Muscarinic and NMDA Receptors in the Substantia Nigra Play a Role in Reward-Related Learning.

BACKGROUND: Reward-related learning, where animals form associations between rewards and stimuli (i.e., conditioned stimuli [CS]) that predict or accompany those rewards, is an essential adaptive function for survival. METHODS: In this study, we investigated the mechanisms underlying the acquisition and performance of conditioned approach learning with a focus on the role of muscarinic acetylcholine (mACh) and NMDA glutamate receptors in the substantia nigra (SN), a brain region implicated in reward and motor processes. RESULTS: Using RNAscope in situ hybridization assays, we found that dopamine neurons of the SN express muscarinic (mACh5), NMDA2a, NMDA2b, and NMDA2d receptor mRNA but not mACh4. NMDA, but not mACh5, receptor mRNA was also found on SN GABA neurons. In a conditioned approach paradigm, rats were exposed to 3 or 7 conditioning sessions during which light/tone (CS) presentations were paired with delivery of food pellets, followed by a test session with CS-only presentations. Intra-SN microinjections of scopolamine (a mACh receptor antagonist) or AP-5 (a NMDA receptor antagonist) were made either prior to each conditioning session (to test their effects on acquisition) or prior to the CS-only test (to test their effects on expression of the learned response). Scopolamine and AP-5 produced dose-dependent significant reductions in the acquisition, but not performance, of conditioned approach. CONCLUSIONS: These results suggest that SN mACh and NMDA receptors are key players in the acquisition, but not the expression, of reward-related learning. Importantly, these findings redefine the role of the SN, which has traditionally been known for its involvement in motor processes, and suggest that the SN possesses attributes consistent with a function as a hub of integration of primary reward and CS signals.

The Impact of Heroin Self-Administration and Environmental Enrichment on Ventral Tegmental CRF1 Receptor Expression.

BACKGROUND: There is a strong link between chronic stress and vulnerability to drug abuse and addiction. Corticotropin releasing factor (CRF) is central to the stress response that contributes to continuation and relapse to heroin abuse. Chronic heroin exposure can exacerbate CRF production, leading to dysregulation of the midbrain CRF-dopamine-glutamate interaction. METHODS: Here we investigated the role of midbrain CRF1 receptors in heroin self-administration and assessed neuroplasticity in CRF1 receptor expression in key opioid addiction brain regions. RESULTS: Infusions of antalarmin (a CRF1 receptor antagonist) into the ventral tegmental area (VTA) dose dependently reduced heroin self-administration in rats but had no impact on food reinforcement or locomotor activity in rats. Using RNAscope in situ hybridization, we found that heroin, but not saline, self-administration upregulated CRF1 receptor mRNA in the VTA, particularly on dopamine neurons. AMPA GluR1 and dopamine reuptake transporter mRNA in VTA neurons were not affected by heroin. The western-blot assay showed that CRF1 receptors were upregulated in the VTA and nucleus accumbens. No significant changes in CRF1 protein expression were detected in the prefrontal cortex, insula, dorsal hippocampus, and substantia nigra. In addition, we found that 15 days of environmental enrichment implemented after heroin self-administration does not reverse upregulation of VTA CRF1 receptor mRNA but it downregulates dopamine transporter mRNA. CONCLUSIONS: Overall, these data suggest that heroin self-administration requires stimulation of VTA CRF1 receptors and upregulates their expression in brain regions involved in reinforcement. Such long-lasting neuroadaptations may contribute to continuation of drug use and relapse due to stress exposure and are not easily reversed by EE exposure.

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.

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.

Differential alterations of insular cortex excitability after adolescent or adult chronic intermittent ethanol administration in male rats.

Adolescent alcohol drinking, primarily in the form of binge-drinking episodes, is a serious public health concern. Binge drinking in laboratory animals has been modeled by a procedure involving chronic intermittent ethanol (CIE) administration, as compared with chronic intermittent water (CIW). The prolonged effects of adolescent binge alcohol exposure in adults, such as high risk of developing alcohol use disorder, are severe but available treatments in the clinic are limited. One reason is the lack of sufficient understanding about the associated neuronal alterations. The involvement of the insular cortex, particularly the anterior agranular insula (AAI), has emerged as a critical region to explain neuronal mechanisms of substance abuse. This study was designed to evaluate the functional output of the AAI by measuring the intrinsic excitability of pyramidal neurons from male rats 2 or 21 days after adolescent or adult CIE treatment. Decreases in intrinsic excitability in AAI pyramidal neurons were detected 21 days, relative to 2 days, after adolescent CIE. Interestingly, the decreased intrinsic excitability in the AAI pyramidal neurons was observed 2 days after adult CIE, compared to adult CIW, but no difference was found between 2 versus 21 days after adult CIE. These data indicate that, although the AAI is influenced within a limited period after adult but not adolescent CIE, neuronal alterations in AAI are affected during the prolonged period of withdrawal from adolescent but not adult CIE. This may explain the prolonged vulnerability to mental disorders of subjects with an alcohol binge history during their adolescent stage.

The role of circTmeff-1 in incubation of context-induced morphine craving.

A progressive increase in drug craving following drug exposure is an important trigger of relapse. CircularRNAs (CircRNAs), key regulators of gene expression, play an important role in neurological diseases. However, the role of circRNAs in drug craving is unclear. In the present study, we trained mice to morphine conditioned place preference (CPP) and collected the nucleus accumbens (NAc) sections on abstinence day 1 (AD1) and day 14 (AD14) for RNA-sequencing. CircTmeff-1, which was highly expressed in the NAc core, was associated with incubation of context-induced morphine craving. The gain- and loss- of function showed that circTmeff-1 was a positive regulator of incubation. Simultaneously, the expression of miR-541-5p and miR-6934-3p were down-regulated in the NAc core during the incubation period. The dual luciferase reporter, RNA pulldown, and fluorescence insitu hybridization assays confirmed that miR-541-5p and miR-6934-3p bind to circTmeff-1 selectively. Furthermore, bioinformatics and western blot analysis suggested that vesicle-associated membrane protein 1 (VAMP1) and neurofascin (NFASC), both overlapping targets of miR-541-5p and miR-6934-3p, were highly expressed during incubation. Lastly, AAV-induced down-regulation of circTmeff-1 decreased VAMP1 and NFASC expression and incubation of morphine craving. These findings suggested that circTmeff-1, a novel circRNA, promotes incubation of context-induced morphine craving by sponging miR-541/miR-6934 in the NAc core. Thus, circTmeff-1 represents a potential therapeutic target for context-induced opioid craving, following prolonged abstinence.

Cell-type specific expression and behavioral impact of galanin and GalR1 in the locus coeruleus during opioid withdrawal.

The neuropeptide galanin is reported to attenuate opioid withdrawal symptoms, potentially by reducing neuronal hyperactivity in the noradrenergic locus coeruleus (LC) via galanin receptor 1 (GalR1). We evaluated this mechanism by using RNAscope in situ hybridization to characterize GalR1 mRNA distribution in the dorsal pons and to compare galanin and GalR1 mRNA expression in tyrosine hydroxylase-positive (TH+) LC cells at baseline and following chronic morphine or precipitated withdrawal. We then used genetically altered mouse lines and pharmacology to test whether noradrenergic galanin (NE-Gal) modulates withdrawal symptoms. RNAscope revealed that, while GalR1 signal was evident in the dorsal pons, 80.7% of the signal was attributable to TH- neurons outside the LC. Galanin and TH mRNA were abundant in LC cells at baseline and were further increased by withdrawal, whereas low basal GalR1 mRNA expression was unaltered by chronic morphine or withdrawal. Naloxone-precipitated withdrawal symptoms in mice lacking NE-Gal (GalcKO-Dbh ) were largely similar to WT littermates, indicating that loss of NE-Gal does not exacerbate withdrawal. Complementary experiments using NE-Gal overexpressor mice (NE-Gal OX) and systemic administration of the galanin receptor agonist galnon revealed that increasing galanin signaling also failed to alter behavioral withdrawal, while suppressing noradrenergic transmission with the alpha-2 adrenergic receptor agonist clonidine attenuated multiple symptoms. These results indicate that galanin does not acutely attenuate precipitated opioid withdrawal via an LC-specific mechanism, which has important implications for the general role of galanin in regulation of somatic and affective opioid responses and LC activity.

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.

Aberrations in Incentive Learning and Responding to Heroin in Male Rats After Adolescent or Adult Chronic Binge-Like Alcohol Exposure.

BACKGROUND AND PURPOSE: Binge drinking is a serious problem among adolescents and young adults despite its adverse consequences on the brain and behavior. One area that remains poorly understood concerns the impact of chronic intermittent ethanol (CIE) exposure on incentive learning. METHODS: Here, we examined the effects of CIE exposure during different developmental stages on conditioned approach and conditioned reward learning in rats experiencing acute or protracted withdrawal from alcohol. Two or 21 days after adolescent or adult CIE exposure, male rats were exposed to pairings of a light stimulus (CS) and food pellets for 3 consecutive daily sessions (30 CS-food pellet pairings per session). This was followed by conditioned approach testing measuring responses (food trough head entries) to the CS-only presentations and by conditioned reward testing measuring responses on a lever producing the CS and on another producing a tone. We then measured behavioral sensitization to repeated injections of heroin (2 mg/kg/d for 9 days). RESULTS: Adolescent and adult alcohol-treated rats showed significantly impaired conditioned reward learning regardless of withdrawal period (acute or prolonged). We found no evidence of changes to conditioned approach learning after adolescent or adult exposure to CIE. Finally, in addition to producing long-term impairments in incentive learning, CIE exposure enhanced locomotor activity in response to heroin and had no effect on behavioral sensitization to heroin regardless of age and withdrawal period. CONCLUSIONS: Our work sets a framework for identifying CIE-induced alterations in incentive learning and inducing susceptibility to subsequent opioid effects.

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.

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.

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.

Repurposing antimalarial artesunate for the prophylactic treatment of depression: Evidence from preclinical research.

INTRODUCTION: Several studies have linked inflammation and oxidative stress with the pathogenesis of depression. Artesunate is a commonly used medication to treat malaria and has been shown to produce antioxidant, anti-inflammatory, and immunomodulatory effects. However, its prophylactic effects on depression and depression-related brain pathology are unknown. METHODS: In Experiment 1, using a PC12 cell line, we investigated whether artesunate can prevent hydrogen peroxide (H2 O2 )-induced oxidative injury that mimics oxidative stress commonly observed in the depressed brain. Next, using lipopolysaccharide (LPS)-induced mouse model of depression, we investigated whether artesunate can prevent behavioral deficits observed in the open field test, novelty-suppressed feeding test, sucrose preference test, forced swimming test, and tail suspension procedure. RESULTS: We found that artesunate significantly prevented a H2 O2 -induced reduction in PC12 cell activity, suggesting its antioxidant potential. We also found that mice pretreated with artesunate (5, 15 mg/kg) intraperitoneally (i.p.) prior to the LPS (.8 mg/kg, i.p.) treatment showed fewer and less severe depression- and anxiety-like behaviors than the LPS-treated control mice. CONCLUSION: Our findings indicate that artesunate produces antioxidant effect, as well as antidepressant and anxiolytic effects. Importantly, our findings first demonstrate that artesunate can prevent LPS-induced depression- and anxiety-like symptoms, strongly suggesting its prophylactic potential in the treatment of depression and, perhaps, other psychiatric disorders associated with inflammation and oxidative stress.

Therapeutic potential of PIMSR, a novel CB1 receptor neutral antagonist, for cocaine use disorder: evidence from preclinical research.

Cannabinoid CB1 receptors (CB1Rs) have been major targets in medication development for the treatment of substance use disorders. However, clinical trials with rimonabant, a CB1R antagonist/inverse agonist, failed due to severe side effects. Here, we evaluated the therapeutic potential of PIMSR, a neutral CB1R antagonist lacking an inverse agonist profile, against cocaine's behavioral effects in experimental animals. We found that systemic administration of PIMSR dose-dependently inhibited cocaine self-administration under fixed-ratio (FR5), but not FR1, reinforcement, shifted the cocaine self-administration dose-response curve downward, decreased incentive motivation to seek cocaine under progressive-ratio reinforcement, and reduced cue-induced reinstatement of cocaine seeking. PIMSR also inhibited oral sucrose self-administration. Importantly, PIMSR alone is neither rewarding nor aversive as assessed by place conditioning. We then used intracranial self-stimulation (ICSS) to explore the possible involvement of the mesolimbic dopamine system in PIMSR's action. We found that PIMSR dose-dependently attenuated cocaine-enhanced ICSS maintained by electrical stimulation of the medial forebrain bundle in rats. PIMSR itself failed to alter electrical ICSS, but dose-dependently inhibited ICSS maintained by optical stimulation of midbrain dopamine neurons in transgenic DAT-Cre mice, suggesting the involvement of dopamine-dependent mechanisms. Lastly, we examined the CB1R mechanisms underlying PIMSR's action. We found that PIMSR pretreatment attenuated Δ9-tetrahydrocannabinol (Δ9-THC)- or ACEA (a selective CB1R agonist)-induced reduction in optical ICSS. Together, our findings suggest that the neutral CB1R antagonist PIMSR deserves further research as a promising pharmacotherapeutic for cocaine use disorder.

CircTmeff-1 in the nucleus accumbens regulates the reconsolidation of cocaine-associated memory.

Reconsolidation of drug memories is the process of restoring unstable memories after unconditioned (UCS; e.g., drugs) or conditioned stimulus (CS; e.g., drug-paired contexts), and provides promise for prevention of drug relapse. Circular RNAs (circRNAs) have important effects on the transcription and post-transcriptional regulation of gene expression. However, the role of circRNAs in the reconsolidation of drug memories is unclear. Here, we observed that cocaine-induced memory retrieval significantly increased circTmeff-1 level in the nucleus accumbens (NAc) core but not shell. Importantly, the disrupted expression of circTmeff-1 using virus in the NAc core damaged the reconsolidation of cocaine-associated memories. The knockdown of circTmeff-1 in the NAc shell or without UCS retrieval or 9 h after UCS retrieval had no such effects. Mechanistically, using bioinformatic analysis and loss- or gain- of function assays, we revealed that antagomiR-206 reversed the inhibitory effect of circTmeff-1 knockdown on the expression of brain-derived neurotrophic factor (BDNF) during the reconsolidation of cocaine-associated memories. Taken together, these results demonstrate the role of circTmeff-1 in the reconsolidation of cocaine-associated memory and that circTmeff-1 may function as a decoy for miR-206 to regulate the expression of BDNF.

Involvement of the ghrelin system in the maintenance and reinstatement of cocaine-motivated behaviors: a role of adrenergic action at peripheral ß1 receptors.

Cocaine addiction is a significant medical and public concern. Despite decades of research effort, development of pharmacotherapy for cocaine use disorder remains largely unsuccessful. This may be partially due to insufficient understanding of the complex biological mechanisms involved in the pathophysiology of this disorder. In the present study, we show that: (1) elevation of ghrelin by cocaine plays a critical role in maintenance of cocaine self-administration and cocaine-seeking motivated by cocaine-conditioned stimuli; (2) acquisition of cocaine-taking behavior is associated with the acquisition of stimulatory effects of cocaine by cocaine-conditioned stimuli on ghrelin secretion, and with an upregulation of ghrelin receptor mRNA levels in the ventral tegmental area (VTA); (3) blockade of ghrelin signaling by pretreatment with JMV2959, a selective ghrelin receptor antagonist, dose-dependently inhibits reinstatement of cocaine-seeking triggered by either cocaine or yohimbine in behaviorally extinguished animals with a history of cocaine self-administration; (4) JMV2959 pretreatment also inhibits brain stimulation reward (BSR) and cocaine-potentiated BSR maintained by optogenetic stimulation of VTA dopamine neurons in DAT-Cre mice; (5) blockade of peripheral adrenergic ß1 receptors by atenolol potently attenuates the elevation in circulating ghrelin induced by cocaine and inhibits cocaine self-administration and cocaine reinstatement triggered by cocaine. These findings demonstrate that the endogenous ghrelin system plays an important role in cocaine-related addictive behaviors and suggest that manipulating and targeting this system may be viable for mitigating cocaine use disorder.

A red nucleus-VTA glutamate pathway underlies exercise reward and the therapeutic effect of exercise on cocaine use.

Physical exercise is rewarding and protective against drug abuse and addiction. However, the neural mechanisms underlying these actions remain unclear. Here, we report that long-term wheel-running produced a more robust increase in c-fos expression in the red nucleus (RN) than in other brain regions. Anatomic and functional assays demonstrated that most RN magnocellular portion (RNm) neurons are glutamatergic. Wheel-running activates a subset of RNm glutamate neurons that project to ventral tegmental area (VTA) dopamine neurons. Optogenetic stimulation of this pathway was rewarding, as assessed by intracranial self-stimulation and conditioned place preference, whereas optical inhibition blocked wheel-running behavior. Running wheel access decreased cocaine self-administration and cocaine seeking during extinction. Last, optogenetic stimulation of the RNm-to-VTA glutamate pathway inhibited responding to cocaine. Together, these findings indicate that physical exercise activates a specific RNm-to-VTA glutamatergic pathway, producing exercise reward and reducing cocaine intake.

A highly D3R-selective and efficacious partial agonist (S)-ABS01-113 compared to its D3R-selective antagonist enantiomer (R)-ABS01-113 as potential treatments for opioid use disorder.

The non-medical use of opioids has become a national crisis in the USA. Developing non-opioid pharmacotherapies for controlling this opioid epidemic is urgent. Dopamine D3 receptor (D3R) antagonists and low efficacy partial agonists have shown promising profiles in animal models of opioid use disorders (OUD). However, to date, advancement to human studies has been limited. Here we report the effects of (S)- and (R)-enantiomers of (±)-ABS01-113, structural analogs of the D3R partial agonist, (±)-VK4-40, in which the 3-OH in the linking chain is replaced by 3-F group. (S)- and (R)-ABS01-113 are identical in chemical structure but with opposite chirality. In vitro receptor binding and functional assays indicate that (S)-ABS01-113 is an efficacious (55%) and potent (EC50 = 7.6 ± 3.9 nM) D3R partial agonist, while the (R)-enantiomer is a potent D3R antagonist (IC50 = 11.4 nM). Both (S)- and (R)-ABS01-113 bind with high affinity to D3R (Ki = 0.84 ± 0.16 and 0.37 ± 0.06 nM, respectively); however, the (S)-enantiomer is more D3/D2-selective (>1000-fold). Pharmacokinetic analyses indicate that both enantiomers display excellent oral bioavailability and high brain penetration. Systemic administration of (S)- or (R)-ABS01-113 alone failed to alter open-field locomotion in male rats and mice. Interestingly, pretreatment with (S)- or (R)-ABS01-113 attenuated heroin-enhanced hyperactivity, heroin self-administration, and (heroin + cue)-induced reinstatement of drug-seeking behavior. Together, these findings reveal that both enantiomers, particularly the highly selective and efficacious D3R partial agonist (S)-ABS01-113, demonstrate promising translational potential for the treatment of OUD.