a specific neurochemical sensitization of DA in the core but not in the shell of the NAC. The number of people seeking treatment for marijuana use in the United States per year (1,243,000) is higher than the number seeking treatment for cocaine or heroin use (787,000 or 507,000, respectively). THC, the main psychoactive ingredient in marijuana, activates brain pathways mediating its reinforcing effects by enhancing the firing of DA neurons in the ventral tegmental area (VTA), resulting in increased release of DA from nerve terminals in the NAC shell. Developing medications that modulate these effects of THC as a reinforcer might provide a therapeutic approach for the treatment of marijuana dependence. For example, previously we found that reward-related behavioral and neurochemical effects of THC could be blocked by methyllycaconitine (MLA), a selective antagonist of alpha7-nAChRs that are present in both the VTA and the NAc shell on glutamatergic nerve terminals. Their activation elicits GLU release, which in turn acts at ionotropic GLUR on DAergic terminals to stimulate DA release. Unfortunately, systemic use of direct antagonists of alpha7-nAChRs is associated with side effects that limit their therapeutic utility. To avoid these unwanted effects, we tested a compound 3,4-dimethoxy-N-4-(3-nitrophenyl)thiazol-2-ylbenzenesulfonamide (Ro 61-8048), a potent, selective, peripherally acting kynurenine 3-monooxygenase (KMO) inhibitor, to indirectly increase brain KYNA, an endogenous negative allosteric modulators of alfa7nAChRs that might be better tolerated than directly acting cholinergic antagonists. Indeed, allosteric modulators change Rs conformations in the presence of orthosteric ligands and often have no effect on their own, acting only when physiologicalRs are activated. Newly formed KYNA is promptly released into the extracellular compartment. Notably, no reuptake processes exist for KYNA, and extracellular KYNA is not degraded enzymatically, but is slowly eliminated from the brain by a non-specific acid transporter. We found that administration of the kynurenine 3-monooxygenase (KMO) inhibitor Ro 61-8048 increases brain KYNA levels and attenuates THC-induced stimulation of DA levels in reward-related brain areas. Administration of Ro 61-8048 also reduced the reinforcing effects of THC measured under self-administration behavioral procedures, also preventing relapse to drug-seeking induced by re-exposure to cannabinoids or cannabinoid-associated cues. The involvement of alpha7-nAChRs was confirmed by administration of positive allosteric modulators of alpha7-nAChRs. These results suggest a therapeutic strategy for treatment of marijuana dependence. We are now studying the effects of these drugs on related GLU levels in the VTA and NAC shell. We found that THC increases GLU levels and decreases KYNA levels in the rat NAc shell via CB1R-dependent mechanism. We are now testing the effects of KMO blockade on THC effects. We have discovered that endogenous cannabinoids possess reinforcing effects. Also, in agreement with positive self-administration behavior, systemic injection of endogenous cannabinoid agonists would increase extracellular levels of DA. One of these endocannabinoids, 2AG, produces a small, transient increase in DA levels in the NAC shell. This is in concordance with reports showing that 2AG is usually very rapidly metabolized in vivo by a specific enzyme, mono-acyl-glyceryl-lipase (MAGL). We have now available a drug, AM-4301, that would selectively block the activity of the MAGL enzyme. By blocking this enzyme we should be able to potentiate the behavioral and neurochemical effects of 2AG. Since we do not know if circulating levels of 2AG can be enhanced to a level sufficient to induce cannabinoid-like behavioral and neurochemical effects, AM-4301 will be tested alone, and then will be tested in combination with 2AG. When tested alone, AM-4301 slightly decreased levels of DA in the shell in rats. When administered in combination with 2AG the results show a larger increase in DA than with 2AG alone. It is interesting to note that our preliminary tests show that AM-4301 might be interacting with the MAGL enzyme with a delayed onset and longer-lasting action. Indeed we found a larger increase in DA after AM-4301 pretreatments in animals injected with 2-AG, when the pretreatment time was 24 hours as compared to 40 or 60 minutes.
