Guo Hua Bi

Brain Cannabinoid CB2 Receptors Modulate Cocaine’s Actions in Mice

The presence and function of cannabinoid CB2 receptors in the brain have been the subjects of much debate. We found that systemic, intranasal or intra-accumbens local administration of JWH133, a selective CB2 receptor agonist, dose-dependently inhibited intravenous cocaine self-administration, cocaine-enhanced locomotion, and cocaine-enhanced accumbens extracellular dopamine in wild-type and CB1 receptor knockout (CB1−/−, also known as Cnr1−/−) mice, but not in CB2−/− (Cnr2−/−) mice. This inhibition was mimicked by GW405833, another CB2 receptor agonist with a different chemical structure, and was blocked by AM630, a selective CB2 receptor antagonist. Intra-accumbens administration of JWH133 alone dose-dependently decreased, whereas intra-accumbens administration of AM630 elevated, extracellular dopamine and locomotion in wild-type and CB1−/− mice, but not in CB2−/− mice. Intra-accumbens administration of AM630 also blocked the reduction in cocaine self-administration and extracellular dopamine produced by systemic administration of JWH133. These findings suggest that brain CB2 receptors modulate cocaines rewarding and locomotor-stimulating effects, likely by a dopamine-dependent mechanism.

Cannabinoid CB2 receptors modulate midbrain dopamine neuronal activity and dopamine-related behavior in mice

Significance Although early studies suggested that cannabinoid CB2 receptors (CB2Rs) are absent in the brain, this view has been challenged by recent findings of significant brain CB2R involvement in several dopamine (DA)-related CNS disorders. The cellular mechanisms underlying these actions are unclear, however. Using multiple approaches, we found that CB2R genes and receptors are expressed in midbrain DA neurons, and that activation of CB2Rs inhibits DA neuronal firing and i.v. cocaine self-administration. These findings not only challenge the long-held view that brain CB2Rs are not expressed in neurons, but also suggest that neuronal CB2Rs modulate DA neuronal activity and DA-regulated behavior. Thus, brain CB2Rs may constitute a new therapeutic target in medication development for treatment of a number of CNS disorders. Cannabinoid CB2 receptors (CB2Rs) have been recently reported to modulate brain dopamine (DA)-related behaviors; however, the cellular mechanisms underlying these actions are unclear. Here we report that CB2Rs are expressed in ventral tegmental area (VTA) DA neurons and functionally modulate DA neuronal excitability and DA-related behavior. In situ hybridization and immunohistochemical assays detected CB2 mRNA and CB2R immunostaining in VTA DA neurons. Electrophysiological studies demonstrated that activation of CB2Rs by JWH133 or other CB2R agonists inhibited VTA DA neuronal firing in vivo and ex vivo, whereas microinjections of JWH133 into the VTA inhibited cocaine self-administration. Importantly, all of the above findings observed in WT or CB1−/− mice are blocked by CB2R antagonist and absent in CB2−/− mice. These data suggest that CB2R-mediated reduction of VTA DA neuronal activity may underlie JWH133s modulation of DA-regulated behaviors.

Increased vulnerability to cocaine in mice lacking dopamine D3 receptors

Neuroimaging studies using positron emission tomography suggest that reduced dopamine D2 receptor availability in the neostriatum is associated with increased vulnerability to drug addiction in humans and experimental animals. The role of D3 receptors (D3Rs) in the neurobiology of addiction remains unclear, however. Here we report that D3R KO (D3−/−) mice display enhanced cocaine self-administration and enhanced motivation for cocaine-taking and cocaine-seeking behavior. This increased vulnerability to cocaine is accompanied by decreased dopamine response to cocaine secondary to increased basal levels of extracellular dopamine in the nucleus accumbens, suggesting a compensatory response to decreased cocaine reward in D3−/− mice. In addition, D3−/− mice also display up-regulation of dopamine transporters in the striatum, suggesting a neuroadaptative attempt to normalize elevated basal extracellular dopamine. These findings suggest that D3R deletion increases vulnerability to cocaine, and that reduced D3R availability in the brain may constitute a risk factor for the development of cocaine addiction.

Species Differences in Cannabinoid Receptor 2 and Receptor Responses to Cocaine Self-Administration in Mice and Rats

The discovery of functional cannabinoid receptors 2 (CB2Rs) in brain suggests a potential new therapeutic target for neurological and psychiatric disorders. However, recent findings in experimental animals appear controversial. Here we report that there are significant species differences in CB2R mRNA splicing and expression, protein sequences, and receptor responses to CB2R ligands in mice and rats. Systemic administration of JWH133, a highly selective CB2R agonist, significantly and dose-dependently inhibited intravenous cocaine self-administration under a fixed ratio (FR) schedule of reinforcement in mice, but not in rats. However, under a progressive ratio (PR) schedule of reinforcement, JWH133 significantly increased breakpoint for cocaine self-administration in rats, but decreased it in mice. To explore the possible reasons for these conflicting findings, we examined CB2R gene expression and receptor structure in the brain. We found novel rat-specific CB2C and CB2D mRNA isoforms in addition to CB2A and CB2B mRNA isoforms. In situ hybridization RNAscope assays found higher levels of CB2R mRNA in different brain regions and cell types in mice than in rats. By comparing CB2R-encoding regions, we observed a premature stop codon in the mouse CB2R gene that truncated 13 amino-acid residues including a functional autophosphorylation site in the intracellular C-terminus. These findings suggest that species differences in the splicing and expression of CB2R genes and receptor structures may in part explain the different effects of CB2R-selective ligands on cocaine self-administration in mice and rats.

Expression of functional cannabinoid CB2 receptor in VTA dopamine neurons in rats

We have recently reported the expression of functional cannabinoid CB2 receptors (CB2Rs) in midbrain dopamine (DA) neurons in mice. However, little is known whether CB2Rs are similarly expressed in rat brain because significant species differences in CB2R structures and expression are found. In situ hybridization and immunohistochemical assays detected CB2 gene and receptors in DA neurons of the ventral tegmental area (VTA), which was up‐regulated in cocaine self‐administration rats. Electrophysiological studies demonstrated that activation of CB2Rs by JWH133 inhibited VTA DA neuronal firing in single dissociated neurons. Systemic administration of JWH133 failed to alter, while local administration of JWH133 into the nucleus accumbens inhibited cocaine‐enhanced extracellular DA and i.v. cocaine self‐administration. This effect was blocked by AM630, a selective CB2R antagonist. These data suggest that CB2Rs are expressed in VTA DA neurons and functionally modulate DA neuronal activities and cocaine self‐administration behavior in rats.

Blockade of D3 receptors by YQA14 inhibits cocaine's rewarding effects and relapse to drug-seeking behavior in rats

Preclinical studies suggest that dopamine D3 receptor (D3R) antagonists are promising for the treatment of drug abuse and addiction. However, few D3R antagonists have potential to be tested in humans due to short half-life, toxicity or limited preclinical research into pharmacotherapeutic efficacy. Here, we report on a novel D3R antagonist YQA14, which has improved half-life and pharmacokinetic profile and which displays potent pharmacotherapeutic efficacy in attenuating cocaine reward and relapse to drug-seeking behavior. Electrical brain-stimulation reward (BSR) in laboratory animals is a highly sensitive experimental approach to evaluate a drugs rewarding effects. We found that cocaine (2 mg/kg) significantly enhanced electrical BSR in rats (i.e., decreased stimulation threshold for BSR), while YQA14 alone had no effect on BSR. Pretreatment with YQA14 significantly and dose-dependently attenuated cocaine-enhanced BSR. YQA14 also facilitated extinction from drug-seeking behavior in rats during early behavioral extinction, and attenuated cocaine- or contextual cue-induced relapse to drug-seeking behavior. YQA14 alone did not maintain self-administration in either naïve rats or in rats experienced at cocaine self-administration. YQA14 also inhibited expression of repeated cocaine-induced behavioral sensitization. These findings suggest that YQA14 may have pharmacotherapeutic potential in attenuating cocaine-taking and cocaine-seeking behavior. Thus, YQA14 deserves further investigation as a promising agent for treatment of cocaine addiction.

Fenobam sulfate inhibits cocaine-taking and cocaine-seeking behavior in rats: implications for addiction treatment in humans.

RationaleThe metabotropic glutamate receptor subtype 5 (mGluR5) has been reported to be critically involved in drug reward and addiction. Because the mGluR5 negative allosteric modulators (NAMs) 2-methyl-6-(phenylethynyl)pyridine (MPEP) and 3-((2-methyl-1,3-thiazol-4-yl)ethynyl)pyridine (MTEP) significantly inhibit addictivelike behaviors of cocaine and other drugs of abuse in experimental animals, it has been suggested that mGluR5 NAMs may have translational potential for treatment of addiction in humans. However, neither MPEP nor MTEP have been evaluated in humans due to their off-target actions and rapid metabolism.ObjectivesHerein, we evaluate a potential candidate for translational addiction research: a new sulfate salt formulation of fenobam, a selective mGluR5 NAM that has been investigated in humans.ResultsIn rats, fenobam sulfate had superior pharmacokinetics compared to the free base, with improved maximal plasma concentration (Cmax) and longer half life. Oral (p.o.) administration of fenobam sulfate (30 or 60xa0mg/kg) inhibited intravenous (i.v.) cocaine self-administration, cocaine-induced reinstatement of drug-seeking behavior, and cocaine-associated cue-induced cocaine-seeking behavior in rats. Fenobam sulfate also inhibited p.o. sucrose self-administration and sucrose-induced reinstatement of sucrose-seeking behavior, but had no effect on locomotion.ConclusionsThis study provides additional support for the role of mGluR5 signaling in cocaine addiction and suggests that fenobam sulfate may have translational potential in medication development for the treatment of cocaine addiction in humans.

A novel mGluR5 antagonist, MFZ 10-7, inhibits cocaine-taking and cocaine-seeking behavior in rats

Pre‐clinical studies suggest that negative allosteric modulators (NAMs) of the metabotropic glutamate receptor subtype 5 (mGluR5), including 2‐methyl‐6‐(phenylethynyl)pyridine (MPEP), 3‐[(2‐methyl‐1,3‐thiazol‐4‐yl)ethynyl]pyridine (MTEP) and fenobam are highly effective in attenuating drug‐taking and drug‐seeking behaviors. However, both MPEP and MTEP have no translational potential for use in humans because of their off‐target effects and short half‐lives. Here, we report that 3‐fluoro‐5‐[(6‐methylpyridin‐2‐yl)ethynyl]benzonitrile (MFZ 10‐7), a novel mGluR5 NAM, is more potent and selective than MPEP, MTEP and fenobam in both in vitro binding and functional assays. Similar to MTEP, intraperitoneal administration of MFZ 10‐7 inhibited intravenous cocaine self‐administration, cocaine‐induced reinstatement of drug‐seeking behavior and cocaine‐associated cue‐induced cocaine‐seeking behavior in rats. Although MFZ 10‐7 and MTEP lowered the rate of oral sucrose self‐administration, they did not alter total sucrose intake. Further, MFZ 10‐7 appeared to be more potent than MTEP in inducing downward shifts in the cocaine dose–response curve, but less effective than MTEP in attenuating sucrose‐induced reinstatement of sucrose‐seeking behavior. MFZ 10‐7 and MTEP had no effect on basal locomotor behavior. These findings not only provide additional evidence supporting an important role for mGluR5 in cocaine reward and addiction, but also introduce a new tool for both in vitro and in vivo investigations with which to further characterize this role.

The Novel Modafinil Analog, JJC8-016, as a Potential Cocaine Abuse Pharmacotherapeutic

(±)Modafinil ((±)MOD) and its R-enantiomer (R-modafinil; R-MOD) have been investigated for their potential as treatments for psychostimulant addiction. We recently reported a series of (±)MOD analogs, of which JJC8-016 (N-(2-((bis(4-fluorophenyl)methyl)thio)ethyl)-3-phenylpropan-1-amine) was selected for further development. JJC8-016 and R-MOD were evaluated for binding across ~70 receptors, transporters, and enzymes. Although at a concentration of 10u2009μM, there were many hits for JJC8-016, binding affinities in the range of its DAT affinity were only observed at the serotonin transporter (SERT), dopamine D2-like, and sigma1 receptors. R-MOD was more selective, but had much lower affinity at the DAT (Ki=3u2009μM) than JJC8-016 (Ki=116u2009nM). In rats, systemic administration of R-MOD alone (10–30u2009mg/kg i.p.) dose-dependently increased locomotor activity and electrical brain-stimulation reward, whereas JJC8-016 (10–30u2009mg/kg i.p.) did not produce these effects. Strikingly, pretreatment with JJC8-016 dose-dependently inhibited cocaine-enhanced locomotion, cocaine self-administration, and cocaine-induced reinstatement of drug-seeking behavior, whereas R-MOD inhibited cocaine-induced reinstatement only at the high dose of 100u2009mg/kg. Notably, JJC8-016 alone neither altered extracellular dopamine in the nucleus accumbens nor maintained self-administration. It also failed to induce reinstatement of drug-seeking behavior. These findings suggest that JJC8-016 is a unique DAT inhibitor that has no cocaine-like abuse potential by itself. Moreover, pretreatment with JJC8-016 significantly inhibits cocaine-taking and cocaine-seeking behavior likely by interfering with cocaine binding to DAT. In addition, off-target actions may also contribute to its potential therapeutic utility in the treatment of cocaine abuse.

T394A Mutation at the μ Opioid Receptor Blocks Opioid Tolerance and Increases Vulnerability to Heroin Self-Administration in Mice

The etiology and pathophysiology underlying opioid tolerance and dependence are still unknown. Because mu opioid receptor (MOR) plays an essential role in opioid action, many vulnerability-related studies have focused on single nucleotide polymorphisms of MOR, particularly on A118G. In this study, we found that a single-point mutation at the MOR T394 phosphorylation site could be another important susceptive factor in the development of opioid tolerance and dependence in mice. T394A mutation, in which a threonine at 394 was replaced by an alanine, did not alter agonist binding to MOR and opioid analgesia, but resulted in loss of etorphine-induced MOR internalization in spinal dorsal horn neurons and opioid analgesic tolerance induced by either morphine or etorphine. In addition, this mutation also caused an increase in intravenous heroin self-administration and in nucleus accumbens dopamine response to heroin. These findings suggest that T394 phosphorylation following MOR activation causes MOR internalization and desensitization, which subsequently contributes to the development of tolerance in both opioid analgesia and opioid reward. Accordingly, T394A mutation blocks opioid tolerance and leads to an increase in brain dopamine response to opioids and in opioid-taking behavior. Thus, the T394 may serve as a new drug target for modulating opioid tolerance and the development of opioid abuse and addiction. SIGNIFICANCE STATEMENT The mechanisms underlying opioid tolerance and susceptibility to opioid addiction remain unclear. The present studies demonstrate that a single-point mutation at the T394 phosphorylation site in the C-terminal of mu opioid receptor (MOR) results in loss of opioid tolerance and enhanced vulnerability to heroin self-administration. These findings suggest that modulation of the MOR-T394 phosphorylation or dephosphorylation status may have therapeutic potential in management of pain, opioid tolerance, and opioid abuse and addiction. Accordingly, MOR-T394 mutation or polymorphisms could be a risk factor in developing opioid abuse and addiction and therefore be used as a new biomarker in prediction and prevention of opioid abuse and addiction.