Basic brain mechanisms underlying drug addiction, craving, and relapse

National Institute On Drug Abuse

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Abstract

During the present reporting period, we continued our prior work on cannabinoids and endocannabinoid signaling in the brain. Systemic administration of the cannabinoid agonists delta-9-THC (THC), WIN55212-2, or XLR11 produced dose-dependent tetrad effects (spontaneous activity, catalepsy, hypothermia, and analgesia) in wild-type (WT) mice. Genetic deletion or pharmacological blockade of CB1 receptors abolished the tetrad effects produced by all three cannabinoids. Genetic deletion of CB2 receptors abolished analgesia and catalepsy produced by THC or WIN55212-2, but not by XLR11. Microinjections of THC into the lateral ventricles also produced tetrad effects in WT, but not in CB1 gene-deleted (CB1-KO) mice. CB2-KO mice displayed a reduction in intraventricular THC-induced analgesia and catalepsy. Genetic deletion of GPR55 receptors caused enhanced responses to THC or WIN55212-2. Antagonism of CB1, CB2, or GPR55 receptors produced alterations similar to those observed in respective gene-deleted mice. These findings suggest that in addition to CB1, both CB2 and GPR55 receptors are involved in pharmacological effects produced by cannabinoids. CB1/CB2, in contrast to GPR55, receptors appears to play opposite roles in cannabinoid action. We also studied beta-Caryophyllene (BCP), a plant-derived terpenoid that is a cannabinoid CB2 receptor agonist. Systemic BCP administration dose-dependently inhibited nicotine self-administration and motivation for nicotine seeking in rats and mice. The reduction in nicotine self-administration was blocked by AM630, a selective CB2 receptor antagonist, but not by AM251, a selective CB1 receptor antagonist. Genetic deletion of CB2 receptors in mice blocked the reduction in nicotine self-administration produced only by low doses, but not by high doses, of BCP, suggesting involvement of both CB2 and non-CB2 receptor mechanisms. Further, BCP attenuated electrical brain-stimulation reward and nicotine-enhanced brain-stimulation reward in rats. BCP also attenuated brain-stimulation reward maintained by optogenetic stimulation of dopaminergic (DA) neurons in the ventral tegmental area (VTA) in DAT-cre mice, suggesting involvement of a DA-dependent mechanism in BCP's action. These findings suggest that BCP has significant anti-nicotine effects via both CB2 and non-CB2 receptor mechanisms and, therefore, deserves further study as a potential new pharmacotherapy for cigarette smoking cessation. We also found that BCP dose-dependently attenuated cocaine self-administration, cocaine-induced place preference, and cocaine-primed relapse to drug seeking in rats. We also found that BCP inhibits methamphetamine-taking and -seeking behaviors via CB2 and non-CB2 receptor mechanisms. In contrast, BCP failed to alter food self-administration or cocaine-induced hyperactivity. It also failed to maintain self-administration in a drug substitution test, suggesting that BCP has no abuse potential. Unexpectedly, pharmacological blockade or genetic deletion of CB1, CB2, or GPR55 receptors in gene-knockout mice failed to alter BCP's action against cocaine self-administration, suggesting the involvement of non-CB1, non-CB2, and non-GPR55 receptor mechanisms. Further, pharmacological blockade of opioid receptor or Toll-like receptors failed to alter, while blockade of peroxisome proliferator-activated receptors (PPARs) reversed BCP-induced reduction in cocaine self-administration, suggesting the involvement of PPARs in BCP's action. We used electrical and optogenetic intracranial self-stimulation (eICSS, oICSS) to study underlying neural substrates, and found that BCP is more effective in attenuating cocaine-enhanced oICSS than eICSS, the former driven by optical activation of midbrain DA neurons in DAT-cre mice. These findings indicate that BCP may be useful for the treatment of cocaine abuse, likely by stimulation of PPARs in the mesolimbic system. Cannabinoids produce both rewarding and aversive effects in humans and experimental animals. We therefore examined the involvement of CB1 and CB2 receptors in cannabinoid action using CB1-KO and CB2-KO mice. We found that THC produced conditioned place preference at low dose in WT mice that was attenuated by deletion of the CB1 receptor. At 5 mg/kg, no subjective effects of THC were detected in WT mice, but CB1-KO mice exhibited a trend towards place aversion and CB2-KO mice developed significant place preferences. These data suggest that CB1 receptor activation is rewarding, while CB2R activation is aversive. We examined nucleus accumbens (NAc) DA response to THC using in vivo microdialysis. Unexpectedly, THC produced a dose-dependent decrease in extracellular DA in WT mice, that was augmented in CB1-KO mice. However, in CB2-KO mice THC produced a dose-dependent increase in extracellular DA, suggesting that activation of the CB2R inhibits DA release in the NAc. In contrast, THC administered systemically or locally into the NAc failed to alter extracellular DA in rats. We also examined locomotor response to THC. WE showed that both CB1 and CB2 receptor mechanisms underlie THC-induced hypolocomotion. These findings indicate that THC's variable subjective effects reflect differential activation of cannabinoid receptors. Specifically, the opposing actions of CB1 and CB2 receptors regulate cannabis reward and aversion, with CB2-mediated effects predominant in mice. The cellular distribution of CB2Rs in brain remain unclear. We have reported that CB2Rs are expressed in VTA dopamine DA neurons and functionally regulate DA-mediated behavior(s). We recently found high densities of CB2-like signaling in a neighboring motor structure - the midbrain red nucleus (RN). We found high densities of CB2R-immunostaining and mRNA signal in RN magnocellular glutamate neurons in WT and CB1-KO, but not CB2-KO, mice. Ex vivo electrophysiological recordings in midbrain slices showed that CB2R activation by JWH133 dose-dependently inhibited firing rates of RN magnocellular neurons in WT, but not CB2-KO, mice, while having no effect on RN GABA neurons in transgenic GAD67-GFP reporter mice, suggesting CB2-mediated effects on glutamatergic neurons. JWH133 microinjection into the RN produced robust ipsilateral rotation in WT, but not CB2-KO mice, which was blocked by pretreatment with either a CB2 or DA D1 or D2 receptor antagonist, suggesting a DA-dependent effect. Fluorescent tract tracing revealed glutamatergic projections from the RN to multiple brain areas including the VTA, thalamus, and cerebellum. These findings suggest that CB2Rs in RN glutamate neurons functionally modulate motor activity, and therefore, constitute a new target in cannabis-based medication development for motor disorders. Finally, we recently turned out attention to ghrelin signaling in the brain and its possible involvement in regulating addiction-like behaviors in laboratory rodents. Using RNAscope neural imaging, we found clear evidence of ghrelin receptors in the VTA and adjacent substantia nigra, and evidence that self-administration of addictive drugs alters the density and cellular distribution of ghrelin receptors in those brain loci.