Purinergic P2X7 receptor antagonist inhibits methamphetamine-induced reward, hyperlocomotion, and cortical IL-7A levels in mice: A role for P2X7/IL-17A crosstalk in methamphetamine behaviors?

P2X7 receptors are dysregulated during psychostimulant exposure. Furthermore, P2X7 receptors enhance endogenous systems (e.g., cytokines, dopamine, and glutamate) that facilitate psychostimulant addiction. Therefore, using mouse locomotor, conditioned place preference (CPP), and intracranial self-stimulation (ICSS) assays, we tested the hypothesis that methamphetamine (METH) reward and acute locomotor activation requires P2X7 receptor activity. We also investigated effects of P2X7 blockade on METH-induced changes in cytokine levels in brain reward regions. A438079 (5, 10, 50 mg/kg), a P2X7 antagonist, did not affect spontaneous locomotor activity but reduced hyperlocomotion caused by acute METH (1 mg/kg) exposure. A438079 (10 mg/kg) also prevented expression of METH CPP without causing aversive or rewarding effects. For ICSS experiments, METH (1 mg/kg) facilitated brain reward function as interpreted from reductions in baseline threshold. In the presence of A438079 (50 mg/kg), METH-induced facilitation of ICSS was reduced. Repeated METH exposure (1 mg/kg × 7 d) caused enhancement of IL-17A levels in the prefrontal cortex (PFC) that was normalized by A438070 (10 mg/kg × 7 d). The present data suggest that P2X7 receptor activity contributes to rewarding and locomotor-stimulant effects of METH through a potential mechanism involving IL-17A, which has recently been implicated in anxiety.

Suvorexant, an orexin/hypocretin receptor antagonist, attenuates motivational and hedonic properties of cocaine.

Orexins ('hypocretins') are peptides produced by neurons of the hypothalamus that project to structures implicated in reward and emotion processing. Converging evidence demonstrates functional roles of orexin signaling in arousal, sleep/wakefulness and motivated behaviors for natural and drug rewards. Suvorexant, a dual orexin receptor antagonist, recently received approval from the US Food and Drug Administration to treat insomnia. In Experiment 1, rats self-administered cocaine under a progressive-ratio schedule of reinforcement and the effects of suvorexant on motivation to self-administer cocaine were measured. In Experiment 2, the effects of suvorexant on cocaine reward were assessed by using a place conditioning paradigm, and 50-kHz ultrasonic vocalizations were also recorded to track changes in hedonic reactivity to cocaine. To rule out potentially confounding effects of suvorexant-induced somnolence, locomotor activity was also measured. In Experiment 3, the effects of suvorexant on cocaine-evoked elevations in ventral striatal dopamine were examined. Data reveal that suvorexant (i) reduced the number of cocaine infusions earned during progressive-ratio self-administration; (ii) attenuated initial positive hedonic reactivity to cocaine and prevented cocaine place preference; (iii) did not affect cocaine-induced hyperlocomotion and (iv) reduced cocaine-induced elevations in extracellular ventral striatal dopamine. The present study examined the therapeutic potential of suvorexant in rodent models of cocaine use disorder. These results contribute toward a growing literature supporting therapeutic roles of orexin receptor antagonists in treating substance use disorders.

Comparing rewarding and reinforcing properties between 'bath salt' 3,4-methylenedioxypyrovalerone (MDPV) and cocaine using ultrasonic vocalizations in rats.

Abuse of synthetic psychostimulants like synthetic cathinones has risen in recent years. 3,4-Methylenedioxypyrovalerone (MDPV) is one such synthetic cathinone that demonstrates a mechanism of action similar to cocaine. Compared to cocaine, MDPV is more potent at blocking dopamine and norepinephrine reuptake and is readily self-administered by rodents. The present study compared the rewarding and reinforcing properties of MDPV and cocaine using systemic injection dose-response and self-administration models. Fifty kilohertz ultrasonic vocalizations (USVs) were recorded as an index of positive affect throughout experiments. In Experiment 1, MDPV and cocaine dose-dependently elicited 50-kHz USVs upon systemic injection, but MDPV increased USVs at greater rates and with greater persistence relative to cocaine. In Experiment 2, latency to begin MDPV self-administration was shorter than latency to begin cocaine self-administration, and self-administered MDPV elicited greater and more persistent rates of 50-kHz USVs versus cocaine. MDPV-elicited 50-kHz USVs were sustained over the course of drug load-up whereas cocaine-elicited USVs waned following initial infusions. Notably, we observed a robust presence of context-elicited 50-kHz USVs from both MDPV and cocaine self-administering rats. Collectively, these data suggest that MDPV has powerfully rewarding and reinforcing effects relative to cocaine at one-tenth doses. Consistent with prior work, we additionally interpret these data in supporting that MDPV has significant abuse risk based on its potency and subjectively positive effects. Future studies will be needed to better refine therapeutic strategies targeted at reducing the rewarding effects of cathinone analogs in efforts to ultimately reduce abuse liability.

Cocaine abuse and midbrain circuits: Functional anatomy of hypocretin/orexin transmission and therapeutic prospect.

Cocaine abuse remains a pervasive public health problem, and treatments thus far have proven ineffective for long-term abstinence maintenance. Intensive research on the neurobiology underlying drug abuse has led to the consideration of many candidate transmitter systems to target for intervention. Among these, the hypocretin/orexin (hcrt/ox) neuropeptide system holds largely untapped yet clinically viable therapeutic potential. Hcrt/ox originates from the hypothalamus and projects widely across the mammalian central nervous system to produce neuroexcitatory actions via two excitatory G-protein coupled receptor subtypes. Functionally, hcrt/ox promotes arousal/wakefulness and facilitates energy homeostasis. In the early 2000s, hcrt/ox transmission was shown to underlie mating behavior in male rats suggesting a novel role in reward-seeking. Soon thereafter, hcrt/ox neurons were shown to respond to drug-associated stimuli, and hcrt/ox transmission was found to facilitate motivated responding for intravenous cocaine. Notably, blocking hcrt/ox transmission using systemic or site-directed pharmacological antagonists markedly reduced motivated drug-taking as well as drug-seeking in tests of relapse. This review will unfold the current state of knowledge implicating hcrt/ox receptor transmission in the context of cocaine abuse and provide detailed background on animal models and underlying midbrain circuits. Specifically, attention will be paid to the mesoaccumbens, tegmental, habenular, pallidal and preoptic circuits. The review will conclude with discussion of recent preclinical studies assessing utility of suvorexant - the first and only FDA-approved hcrt/ox receptor antagonist - against cocaine-associated behaviors.

The kappa opioid receptor agonist U50,488H did not affect brain-stimulation reward while it elicited conditioned place aversion in mice.

OBJECTIVE: Selective kappa opioid receptor (KOR) agonists were shown to produce a dose-dependent depression of brain-stimulation reward (BSR) in the rat intracranial self-stimulation (ICSS) tests. However, limited studies using mice produced less conclusive results. Here the effects of U50,488H were re-examined on BSR in mice with a larger cohort of animals. RESULTS: Forty C57BL/6J male mice were implanted with the electrodes in medial forebrain bundle. About a week after surgery, mice were subject to ICSS training. Only eighteen passed the two-phase procedures, at which point they readily spun the wheels to obtain reinforcing effect of BSR, and were used for the ICSS tests. Compared with saline (s.c.), U50,488H (2 mg/kg, s.c.) did not have effects on the BSR thresholds within 1 h post-treatment, while it decreased the maximum wheel-spinning rates in a time-dependent manner. In contrast, cocaine (5 mg/kg, s.c.) decreased the BSR thresholds time-dependently without affecting the maximum wheel-spinning rates in the same cohort of mice, demonstrating the validity of our mouse ICSS models. For comparison, U50,488H (2 mg/kg, s.c.) induced significant conditioned place aversion (CPA) in a different cohort of mice without surgeries. Thus, ICSS may not be an appropriate test for KOR agonist-induced aversion in mice.

Synthetic cathinone MDPV enhances reward function through purinergic P2X7 receptor-dependent pathway and increases P2X7 gene expression in nucleus accumbens.

BACKGROUND AND PURPOSE: Purinergic P2X7 receptors are present on neurons, astrocytes and microglia and activated by extracellular ATP. Since P2X7 receptor activation releases endogenous substrates (e.g., pro-inflammatory cytokines, dopamine, and glutamate) that facilitate psychostimulant reward and reinforcement, we investigated the hypothesis that the synthetic cathinone 3,4-methylenedioxypyrovalerone (MDPV) produces rewarding effects that are dependent on active P2X7 receptors. METHODS: Reward function was measured in male mice using intracranial self-stimulation (ICSS). MDPV (0.1, 0.3, 0.5 mg/kg, SC) and a selective P2X7 antagonist (A438079) (5, 10, 50 mg/kg, IP) were tested alone and in combination. In separate mice, gene and protein expression of P2X7 and mitochondrial adenosine triphosphate (ATP) synthase (an enzyme that catalyzes synthesis of ATP, an endogenous ligand for P2X7 receptors) in the nucleus accumbens (NAcc) were quantified following MDPV exposure (0.1, 0.5, 5 mg/kg, SC). KEY RESULTS: MDPV (0.5 mg/kg, SC) facilitated ICSS as quantified by a significant reduction in brain reward threshold. A438079 (5, 10, 50 mg/kg, IP) did not affect ICSS by itself; however, for combined administration, A438079 (10 mg/kg, IP) inhibited facilitation of ICSS by MDPV (0.5 mg/kg, SC). At the cellular level, MDPV exposure increased gene and protein expression of P2X7 and ATP synthase in the NAcc. CONCLUSION AND IMPLICATION: We provide evidence that a psychostimulant drug produces reward enhancement that is influenced by P2X7 receptor activity and enhances P2X7 receptor expression in the brain reward circuit.

Phosphoproteomic approach for agonist-specific signaling in mouse brains: mTOR pathway is involved in κ opioid aversion.

Kappa opioid receptor (KOR) agonists produce analgesic and anti-pruritic effects, but their clinical application was limited by dysphoria and hallucinations. Nalfurafine, a clinically used KOR agonist, does not cause dysphoria or hallucinations at therapeutic doses in humans. We found that in CD-1 mice nalfurafine produced analgesic and anti-scratch effects dose-dependently, like the prototypic KOR agonist U50,488H. In contrast, unlike U50,488H, nalfurafine caused no aversion, anhedonia, or sedation or and a low level of motor incoordination at the effective analgesia and anti-scratch doses. Thus, we established a mouse model that recapitulated important aspects of the clinical observations. We then employed a phosphoproteomics approach to investigate mechanisms underlying differential KOR-mediated effects. A large-scale mass spectrometry (MS)-based analysis on brains revealed that nalfurafine perturbed phosphoproteomes differently from U50,488H in a brain-region specific manner after 30-min treatment. In particular, U50,488H and nalfurafine imparted phosphorylation changes to proteins found in different cellular components or signaling pathways in different brain regions. Notably, we observed that U50,488H, but not nalfurafine, activated the mammalian target of rapamycin (mTOR) pathway in the striatum and cortex. Inhibition of the mTOR pathway by rapamycin abolished U50,488H-induced aversion, without affecting analgesic, anti-scratch, and sedative effects and motor incoordination. The results indicate that the mTOR pathway is involved in KOR agonist-induced aversion. This is the first demonstration that phosphoproteomics can be applied to agonist-specific signaling of G protein-coupled receptors (GPCRs) in mouse brains to unravel pharmacologically important pathways. Furthermore, this is one of the first two reports that the mTOR pathway mediates aversion caused by KOR activation.

Effects of Suvorexant, a Dual Orexin/Hypocretin Receptor Antagonist, on Impulsive Behavior Associated with Cocaine.

Hypothalamic hypocretin (orexin) peptides mediate arousal, attention, and reward processing. Fibers containing orexins project to brain structures that govern motivated behavior, including the ventral tegmental area (VTA). A number of psychiatric conditions, including attention deficit hyperactivity disorder (ADHD) and substance use disorders, are characterized by deficits in impulse control, however the relationship between orexin and impulsive behavior is incompletely characterized. The effects of systemic or centrally administered orexin receptor (OXR) antagonists on measures of impulsive-like behavior in rats were evaluated using the five-choice serial reaction time task (5-CSRTT) and delay discounting procedures. These paradigms were also used to test the capacity of OXR antagonists to attenuate acute cocaine-evoked impulsivity. Finally, immunohistochemistry and calcium imaging were used to assess potential cellular mechanisms by which OXR blockade may influence motor impulsivity. Suvorexant, a dual (OX1/2R) orexin receptor antagonist, reduced cocaine-evoked premature responses in 5-CSRTT when administered systemically or directly into VTA. Neither suvorexant nor OX1R- or OX2R-selective compounds (SB334867 or TCS-OX2-29, respectively) altered delay discounting. Finally, suvorexant did not alter Fos-immunoreactivity within tyrosine hydroxylase-immunolabeled neurons of VTA, but did attenuate cocaine- and orexin-induced increases in calcium transient amplitude within neurons of VTA. Results from the present studies suggest potential therapeutic utility of OXR antagonists in reducing psychostimulant-induced motor impulsivity. These findings also support the view that orexin transmission is closely involved in executive function in normal and pathological conditions.

Role of hypocretin/orexin receptor blockade on drug-taking and ultrasonic vocalizations (USVs) associated with low-effort self-administration of cathinone-derived 3,4-methylenedioxypyrovalerone (MDPV) in rats.

RATIONALE: Synthetic psychostimulant abuse, including cathinone-derived 3,4-methylenedioxypyrovalerone (MDPV), continues to increase in many countries. Similar to cocaine but with greater potency, MDPV elicits a transient sympathomimetic response by blocking cellular uptake of dopamine (DA) and norepinephrine (NE)-administration in some users is reported as euphoria-inducing much like cocaine and amphetamine. Pharmacological agents that disrupt excitatory transmission onto midbrain DA-producing neurons, including hypothalamic hypocretin/orexin (hcrt/ox) receptor antagonists, present attractive targets to aide abstinence maintenance by reducing psychostimulant-associated reward and reinforcement. OBJECTIVE: The present study sought to assess the degree to which suvorexant, a dual hcrt/ox receptor antagonist, influences drug-taking as well as ultrasonic vocalizations (USVs) associated with MDPV self-administration. METHODS: Rats were trained to self-administer MDPV (~0.03 mg/kg/inf, 3-s) for 14 days under a fixed-ratio 1 schedule of reinforcement, and effects of suvorexant (0, 3, 10, 30 mg/kg, i.p.) on drug-taking was assessed. USVs were recorded during a 30-min pre-lever period as well as during 2-h of MDPV self-administration. RESULTS: We observed that suvorexant modestly suppressed the number of MDPV infusions earned. Notably, we observed that suvorexant reduced 50-kHz USVs associated with pre- and post-lever time-points but did not noticeably alter call type profiles. Upon comparison of the two measures, we observed trending positive associations between suvorexant-induced changes in drug-taking and 50-kHz USVs. CONCLUSIONS: Results from this exploratory study provide support for the following: (1) studying how suvorexant may provide benefit to humans with stimulant use disorders, (2) identifying a potential role for orexin transmission in cathinone abuse, and (3) further interrogating the potential utility of rat USVs to predict drug consumption in preclinical models of substance use disorders.

Stereoselective Differences between the Reinforcing and Motivational Effects of Cathinone-Derived 4-Methylmethcathinone (Mephedrone) In Self-Administering Rats.

Mephedrone (4-methylmethcathinone (4-MMC)) (MEPH) is a new psychoactive substance (NPS) of the synthetic cathinone class. MEPH has a chiral center and exists as two enantiomers (R-,S-MEPH), yet stereospecific effects of MEPH have not been extensively investigated in preclinical assays. Because significant behavioral and neurochemical differences can exist between enantiomers, probing effects of stereochemistry on biological activity enables separation of adverse and therapeutic effects. Our prior work showed that R-MEPH, relative to S-MEPH, produced greater locomotor activation, place preference, and facilitation of brain reward thresholds in rodents. The present study sought to determine if MEPH enantiomers display stereospecific reward and reinforcement in rat self-administration assays. In Experiment 1, rats were trained to self-administer racemic MEPH (0.50 mg/kg/inf), and dose substitution effects of R-MEPH (0.50 mg/kg/inf) and S-MEPH (0.25, 0.50, 2.00 mg/kg/inf) were examined. In Experiment 2, separate rats were trained to self-administer R-MEPH (0.25, 0.50, 2.00 mg/kg/inf) or S-MEPH (0.25, 0.50, 2.00 mg/kg/inf) and were thereafter evaluated under progressive-ratio access conditions. Within this cohort, 50 kHz ultrasonic vocalizations (USVs) were recorded to measure potential differences in subjective positive affect associated with MEPH enantiomer self-administration. We identified enantiomer- and dose-dependent effects on infusions earned during self-administration following acquisition of racemic MEPH, with greatest infusions under low-effort, fixed-ratio 1 access conditions from low-dose S-MEPH self-administration. When taxed with progressive-ratio access conditions, rats trained to self-administer R-MEPH showed higher break points than those of rats trained to self-administer S-MEPH. Additionally, R-MEPH elicited greatest rates of 50 kHz USVs compared to S-MEPH. Taken together, these data suggest that the R-enantiomer of MEPH is primarily responsible for the rewarding, reinforcing, and motivational properties of racemic MEPH, which increases our understanding of stereospecific preferences pertaining to MEPH abuse.

Nicotinic receptor blockade decreases fos immunoreactivity within orexin/hypocretin-expressing neurons of nicotine-exposed rats.

Tobacco smoking is the leading cause of preventable death in the United States. Nicotine is the principal psychoactive ingredient in tobacco that causes addiction. The structures governing nicotine addiction, including those underlying withdrawal, are still being explored. Nicotine withdrawal is characterized by negative affective and cognitive symptoms that enhance relapse susceptibility, and suppressed dopaminergic transmission from ventral tegmental area (VTA) to target structures underlies behavioral symptoms of nicotine withdrawal. Agonist and partial agonist therapies help 1 in 4 treatment-seeking smokers at one-year post-cessation, and new targets are needed to more effectively aid smokers attempting to quit. Hypothalamic orexin/hypocretin neurons send excitatory projections to dopamine (DA)-producing neurons of VTA and modulate mesoaccumbal DA release. The effects of nicotinic receptor blockade, which is commonly used to precipitate withdrawal, on orexin neurons remain poorly investigated and present an attractive target for intervention. The present study sought to investigate the effects of nicotinic receptor blockade on hypothalamic orexin neurons using mecamylamine to precipitate withdrawal in rats. Separate groups of rats were treated with either chronic nicotine or saline for 7-days at which point effects of mecamylamine or saline on somatic signs and anxiety-like behavior were assessed. Finally, tissue from rats was harvested for immunofluorescent analysis of Fos within orexin neurons. Results demonstrate that nicotinic receptor blockade leads to reduced orexin cell activity, as indicated by lowered Fos-immunoreactivity, and suggest that this underlying cellular activity may be associated with symptoms of nicotine withdrawal as effects were most prominently observed in rats given chronic nicotine. We conclude from this study that orexin transmission becomes suppressed in rats upon nicotinic receptor blockade, and that behavioral symptoms associated with nicotine withdrawal may be aided by intervention upon orexinergic transmission.