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.

New Drugs, Old Targets: Tweaking the Dopamine System to Treat Psychostimulant Use Disorders.

The abuse of illicit psychostimulants such as cocaine and methamphetamine continues to pose significant health and societal challenges. Despite considerable efforts to develop medications to treat psychostimulant use disorders, none have proven effective, leaving an underserved patient population and unanswered questions about what mechanism(s) of action should be targeted for developing pharmacotherapies. As both cocaine and methamphetamine rapidly increase dopamine (DA) levels in mesolimbic brain regions, leading to euphoria that in some can lead to addiction, targets in which this increased dopaminergic tone may be mitigated have been explored. Further, understanding and targeting mechanisms underlying relapse are fundamental to the success of discovering medications that reduce the reinforcing effects of the drug of abuse, decrease the negative reinforcement or withdrawal/negative affect that occurs during abstinence, or both. Atypical inhibitors of the DA transporter and partial agonists/antagonists at DA D3 receptors are described as two promising targets for future drug development.

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.

Optical Intracranial Self-Stimulation (oICSS): A New Behavioral Model for Studying Drug Reward and Aversion in Rodents.

Brain-stimulation reward, also known as intracranial self-stimulation (ICSS), is a commonly used procedure for studying brain reward function and drug reward. In electrical ICSS (eICSS), an electrode is surgically implanted into the medial forebrain bundle (MFB) in the lateral hypothalamus or the ventral tegmental area (VTA) in the midbrain. Operant lever responding leads to the delivery of electrical pulse stimulation. The alteration in the stimulation frequency-lever response curve is used to evaluate the impact of pharmacological agents on brain reward function. If a test drug induces a leftward or upward shift in the eICSS response curve, it implies a reward-enhancing or abuse-like effect. Conversely, if a drug causes a rightward or downward shift in the functional response curve, it suggests a reward-attenuating or aversive effect. A significant drawback of eICSS is the lack of cellular selectivity in understanding the neural substrates underlying this behavior. Excitingly, recent advancements in optical ICSS (oICSS) have facilitated the development of at least three cell type-specific oICSS models-dopamine-, glutamate-, and GABA-dependent oICSS. In these new models, a comparable stimulation frequency-lever response curve has been established and employed to study the substrate-specific mechanisms underlying brain reward function and a drug's rewarding versus aversive effects. In this review article, we summarize recent progress in this exciting research area. The findings in oICSS have not only increased our understanding of the neural mechanisms underlying drug reward and addiction but have also introduced a novel behavioral model in preclinical medication development for treating substance use disorders.

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.

Pharmacological targeting of G protein-coupled receptor heteromers.

A main rationale for the role of G protein-coupled receptor (GPCR) heteromers as targets for drug development is the putative ability of selective ligands for specific GPCRs to change their pharmacological properties upon GPCR heteromerization. The present study provides a proof of concept for this rationale by demonstrating that heteromerization of dopamine D1 and D3 receptors (D1R and D3R) influences the pharmacological properties of three structurally similar selective dopamine D3R ligands, the phenylpiperazine derivatives PG01042, PG01037 and VK4-116. By using D1R-D3R heteromer-disrupting peptides, it could be demonstrated that the three D3R ligands display different D1R-D3R heteromer-dependent pharmacological properties: PG01042, acting as G protein-biased agonist, counteracted D1R-mediated signaling in the D1R-D3R heteromer; PG01037, acting as a D3R antagonist cross-antagonized D1R-mediated signaling in the D1R-D3R heteromer; and VK4-116 specifically acted as a ß-arrestin-biased agonist in the D1R-D3R heteromer. Molecular dynamics simulations predicted potential molecular mechanisms mediating these qualitatively different pharmacological properties of the selective D3R ligands that are dependent on D1R-D3R heteromerization. The results of in vitro experiments were paralleled by qualitatively different pharmacological properties of the D3R ligands in vivo. The results supported the involvement of D1R-D3R heteromers in the locomotor activation by D1R agonists in reserpinized mice and L-DOPA-induced dyskinesia in rats, highlighting the D1R-D3R heteromer as a main pharmacological target for L-DOPA-induced dyskinesia in Parkinson's disease. More generally, the present study implies that when suspecting its pathogenetic role, a GPCR heteromer, and not its individual GPCR units, should be considered as main target for drug development.

Repeated cocaine administration upregulates CB2 receptor expression in striatal medium-spiny neurons that express dopamine D1 receptors in mice.

Cannabinoid CB2 receptors (CB2R) are importantly involved in drug reward and addiction. However, the cellular mechanisms underlying CB2R action remain unclear. We have previously reported that cocaine self-administration upregulates CB2R expression in midbrain dopamine (DA) neurons. In the present study, we investigated whether cocaine or heroin also alters CB2R expression in striatal medium-spiny neurons that express dopamine D1 or D2 receptors (D1-MSNs, D2-MSNs) and microglia. Due to the concern of CB2R antibody specificity, we developed three mouse CB2-specific probes to detect CB2R mRNA using quantitative RT-PCR and RNAscope in situ hybridization (ISH) assays. We found that a single injection of cocaine failed to alter, while repeated cocaine injections or self-administration dose-dependently upregulated CB2R gene expression in both brain (cortex and striatum) and periphery (spleen). In contrast, repeated administration of heroin produced a dose-dependent reduction in striatal CB2 mRNA expression. RNAscope ISH assays detected CB2R mRNA in striatal D1- and D2-MSNs, not in microglia. We then used transgenic CX3CR1eGFP/+ microglia reporter mice and D1- or D2-Cre-RiboTag mice to purify striatal microglia or ribosome-associated mRNAs from CX3CR1eGFP/+, D1-MSNs, or D2-MSNs, respectively. We found that CB2R upregulation occurred mainly in D1-MSNs, not in D2-MSNs or microglia, in the nucleus accumbens rather than the dorsal striatum. These findings indicate that repeated cocaine exposure may upregulate CB2R expression in both brain and spleen, with regional and cell type-specific profiles. In the striatum, CB2R upregulation occurs mainly in D1-MSNs in the nucleus accumbens. Given the important role of D1-MSNs in brain reward function, the present findings provide new insight into mechanisms by which brain CB2Rs modulate cocaine action.

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.

Possible Receptor Mechanisms Underlying Cannabidiol Effects on Addictive-like Behaviors in Experimental Animals.

Substance use disorder (SUD) is a serious public health problem worldwide for which available treatments show limited effectiveness. Since the legalization of cannabis and the approval of cannabidiol (CBD) by the US Food and Drug Administration, therapeutic potential of CBD for the treatment of SUDs and other diseases has been widely explored. In this mini-review article, we first review the history and evidence supporting CBD as a potential pharmacotherapeutic. We then focus on recent progress in preclinical research regarding the pharmacological efficacy of CBD and the underlying receptor mechanisms on addictive-like behavior. Growing evidence indicates that CBD has therapeutic potential in reducing drug reward, as assessed in intravenous drug self-administration, conditioned place preference and intracranial brain-stimulation reward paradigms. In addition, CBD is effective in reducing relapse in experimental animals. Both in vivo and in vitro receptor mechanism studies indicate that CBD may act as a negative allosteric modulator of type 1 cannabinoid (CB1) receptor and an agonist of type 2 cannabinoid (CB2), transient receptor potential vanilloid 1 (TRPV1), and serotonin 5-HT1A receptors. Through these multiple-receptor mechanisms, CBD is believed to modulate brain dopamine in response to drugs of abuse, leading to attenuation of drug-taking and drug-seeking behavior. While these findings suggest that CBD is a promising therapeutic candidate, further investigation is required to verify its safety, pharmacological efficacy and the underlying receptor mechanisms in both experimental animals and humans.

Newly Developed Dopamine D3 Receptor Antagonists, R-VK4-40 and R-VK4-116, Do Not Potentiate Cardiovascular Effects of Cocaine or Oxycodone in Rats.

Opioid and cocaine abuse are major public health burdens. Existing medications for opioid use disorder are limited by abuse liability and side effects, whereas no treatments are currently approved in the United States for cocaine use disorder. Dopamine D3 receptor (D3R) antagonists have shown promise in attenuating opioid and cocaine reward and mitigating relapse in preclinical models. However, translation of D3R antagonists to the clinic has been hampered by reports that the D3R antagonists GSK598,809 (5-(5-((3-((1S,5R)-1-(2-fluoro-4-(trifluoromethyl)phenyl)-3-azabicyclo[3.1.0]hexan-3-yl)propyl)thio)-4-methyl-4H-1,2,4-triazol-3-yl)-4-methyloxazole) and SB-277,011A (2-(2-((1r,4r)-4-(2-oxo-2-(quinolin-4-yl)ethyl)cyclohexyl)ethyl)-1,2,3,4-tetrahydroisoquinoline-6-carbonitrile) have adverse cardiovascular effects in the presence of cocaine. Recently, we developed two structurally novel D3R antagonists, R-VK4-40 and R-VK4-116, which are highly selective for D3R and display translational potential for treatment of opioid use disorder. Here, we tested whether R-VK4-40 ((R)-N-(4-(4-(2-Chloro-3-ethylphenyl)piperazin-1-yl)-3-hydroxybutyl)-1H-indole-2-carboxamide) and R-VK4-116 ((R)-N-(4-(4-(3-Chloro-5-ethyl-2-methoxyphenyl)piperazin-1-yl)-3-hydroxybutyl)-1H-indole-2-carboxamide) have unwanted cardiovascular effects in the presence of oxycodone, a prescription opioid, or cocaine in freely moving rats fitted with surgically implanted telemetry transmitters. We also examined cardiovascular effects of the D3R antagonist, SB-277,011A, and L-741,626 (1-((1H-indol-3-yl)methyl)-4-(4-chlorophenyl)piperidin-4-ol), a dopamine D2 receptor-selective antagonist, for comparison. Consistent with prior reports, SB-277,011A increased blood pressure, heart rate, and locomotor activity alone and in the presence of cocaine. L-741,626 increased blood pressure and heart rate. In contrast, R-VK4-40 alone dose-dependently reduced blood pressure and heart rate and attenuated oxycodone-induced increases in blood pressure and oxycodone or cocaine-induced increases in heart rate. Similarly, R-VK4-116 alone dose-dependently reduced cocaine-induced increases in blood pressure and heart rate. These results highlight the safety of new D3R antagonists and support the continued development of R-VK4-40 and R-VK4-116 for the treatment of opioid and cocaine use disorders. SIGNIFICANCE STATEMENT: Opioid and cocaine abuse are major public health challenges and new treatments that do not adversely impact the cardiovascular system are needed. Here, we show that two structurally novel dopamine D3 receptor antagonists, R-VK4-40 and R-VK4-116, do not potentiate, and may even protect against, oxycodone- or cocaine-induced changes in blood pressure and heart rate, supporting their further development for the treatment of opioid and/or cocaine use disorders.

CB2 receptor antibody signal specificity: correlations with the use of partial CB2-knockout mice and anti-rat CB2 receptor antibodies.

Cannabinoid CB1 receptors are highly expressed in the brain and functionally modulate presynaptic neurotransmitter release, while cannabinoid CB2 receptors (CB2Rs) were initially identified in the spleen and regarded as peripheral cannabinoid receptors. Recently, growing evidence indicates the presence of functional CB2Rs in the brain. However, this finding is disputed because of the specificity of CB2R antibody signals. We used two strains of currently available partial CB2-knockout (CB2-KO) mice as controls, four anti-rat or anti-mouse CB2R antibodies, and mRNA quantification to further address this issue. Western blot assays using the four antibodies detected a CB2R-like band at ~40 kD in both the brain and spleen. Notably, more bands were detected in the brain than in the spleen, and specific immune peptides blocked band detection. Immunohistochemical assays also detected CB2-like immunostaining in mouse midbrain dopamine neurons. CB2R deletion in CB2-KO mice may reduce or leave CB2R-like immunoreactivity unaltered depending on antibody epitope. Antibodies with epitopes at the receptor-deleted region detected a significant reduction in CB2R band density and immunostaining in N-terminal-deleted Deltagen and C-terminal-deleted Zimmer strain CB2-KO mice. Other antibodies with epitopes at the predicted receptor-undeleted regions detected similar band densities and immunostaining in wild-type and CB2-KO mice. Quantitative RT-PCR assays detected CB2 mRNA expression using probes that targeted upstream or downstream gene sequences but not the probe that targeted the gene-deleted sequence in Deltagen or Zimmer CB2-KO mice. These findings suggest that none of the tested four polyclonal antibodies are highly mouse CB2R-specific. Non-specific binding may be related to the expression of mutant or truncated CB2R-like proteins in partial CB2-KO mice and the use of anti-rat CB2 antibodies because the epitopes are different between rat and mouse CB2Rs.

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.

Identification of novel mouse and rat CB1R isoforms and in silico modeling of human CB1R for peripheral cannabinoid therapeutics.

Targeting peripheral CB1R is desirable for the treatment of metabolic syndromes without adverse neuropsychiatric effects. We previously reported a human hCB1b isoform that is selectively enriched in pancreatic beta-cells and hepatocytes, providing a potential peripheral therapeutic hCB1R target. It is unknown whether there are peripherally enriched mouse and rat CB1R (mCB1 and rCB1, respectively) isoforms. In this study, we found no evidence of peripherally enriched rodent CB1 isoforms; however, some mCB1R isoforms are absent in peripheral tissues. We show that the mouse Cnr1 gene contains six exons that are transcribed from a single promoter. We found that mCB1A is a spliced variant of extended exon 1 and protein-coding exon 6; mCB1B is a novel spliced variant containing unspliced exon 1, intron 1, and exon 2, which is then spliced to exon 6; and mCB1C is a spliced variant including all 6 exons. Using RNAscope in situ hybridization, we show that the isoforms mCB1A and mCB1B are expressed at a cellular level and colocalized in GABAergic neurons in the hippocampus and cortex. RT-qPCR reveals that mCB1A and mCB1B are enriched in the brain, while mCB1B is not expressed in the pancreas or the liver. Rat rCB1R isoforms are differentially expressed in primary cultured neurons, astrocytes, and microglia. We also investigated modulation of Cnr1 expression by insulin in vivo and carried out in silico modeling of CB1R with JD5037, a peripherally restricted CB1R inverse agonist, using the published crystal structure of hCB1R. The results provide models for future CB1R peripheral targeting.

Sigma-1 receptor mediates cocaine-induced transcriptional regulation by recruiting chromatin-remodeling factors at the nuclear envelope.

The sigma-1 receptor (Sig-1R) chaperone at the endoplasmic reticulum (ER) plays important roles in cellular regulation. Here we found a new function of Sig-1R, in that it translocates from the ER to the nuclear envelope (NE) to recruit chromatin-remodeling molecules and regulate the gene transcription thereof. Sig-1Rs mainly reside at the ER-mitochondrion interface. However, on stimulation by agonists such as cocaine, Sig-1Rs translocate from ER to the NE, where Sig-1Rs bind NE protein emerin and recruit chromatin-remodeling molecules, including lamin A/C, barrier-to-autointegration factor (BAF), and histone deacetylase (HDAC), to form a complex with the gene repressor specific protein 3 (Sp3). Knockdown of Sig-1Rs attenuates the complex formation. Cocaine was found to suppress the gene expression of monoamine oxidase B (MAOB) in the brain of wild-type but not Sig-1R knockout mouse. A single dose of cocaine (20 mg/kg) in rats suppresses the level of MAOB at nuclear accumbens without affecting the level of dopamine transporter. Daily injections of cocaine in rats caused behavioral sensitization. Withdrawal from cocaine in cocaine-sensitized rats induced an apparent time-dependent rebound of the MAOB protein level to about 200% over control on day 14 after withdrawal. Treatment of cocaine-withdrawn rats with the MAOB inhibitor deprenyl completely alleviated the behavioral sensitization to cocaine. Our results demonstrate a role of Sig-1R in transcriptional regulation and suggest cocaine may work through this newly discovered genomic action to achieve its addictive action. Results also suggest the MAOB inhibitor deprenyl as a therapeutic agent to block certain actions of cocaine during withdrawal.

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.