Cannabinoid CB1 Receptors Are Expressed in a Subset of Dopamine Neurons and Underlie Cannabinoid-Induced Aversion, Hypoactivity, and Anxiolytic Effects in Mice.

Cannabinoids modulate dopamine (DA) transmission and DA-related behavior, which has been thought to be mediated initially by activation of cannabinoid CB1 receptors (CB1Rs) on GABA neurons. However, there is no behavioral evidence supporting it. In contrast, here we report that CB1Rs are also expressed in a subset of DA neurons and functionally underlie cannabinoid action in male and female mice. RNAscope in situ hybridization (ISH) assays demonstrated CB1 mRNA in tyrosine hydroxylase (TH)-positive DA neurons in the ventral tegmental area (VTA) and glutamate decarboxylase 1 (GAD1)-positive GABA neurons. The CB1R-expressing DA neurons were located mainly in the middle portion of the VTA with the number of CB1-TH colocalization progressively decreasing from the medial to the lateral VTA. Triple-staining assays indicated CB1R mRNA colocalization with both TH and vesicular glutamate transporter 2 (VgluT2, a glutamate neuronal marker) in the medial VTA close to the midline of the brain. Optogenetic activation of this population of DA neurons was rewarding as assessed by optical intracranial self-stimulation. Δ9-tetrahydrocannabinol (Δ9-THC) or ACEA (a selective CB1R agonist) dose-dependently inhibited optical intracranial self-stimulation in DAT-Cre control mice, but not in conditional knockout mice with the CB1R gene absent in DA neurons. In addition, deletion of CB1Rs from DA neurons attenuated Δ9-THC-induced reduction in DA release in the NAc, locomotion, and anxiety. Together, these findings indicate that CB1Rs are expressed in a subset of DA neurons that corelease DA and glutamate, and functionally underlie cannabinoid modulation of DA release and DA-related behavior.SIGNIFICANCE STATEMENT Cannabinoids produce a series of psychoactive effects, such as aversion, anxiety, and locomotor inhibition in rodents. However, the cellular and receptor mechanisms underlying these actions are not fully understood. Here we report that CB1 receptors are expressed not only in GABA neurons but also in a subset of dopamine neurons, which are located mainly in the medial VTA close to the midline of the midbrain and corelease dopamine and glutamate. Optogenetic activation of these dopamine neurons is rewarding, which is dose-dependently inhibited by cannabinoids. Selective deletion of CB1 receptor from dopamine neurons blocked cannabinoid-induced aversion, hypoactivity, and anxiolytic effects. These findings demonstrate that dopaminergic CB1 receptors play an important role in mediating cannabinoid action.

PPARα and PPARγ are expressed in midbrain dopamine neurons and modulate dopamine- and cannabinoid-mediated behavior in mice.

Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear receptors that regulate gene expression. Δ9-tetrahydrocannabinol (Δ9-THC) is a PPARγ agonist and some endocannabinoids are natural activators of PPARα and PPARγ. However, little is known regarding their cellular distributions in the brain and functional roles in cannabinoid action. Here, we first used RNAscope in situ hybridization and immunohistochemistry assays to examine the cellular distributions of PPARα and PPARγ expression in the mouse brain. We found that PPARα and PPARγ are expressed in ~70% of midbrain dopamine (DA) neurons. In the amygdala, PPARα is expressed in ~60% of glutamatergic neurons, while PPARγ is expressed in ~60%  of GABA neurons. However, no PPARα/γ signal was detected in GABA neurons in the nucleus accumbens. We then used a series of behavioral assays to determine the functional roles of PPARα/γ in the CNS effects of Δ9-THC. We found that optogenetic stimulation of midbrain DA neurons was rewarding as assessed by optical intracranial self-stimulation (oICSS) in DAT-cre mice. Δ9-THC and a PPARγ (but not PPARα) agonist dose-dependently inhibited oICSS. Pretreatment with PPARα or PPARγ antagonists attenuated the Δ9-THC-induced reduction in oICSS and Δ9-THC-induced anxiogenic effects. In addition, a PPARγ agonist increased, while PPARα or PPARγ antagonists decreased open-field locomotion. Pretreatment with PPARα or PPARγ antagonists potentiated Δ9-THC-induced hypoactivity and catalepsy but failed to alter Δ9-THC-induced analgesia, hypothermia and immobility. These findings provide the first anatomical and functional evidence supporting an important role of PPARα/γ in DA-dependent behavior and cannabinoid action.

Elevation of Extracellular Glutamate by Blockade of Astrocyte Glutamate Transporters Inhibits Cocaine Reinforcement in Rats via a NMDA-GluN2B Receptor Mechanism.

It is well established that glutamate plays an important role in drug-induced and cue-induced reinstatement of drug seeking. However, the role of glutamate in drug reward is unclear. In this study, we systemically evaluated the effects of multiple glutamate transporter (GLT) inhibitors on extracellular glutamate and dopamine (DA) in the nucleus accumbens (NAc), intravenous cocaine self-administration, intracranial brain-stimulation reward (BSR), and reinstatement of cocaine seeking in male and female rats. Among the five GLT inhibitors we tested, TFB-TBOA was the most potent. Microinjections of TFB-TBOA into the NAc, but not the ventral tegmental area (VTA), or dorsal striatum (DS), dose-dependently inhibited cocaine self-administration under fixed-ratio and progressive-ratio (PR) reinforcement schedules, shifted the cocaine dose-response curve downward, and inhibited intracranial BSR. Selective downregulation of astrocytic GLT-1 expression in the NAc by GLT-1 antisense oligonucleotides also inhibited cocaine self-administration. The reduction in cocaine self-administration following TFB-TBOA administration was NMDA GluN2B receptor dependent, and rats self-administering cocaine showed upregulation of GluN2B expression in NAc DA- and cAMP-regulated phosphoprotein 32 (DARPP-32)-positive medium-spiny neurons (MSNs). In contrast, TFB-TBOA, when locally administered into the NAc, VTA, or ventral pallidum (VP), dose-dependently reinstated cocaine-seeking behavior. Intra-NAc TFB-TBOA-evoked drug-seeking was long-lasting and NMDA/AMPA receptor dependent. These findings, for the first time, indicate that glutamate in the NAc negatively regulates cocaine's rewarding effects, while an excess of glutamate in multiple brain regions can trigger reinstatement of drug-seeking behavior.SIGNIFICANCE STATEMENT It is well known that glutamate plays an important role in relapse to drug seeking. However, the role of glutamate in drug reward is less clear. Here, we report that TFB-TBOA, a highly potent glutamate transporter (GLT) inhibitor, dose-dependently elevates extracellular glutamate and inhibits cocaine self-administration and brain-stimulation reward (BSR), when administered locally into the nucleus accumbens (NAc), but not other brain regions. Mechanistic assays indicate that cocaine self-administration upregulates NMDA-GluN2B receptor subtype expression in striatal dopaminoceptive neurons and activation of GluN2B by TFB-TBOA-enhanced glutamate inhibits cocaine self-administration. TFB-TBOA also reinstates cocaine-seeking behavior when administered into the NAc, ventral tegmental area (VTA), and ventral pallidum (VP). These findings demonstrate that glutamate differentially regulates cocaine reward versus relapse, reducing cocaine reward, while potentiating relapse to cocaine seeking.

Involvement of the ghrelin system in the maintenance of oxycodone self-administration: converging evidence from endocrine, pharmacologic and transgenic approaches.

Ghrelin, an orexigenic hormone, has emerged as a critical biological substrate implicated in drug reward. However, the response of the ghrelin system to opioid-motivated behaviors and the role of ghrelin in oxycodone self-administration remain to be studied. Here, we investigated the reciprocal interactions between the endogenous ghrelin system and oxycodone self-administration behaviors in rats and the role of the ghrelin system in brain stimulation reward (BSR) driven by optogenetic stimulation of midbrain reward circuits in mice. Oxycodone self-administration significantly elevated plasma ghrelin, des-acyl ghrelin and growth hormone and showed no effect on plasma LEAP2, a newly identified endogenous ghrelin receptor (GHS-R1a) antagonist. Oxycodone self-administration produced significant decreases in plasma gastric inhibitory polypeptide and insulin. Acquisition of oxycodone self-administration significantly upregulated GHS-R1a mRNA levels in dopamine neurons in the ventral tegmental area (VTA), a brain region critical in drug reward. Pretreatment with JMV2959, a selective GHS-R1a antagonist, dose-dependently reduced oxycodone self-administration and decreased the breakpoint for oxycodone under a progressive ratio reinforcement in Long-Evans rats. The inhibitory effects of JMV2959 on oxycodone self-administration is selectively mediated by GHS-R1a as JMV2959 showed a similar effect in Wistar wildtype but not in GHS-R knockout rats. JMV2959 pretreatment significantly inhibited BSR driven by selective stimulation of VTA dopamine neurons, but not by stimulation of striatal GABA neurons projecting to the VTA in mice. These findings suggest that elevation of ghrelin signaling by oxycodone or oxycodone-associated stimuli is a causal process by which oxycodone motivates oxycodone drug-taking and targeting the ghrelin system may be a viable treatment approach for opioid use disorders.

Dissecting the Role of GABA Neurons in the VTA versus SNr in Opioid Reward.

Opioid reward has traditionally been thought to be mediated by GABA-induced disinhibition of dopamine (DA) neurons in the VTA. However, direct behavioral evidence supporting this hypothesis is still lacking. In this study, we found that the µ opioid receptor (MOR) gene, Oprm1, is highly expressed in GABA neurons, with ∼50% of GABA neurons in the substantia nigra pars reticulata (SNr), ∼30% in the VTA, and ∼70% in the tail of the VTA (also called the rostromedial tegmental nucleus) in male rats. No Oprm1 mRNA was detected in midbrain DA neurons. We then found that optogenetic inhibition of VTA DA neurons reduced intravenous heroin self-administration, whereas activation of these neurons produced robust optical intracranial self-stimulation in DAT-Cre mice, supporting an important role of DA neurons in opioid reward. Unexpectedly, pharmacological blockade of MORs in the SNr was more effective than in the VTA in reducing heroin reward. Optogenetic activation of VTA GABA neurons caused place aversion and inhibited cocaine, but not heroin, self-administration, whereas optogenetic activation of SNr GABA neurons caused a robust increase in heroin self-administration with an extinction pattern, suggesting a compensatory response in drug intake due to reduced heroin reward. In addition, activation of SNr GABA neurons attenuated heroin-primed, but not cue-induced, reinstatement of drug-seeking behavior, whereas inhibition of SNr GABA neurons produced optical intracranial self-stimulation and place preference. Together, these findings suggest that MORs on GABA neurons in the SNr play more important roles in opioid reward and relapse than MORs on VTA GABA neurons.SIGNIFICANCE STATEMENT Opioid reward has long been believed to be mediated by inhibition of GABA interneurons in the VTA that subsequently leads to disinhibition of DA neurons. In this study, we found that more µ opioid receptors (MORs) are expressed in GABA neurons in the neighboring SNr than in the VTA, and that pharmacological blockade of MORs in the SNr is more effective in reducing heroin reward than blockade of MORs in the VTA. Furthermore, optogenetic activation of VTA GABA neurons inhibited cocaine, but not heroin, self-administration, whereas activation of SNr GABA neurons inhibited heroin reward and relapse. These findings suggest that opioid reward is more likely mediated by stimulation of MORs in GABA afferents from other brain regions than in VTA GABA neurons.

Optogenetic brain-stimulation reward: A new procedure to re-evaluate the rewarding versus aversive effects of cannabinoids in dopamine transporter-Cre mice.

Despite extensive research, the rewarding effects of cannabinoids are still debated. Here, we used a newly established animal procedure called optogenetic intracranial self-stimulation (ICSS) (oICSS) to re-examine the abuse potential of cannabinoids in mice. A specific adeno-associated viral vector carrying a channelrhodopsin gene was microinjected into the ventral tegmental area (VTA) to express light-sensitive channelrhodopsin in dopamine (DA) neurons of transgenic dopamine transporter (DAT)-Cre mice. Optogenetic stimulation of VTA DA neurons was highly reinforcing and produced a classical "sigmoidal"-shaped stimulation-response curve dependent upon the laser pulse frequency. Systemic administration of cocaine dose-dependently enhanced oICSS and shifted stimulation-response curves upward, in a way similar to previously observed effects of cocaine on electrical ICSS. In contrast, Δ9 -tetrahydrocannabinol (Δ9 -THC), but not cannabidiol, dose-dependently decreased oICSS responding and shifted oICSS curves downward. WIN55,212-2 and ACEA, two synthetic cannabinoids often used in laboratory settings, also produced dose-dependent reductions in oICSS. We then examined several new synthetic cannabinoids, which are used recreationally. XLR-11 produced a cocaine-like increase, AM-2201 produced a Δ9 -THC-like reduction, while 5F-AMB had no effect on oICSS responding. Immunohistochemistry and RNAscope in situ hybridization assays indicated that CB1 Rs are expressed mainly in VTA GABA and glutamate neurons, while CB2 Rs are expressed mainly in VTA DA neurons. Together, these findings suggest that most cannabinoids are not reward enhancing, but rather reward attenuating or aversive in mice. Activation of CB1 R and/or CB2 R in different populations of neurons in the brain may underlie the observed actions.

Cocaine reward is reduced by decreased expression of receptor-type protein tyrosine phosphatase D (PTPRD) and by a novel PTPRD antagonist.

Receptor-type protein tyrosine phosphatase D (PTPRD) is a neuronal cell-adhesion molecule/synaptic specifier that has been implicated in addiction vulnerability and stimulant reward by human genomewide association and mouse cocaine-conditioned place-preference data. However, there have been no reports of effects of reduced expression on cocaine self-administration. There have been no reports of PTPRD targeting by any small molecule. There are no data about behavioral effects of any PTPRD ligand. We now report (i) robust effects of heterozygous PTPRD KO on cocaine self-administration (These data substantially extend prior conditioned place-preference data and add to the rationale for PTPRD as a target for addiction therapeutics.); (ii) identification of 7-butoxy illudalic acid analog (7-BIA) as a small molecule that targets PTPRD and inhibits its phosphatase with some specificity; (iii) lack of toxicity when 7-BIA is administered to mice acutely or with repeated dosing; (iv) reduced cocaine-conditioned place preference when 7-BIA is administered before conditioning sessions; and (v) reductions in well-established cocaine self-administration when 7-BIA is administered before a session (in WT, not PTPRD heterozygous KOs). These results add to support for PTPRD as a target for medications to combat cocaine use disorders. 7-BIA provides a lead compound for addiction therapeutics.

Genetic deletion of vesicular glutamate transporter in dopamine neurons increases vulnerability to MPTP-induced neurotoxicity in mice.

A subset of midbrain dopamine (DA) neurons express vesicular glutamate transporter 2 (VgluT2), which facilitates synaptic vesicle loading of glutamate. Recent studies indicate that such expression can modulate DA-dependent reward behaviors, but little is known about functional consequences of DA neuron VgluT2 expression in neurodegenerative diseases like Parkinson's disease (PD). Here, we report that selective deletion of VgluT2 in DA neurons in conditional VgluT2-KO (VgluT2-cKO) mice abolished glutamate release from DA neurons, reduced their expression of brain-derived neurotrophic factor (BDNF) and tyrosine receptor kinase B (TrkB), and exacerbated the pathological effects of exposure to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Furthermore, viral rescue of VgluT2 expression in DA neurons of VglutT2-cKO mice restored BDNF/TrkB expression and attenuated MPTP-induced DA neuron loss and locomotor impairment. Together, these findings indicate that VgluT2 expression in DA neurons is neuroprotective. Genetic or environmental factors causing reduced expression or function of VgluT2 in DA neurons may place some individuals at increased risk for DA neuron degeneration. Therefore, maintaining physiological expression and function of VgluT2 in DA neurons may represent a valid molecular target for the development of preventive therapeutic interventions for PD.

Cannabidiol inhibits sucrose self-administration by CB1 and CB2 receptor mechanisms in rodents.

A growing number of studies suggest therapeutic applications of cannabidiol (CBD), a recently U.S. Food and Drug Administration (FDA)-approved medication for epilepsy, in treatment of many other neuropsychological disorders. However, pharmacological action and the mechanisms by which CBD exerts its effects are not fully understood. Here, we examined the effects of CBD on oral sucrose self-administration in rodents and explored the receptor mechanisms underlying CBD-induced behavioral effects using pharmacological and transgenic approaches. Systemic administration of CBD (10, 20, and 40 mg/kg, ip) produced a dose-dependent reduction in sucrose self-administration in rats and in wild-type (WT) and CB1-/- mice but not in CB2-/- mice. CBD appeared to be more efficacious in CB1-/- mice than in WT mice. Similarly, pretreatment with AM251, a CB1R antagonist, potentiated, while AM630, a selective CB2R antagonist, blocked CBD-induced reduction in sucrose self-administration, suggesting the involvement of CB1 and CB2 receptors. Furthermore, systemic administration of JWH133, a selective CB2R agonist, also produced a dose-dependent reduction in sucrose self-administration in WT and CB1-/- mice, but not in CB2-/- mice. Pretreatment with AM251 enhanced, while AM630 blocked JWH133-induced reduction in sucrose self-administration in WT mice, suggesting that CBD inhibits sucrose self-administration likely by CB1 receptor antagonism and CB2 receptor agonism. Taken together, the present findings suggest that CBD may have therapeutic potential in reducing binge eating and the development of obesity.

Cannabinoid CB1 receptor neutral antagonist AM4113 inhibits heroin self-administration without depressive side effects in rats.

Cannabinoid CB1 receptors (CB1Rs) have been shown to be a promising target in medication development for the treatment of addiction. However, clinical trials with SR141716A (rimonabant, a selective CB1R antagonist/inverse agonist) for the treatment of obesity and smoking cessation failed due to unwanted side effects, such as depression, anxiety, and suicidal tendencies. Recent preclinical studies suggest that the neutral CB1R antagonist AM4113 may retain the therapeutic anti-addictive effects of SR141716A in nicotine self-administration models and possibly has fewer unwanted side effects. However, little is known about whether AM4113 is also effective for other drugs of abuse, such as opioids and psychostimulants, and whether it produces depressive side effects similar to SR141716A in experimental animals. In this study, we demonstrated that systemic administration of AM4113 (3 and 10 mg/kg) dose-dependently inhibited the self-administration of intravenous heroin but not cocaine or methamphetamine, whereas SR141716A (3 and 10 mg/kg) dose-dependently inhibited the self-administration of heroin and methamphetamine but not cocaine. In the electrical brain-stimulation reward (BSR) paradigm, SR141716A (3 and 10 mg/kg) dose-dependently increased the BSR stimulation threshold (i.e., decreased the stimulation reward), but AM4113 had no effect on BSR at the same doses, suggesting that SR141716A may produce aversive effects while AM4113 may not. Together, these findings show that neutral CB1R antagonists such as AM4113 deserve further research as a new class of CB1R-based medications for the treatment of opioid addiction without SR141716A-like aversive effects.

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.

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

We have recently reported the expression of functional cannabinoid CB2 receptors (CB2 Rs) in midbrain dopamine (DA) neurons in mice. However, little is known whether CB2 Rs are similarly expressed in rat brain because significant species differences in CB2 R 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 CB2 Rs 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 CB2 R antagonist. These data suggest that CB2 Rs are expressed in VTA DA neurons and functionally modulate DA neuronal activities and cocaine self-administration behavior in rats.

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

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 JWH133's 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 D(2) receptor availability in the neostriatum is associated with increased vulnerability to drug addiction in humans and experimental animals. The role of D(3) receptors (D(3)Rs) in the neurobiology of addiction remains unclear, however. Here we report that D(3)R KO (D(3)(-/-)) 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 D(3)(-/-) mice. In addition, D(3)(-/-) 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 D(3)R deletion increases vulnerability to cocaine, and that reduced D(3)R availability in the brain may constitute a risk factor for the development of cocaine addiction.

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.

ß-caryophyllene inhibits heroin self-administration, but does not alter opioid-induced antinociception in rodents.

A growing body of research indicates that ß-caryophyllene (BCP), a constituent present in a large number of plants, possesses significant therapeutic properties against CNS disorders, including alcohol and psychostimulant use disorders. However, it is unknown whether BCP has similar therapeutic potential for opioid use disorders. In this study, we found that systemic administration of BCP dose-dependently reduced heroin self-administration in rats under an FR2 schedule of reinforcement and partially blocked heroin-enhanced brain stimulation reward in DAT-cre mice, maintained by optical stimulation of midbrain dopamine neurons at high frequencies. Acute administration of BCP failed to block heroin conditioned place preference (CPP) in male mice, but attenuated heroin-induced CPP in females. Furthermore, repeated dosing with BCP for 5 days facilitated the extinction of CPP in female but not male mice. In the hot plate assay, pretreatment with the same doses of BCP failed to enhance or prolong opioid antinociception. Lastly, in a substitution test, BCP replacement for heroin failed to maintain intravenous BCP self-administration, suggesting that BCP itself has no reinforcing properties. These findings suggest that BCP may have certain therapeutic effects against opioid use disorders with fewer unwanted side-effects by itself.

AM6527, a neutral CB1 receptor antagonist, suppresses opioid taking and seeking, as well as cocaine seeking in rodents without aversive effects.

Preclinical research has demonstrated the efficacy of CB1 receptor (CB1R) antagonists in reducing drug-taking behavior. However, clinical trials with rimonabant, a CB1R antagonist with inverse agonist profile, failed due to severe adverse effects, such as depression and suicidality. As a result, efforts have shifted towards developing novel neutral CB1R antagonists without an inverse agonist profile for treating substance use disorders. Here, we assessed AM6527, a CB1R neutral antagonist, in addiction animal models. Our findings revealed that AM6527 did not affect cocaine self-administration under fixed-ratio reinforcement schedules but dose-dependently inhibited it under progressive-ratio reinforcement schedules. Additionally, AM6527 dose-dependently inhibited heroin self-administration under both fixed-ratio and progressive-ratio reinforcement schedules and oral sucrose self-administration under a fixed-ratio reinforcement schedule, as well as cocaine- or heroin-triggered reinstatement of drug-seeking behavior in rats. However, chronic AM6527 administration for five consecutive days significantly inhibited heroin self-administration only during the initial two days, indicating tolerance development. Notably, AM6527 did not produce rewarding or aversive effects by itself in classical electrical intracranial self-stimulation and conditioned place preference tests. However, in optical intracranial self-stimulation (oICSS) maintained by optogenetic stimulation of midbrain dopamine neurons in DAT-cre mice, both AM6527 and rimonabant dose-dependently inhibited dopamine-dependent oICSS behavior. Together, these findings suggest that AM6527 effectively reduces drug-taking and seeking behaviors without rimonabant-like adverse effects. Thus, AM6527 warrants further investigation as a potential pharmacotherapy for opioid and cocaine use disorders.

GPR55 is expressed in glutamate neurons and functionally modulates drug taking and seeking in rats and mice.

G protein-coupled receptor 55 (GPR55) has been thought to be a putative cannabinoid receptor. However, little is known about its functional role in cannabinoid action and substance use disorders. Here we report that GPR55 is predominantly found in glutamate neurons in the brain, and its activation reduces self-administration of cocaine and nicotine in rats and mice. Using RNAscope in situ hybridization, GPR55 mRNA was identified in cortical vesicular glutamate transporter 1 (VgluT1)-positive and subcortical VgluT2-positive glutamate neurons, with no detection in midbrain dopamine (DA) neurons. Immunohistochemistry detected a GPR55-like signal in both wildtype and GPR55-knockout mice, suggesting non-specific staining. However, analysis using a fluorescent CB1/GPR55 ligand (T1117) in CB1-knockout mice confirmed GPR55 binding in glutamate neurons, not in midbrain DA neurons. Systemic administration of the GPR55 agonist O-1602 didnt impact ∆9-THC-induced analgesia, hypothermia and catalepsy, but significantly mitigated cocaine-enhanced brain-stimulation reward caused by optogenetic activation of midbrain DA neurons. O-1602 alone failed to alter extracellar DA, but elevated extracellular glutamate, in the nucleus accumbens. In addition, O-1602 also demonstrated inhibitory effects on cocaine or nicotine self-administration under low fixed-ratio and/or progressive-ratio reinforcement schedules in rats and wildtype mice, with no such effects observed in GPR55-knockout mice. Together, these findings suggest that GPR55 activation may functionally modulate drug-taking and drug-seeking behavior possibly via a glutamate-dependent mechanism, and therefore, GPR55 deserves further study as a new therapeutic target for treating substance use disorders.

Involvement of dopamine D3 receptor in impulsive choice decision-making in male rats.

Impulsive decision-making has been linked to impulse control disorders and substance use disorders. However, the neural mechanisms underlying impulsive choice are not fully understood. While previous PET imaging and autoradiography studies have shown involvement of dopamine and D2/3 receptors in impulsive behavior, the roles of distinct D1, D2, and D3 receptors in impulsive decision-making remain unclear. In this study, we used a food reward delay-discounting task (DDT) to identify low- and high-impulsive rats, in which low-impulsive rats exhibited preference for large delayed reward over small immediate rewards, while high-impulsive rats showed the opposite preference. We then examined D1, D2, and D3 receptor gene expression using RNAscope in situ hybridization assays. We found that high-impulsive male rats exhibited lower levels of D2 and D3, and particularly D3, receptor expression in the nucleus accumbens (NAc), with no significant changes in the insular, prelimbic, and infralimbic cortices. Based on these findings, we further explored the role of the D3 receptor in impulsive decision-making. Systemic administration of a selective D3 receptor agonist (FOB02-04) significantly reduced impulsive choices in high-impulsive rats but had no effects in low-impulsive rats. Conversely, a selective D3 receptor antagonist (VK4-116) produced increased both impulsive and omission choices in both groups of rats. These findings suggest that impulsive decision-making is associated with a reduction in D3 receptor expression in the NAc. Selective D3 receptor agonists, but not antagonists, may hold therapeutic potentials for mitigating impulsivity in high-impulsive subjects.

Presynaptic and Postsynaptic Mesolimbic Dopamine D3 Receptors Play Distinct Roles in Cocaine Versus Opioid Reward in Mice.

BACKGROUND: Past research has illuminated pivotal roles of dopamine D3 receptors (D3R) in the rewarding effects of cocaine and opioids. However, the cellular and neural circuit mechanisms that underlie these actions remain unclear. METHODS: We employed Cre-LoxP techniques to selectively delete D3R from presynaptic dopamine neurons or postsynaptic dopamine D1 receptor (D1R)-expressing neurons in male and female mice. We utilized RNAscope in situ hybridization, immunohistochemistry, real-time polymerase chain reaction, voltammetry, optogenetics, microdialysis, and behavioral assays (n ≥ 8 animals per group) to functionally characterize the roles of presynaptic versus postsynaptic D3R in cocaine and opioid actions. RESULTS: Our results revealed D3R expression in ∼25% of midbrain dopamine neurons and ∼70% of D1R-expressing neurons in the nucleus accumbens. While dopamine D2 receptors (D2R) were expressed in ∼80% dopamine neurons, we found no D2R and D3R colocalization among these cells. Selective deletion of D3R from dopamine neurons increased exploratory behavior in novel environments and enhanced pulse-evoked nucleus accumbens dopamine release. Conversely, deletion of D3R from D1R-expressing neurons attenuated locomotor responses to D1-like and D2-like agonists. Strikingly, deletion of D3R from either cell type reduced oxycodone self-administration and oxycodone-enhanced brain-stimulation reward. In contrast, neither of these D3R deletions impacted cocaine self-administration, cocaine-enhanced brain-stimulation reward, or cocaine-induced hyperlocomotion. Furthermore, D3R knockout in dopamine neurons reduced oxycodone-induced hyperactivity and analgesia, while deletion from D1R-expressing neurons potentiated opioid-induced hyperactivity without affecting analgesia. CONCLUSIONS: We dissected presynaptic versus postsynaptic D3R function in the mesolimbic dopamine system. D2R and D3R are expressed in different populations of midbrain dopamine neurons, regulating dopamine release. Mesolimbic D3R are critically involved in the actions of opioids but not cocaine.