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.

Chiral Resolution of the Enantiomers of the Slow-Onset Dopamine Reuptake Inhibitor CTDP-32476 and Their Activities.

An improved synthesis was developed for CDTP-32476, a potent slow-onset dopamine reuptake blocker that may have utility as a treatment for cocaine abuse. The enantiomers of the compound were separated by fractional crystallization with N-acetylleucine enantiomers. An X-ray crystal structure was obtained of the RR enantiomer paired with N-acetyl-d-leucine. Chiral chromatography showed that the resolved enantiomers were pure with little contamination by the other enantiomer. The enantiomers were tested for their ability to block the reuptake of monoamines at their respective transporters and to stimulate locomotor activity in mice. Both enantiomers potently blocked the reuptake of dopamine and stimulated locomotor activity in mice. The RR enantiomer that corresponds to the active RR enantiomer of methylphenidate was slightly more potent at the dopamine reuptake site. The RR enantiomer also was found to be about twice as selective for the dopamine transporter relative to the norepinephrine transporter, which may have clinical implications. A method for designing slow-onset stimulants is proposed since there is increasing evidence that such activity is an important factor in stimulants that may have limited abuse potential.

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.

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.

Frequency of the Dopamine Receptor D3 (rs6280) vs. Opioid Receptor µ1 (rs1799971) Polymorphic Risk Alleles in Patients with Opioid Use Disorder: A Preponderance of Dopaminergic Mechanisms?

While opioids are a powerful class of drugs that inhibit transmission of pain signals, their use is tarnished by the current epidemic of opioid use disorder (OUD) and overdose deaths. Notwithstanding published reports, there remain gaps in our knowledge of opioid receptor mechanisms and their role in opioid seeking behavior. Thus, novel insights into molecular, neurogenetic and neuropharmacological bases of OUD are needed. We propose that an addictive endophenotype may not be entirely specific to the drug of choice but rather may be generalizable to altered brain reward circuits impacting net mesocorticolimbic dopamine release. We suggest that genetic or epigenetic alterations across dopaminergic reward systems lead to uncontrollable self-administration of opioids and other drugs. For instance, diminished availability via knockout of dopamine D3 receptor (DRD3) increases vulnerability to opioids. Building upon this concept via the use of a sophisticated polymorphic risk analysis in a human cohort of chronic opioid users, we found evidence for a higher frequency of polymorphic DRD3 risk allele (rs6280) than opioid receptor µ1 (rs1799971). In conclusion, while opioidergic mechanisms are involved in OUD, dopamine-related receptors may have primary influence on opioid-seeking behavior in African Americans. These findings suggest OUD-targeted novel and improved neuropharmacological therapies may require focus on DRD3-mediated regulation of dopaminergic homeostasis.

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.

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.

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.

A closer look at alcohol-induced changes in the ghrelin system: novel insights from preclinical and clinical data.

Ghrelin is a gastric-derived peptide hormone with demonstrated impact on alcohol intake and craving, but the reverse side of this bidirectional link, that is, the effects of alcohol on the ghrelin system, remains to be fully established. To further characterize this relationship, we examined (1) ghrelin levels via secondary analysis of human laboratory alcohol administration experiments with heavy-drinking participants; (2) expression of ghrelin, ghrelin receptor, and ghrelin-O-acyltransferase (GOAT) genes (GHRL, GHSR, and MBOAT4, respectively) in post-mortem brain tissue from individuals with alcohol use disorder (AUD) versus controls; (3) ghrelin levels in Ghsr knockout and wild-type rats following intraperitoneal (i.p.) alcohol administration; (4) effect of alcohol on ghrelin secretion from gastric mucosa cells ex vivo and GOAT enzymatic activity in vitro; and (5) ghrelin levels in rats following i.p. alcohol administration versus a calorically equivalent non-alcoholic sucrose solution. Acyl- and total-ghrelin levels decreased following acute alcohol administration in humans, but AUD was not associated with changes in central expression of ghrelin system genes in post-mortem tissue. In rats, alcohol decreased acyl-ghrelin, but not des-acyl-ghrelin, in both Ghsr knockout and wild-type rats. No dose-dependent effects of alcohol were observed on acyl-ghrelin secretion from gastric mucosa cells or on GOAT acylation activity. Lastly, alcohol and sucrose produced distinct effects on ghrelin in rats despite equivalent caloric value. Our findings suggest that alcohol acutely decreases peripheral ghrelin concentrations in vivo, but not in proportion to alcohol's caloric value or through direct interaction with ghrelin-secreting gastric mucosal cells, the ghrelin receptor, or the GOAT enzyme.

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.

ß-caryophyllene, an FDA-Approved Food Additive, Inhibits Methamphetamine-Taking and Methamphetamine-Seeking Behaviors Possibly via CB2 and Non-CB2 Receptor Mechanisms.

Recent research indicates that brain cannabinoid CB2 receptors are involved in drug reward and addiction. However, it is unclear whether ß-caryophyllene (BCP), a natural product with a CB2 receptor agonist profile, has therapeutic effects on methamphetamine (METH) abuse and dependence. In this study, we used animal models of self-administration, electrical brain-stimulation reward (BSR) and in vivo microdialysis to explore the effects of BCP on METH-taking and METH-seeking behavior. We found that systemic administration of BCP dose-dependently inhibited METH self-administration under both fixed-ratio and progressive-ratio reinforcement schedules in rats, indicating that BCP reduces METH reward, METH intake, and incentive motivation to seek and take METH. The attenuating effects of BCP were partially blocked by AM 630, a selective CB2 receptor antagonist. Genetic deletion of CB2 receptors in CB2-knockout (CB2-KO) mice also blocked low dose BCP-induced reduction in METH self-administration, suggesting possible involvement of a CB2 receptor mechanism. However, at high doses, BCP produced a reduction in METH self-administration in CB2-KO mice in a manner similar as in WT mice, suggesting that non-CB2 receptor mechanisms underlie high dose BCP-produced effects. In addition, BCP dose-dependently attenuated METH-enhanced electrical BSR and inhibited METH-primed and cue-induced reinstatement of drug-seeking in rats. In vivo microdialysis assays indicated that BCP alone did not produce a significant reduction in extracellular dopamine (DA) in the nucleus accumbens (NAc), while BCP pretreatment significantly reduced METH-induced increases in extracellular NAc DA in a dose-dependent manner, suggesting a DA-dependent mechanism involved in BCP action. Together, the present findings suggest that BCP might be a promising therapeutic candidate for the treatment of METH use disorder.

Cannabinoid CB2 receptors are expressed in glutamate neurons in the red nucleus and functionally modulate motor behavior in mice.

Cannabinoids produce a number of central nervous system effects via the CB2 receptor (CB2R), including analgesia, antianxiety, anti-reward, hypoactivity and attenuation of opioid-induced respiratory depression. However, the cellular distributions of the CB2Rs in the brain remain unclear. We have reported that CB2Rs are expressed in midbrain dopamine (DA) neurons and functionally regulate DA-mediated behavior(s). Unexpectedly, high densities of CB2-like signaling were also found in a neighboring motor structure - the red nucleus (RN) of the midbrain. In the present study, we systematically explored CB2R expression and function in the RN. Immunohistochemistry and in situ hybridization assays showed high densities of CB2R-immunostaining and mRNA signal in RN magnocellular glutamate neurons in wildtype and CB1-knockout, but not CB2-knockout, mice. Ex vivo electrophysiological recordings in midbrain slices demonstrated that CB2R activation by JWH133 dose-dependently inhibited firing rates of RN magnocellular neurons in wildtype, but not CB2-knockout, mice, while having no effect on RN GABA neurons in transgenic GAD67-GFP reporter mice, suggesting CB2-mediated effects on glutamatergic neurons. In addition, microinjection of JWH133 into the RN produced robust ipsilateral rotations in wildtype, but not CB2-knockout mice, which was blocked by pretreatment with either a CB2 or DA D1 or D2 receptor antagonist, suggesting a DA-dependent effect. Finally, fluorescent tract tracing revealed glutamatergic projections from the RN to multiple brain areas including the ventral tegmental area, thalamus, and cerebellum. These findings suggest that CB2Rs in RN glutamate neurons functionally modulate motor activity, and therefore, constitute a new target in cannabis-based medication development for motor disorders.

Dissecting the role of CB1 and CB2 receptors in cannabinoid reward versus aversion using transgenic CB1- and CB2-knockout mice.

Cannabinoids produce both rewarding and aversive effects in humans and experimental animals. However, the mechanisms underlying these conflicting findings are unclear. Here we examined the potential involvement of CB1 and CB2 receptors in cannabinoid action using transgenic CB1-knockout (CB1-KO) and CB2-knockout (CB2-KO) mice. We found that Δ9-tetrahydrocannabinol (Δ9-THC) induced conditioned place preference at a low dose (1 mg/kg) in WT mice that was attenuated by deletion of the CB1 receptor. At 5 mg/kg, no subjective effects of Δ9-THC were detected in WT mice, but CB1-KO mice exhibited a trend towards place aversion and CB2-KO mice developed significant place preferences. This data suggests that activation of the CB1 receptor is rewarding, while CB2R activation is aversive. We then examined the nucleus accumbens (NAc) dopamine (DA) response to Δ9-THC using in vivo microdialysis. Unexpectedly, Δ9-THC produced a dose-dependent decrease in extracellular DA in WT mice, that was potentiated in CB1-KO mice. However, in CB2-KO mice Δ9-THC produced a dose-dependent increase in extracellular DA, suggesting that activation of the CB2R inhibits DA release in the NAc. In contrast, Δ9-THC, when administered systemically or locally into the NAc, failed to alter extracellular DA in rats. Lastly, we examined the locomotor response to Δ9-THC. Both CB1 and CB2 receptor mechanisms were shown to underlie Δ9-THC-induced hypolocomotion. These findings indicate that Δ9-THC's variable subjective effects reflect differential activation of cannabinoid receptors. Specifically, the opposing actions of CB1 and CB2 receptors regulate cannabis reward and aversion, with CB2-mediated effects predominant in mice.

Beta-caryophyllene inhibits cocaine addiction-related behavior by activation of PPARα and PPARγ: repurposing a FDA-approved food additive for cocaine use disorder.

Cocaine abuse continues to be a serious health problem worldwide. Despite intense research, there is still no FDA-approved medication to treat cocaine use disorder (CUD). In this report, we explored the potential utility of beta-caryophyllene (BCP), an FDA-approved food additive for the treatment of CUD. We found that BCP, when administered intraperitoneally or intragastrically, dose-dependently attenuated cocaine self-administration, cocaine-conditioned place preference, and cocaine-primed reinstatement of drug seeking in rats. In contrast, BCP failed to alter food self-administration or cocaine-induced hyperactivity. It also failed to maintain self-administration in a drug substitution test, suggesting that BCP has no abuse potential. BCP was previously reported to be a selective CB2 receptor agonist. Unexpectedly, pharmacological blockade or genetic deletion of CB1, CB2, or GPR55 receptors in gene-knockout mice failed to alter BCP's action against cocaine self-administration, suggesting the involvement of non-CB1, non-CB2, and non-GPR55 receptor mechanisms. Furthermore, pharmacological blockade of µ opioid receptor or Toll-like receptors complex failed to alter, while blockade of peroxisome proliferator-activated receptors (PPARα, PPARγ) reversed BCP-induced reduction in cocaine self-administration, suggesting the involvement of PPARα and PPARγ in BCP's action. Finally, we used electrical and optogenetic intracranial self-stimulation (eICSS, oICSS) paradigms to study the underlying neural substrate mechanisms. We found that BCP is more effective in attenuation of cocaine-enhanced oICSS than eICSS, the former driven by optical activation of midbrain dopamine neurons in DAT-cre mice. These findings indicate that BCP may be useful for the treatment of CUD, likely by stimulation of PPARα and PPARγ in the mesolimbic system.

Neurochemical and behavioral comparisons of contingent and non-contingent methamphetamine exposure following binge or yoked long-access self-administration paradigms.

RATIONALE: Abuse of the psychostimulant methamphetamine (METH) can cause long-lasting damage to brain monoaminergic systems and is associated with profound mental health problems for users, including lasting cognitive impairments. Animal models of METH exposure have been useful in dissecting the molecular effects of the drug on cognition, but many studies use acute, non-contingent "binge" administrations of METH which do not adequately approximate human METH use. Long-term METH exposure via long-access (LgA) self-administration paradigms has been proposed to more closely reflect human use and induce cognitive impairments. OBJECTIVE: To better understand the role of contingency and patterns of exposure in METH-induced cognitive impairments, we analyzed behavioral and neurochemical outcomes in adult male rats, comparing non-contingent "binge" METH administration with contingent (LgA) METH self-administration and non-contingent yoked partners. RESULTS: Binge METH (40 mg/kg, i.p., over 1 day) dramatically altered striatal and hippocampal dopamine, DOPAC, 5-HT, 5-HIAA, BDNF, and TrkB 75 days after drug exposure. In contrast, 6-h LgA METH self-administration (cumulative 24.8-48.9 mg METH, i.v., over 16 days) altered hippocampal BDNF in both contingent and yoked animals but reduced striatal 5-HIAA in only contingent animals. Neurochemical alterations following binge METH administration were not accompanied by cognitive deficits in Morris water maze, novel object recognition, or Y-maze tests. However, contingent LgA METH self-administration resulted in impaired spatial memory in the water maze. CONCLUSIONS: Overall, substantial differences in neurochemical markers between METH exposure and self-administration paradigms did not consistently translate to deficits in cognitive tasks, highlighting the complexity of correlating METH-induced neurochemical changes with cognitive outcomes.

ß-Caryophyllene, a dietary terpenoid, inhibits nicotine taking and nicotine seeking in rodents.

BACKGROUND AND PURPOSE: ß-Caryophyllene (BCP) is a plant-derived terpenoid used as a food additive for many decades. Recent studies indicate that BCP is a cannabinoid CB2 receptor agonist with medical benefits for a number of human diseases. However, little is known about its therapeutic potential for drug abuse and addiction. EXPERIMENT APPROACH: We used pharmacological, transgenic, and optogenetic approaches to systematically evaluate the effects of BCP on nicotine-taking and nicotine-seeking behaviour in animal models of drug self-administration, electrical, and optical brain-stimulation reward. KEY RESULTS: Systemic administration of BCP dose-dependently inhibited nicotine self-administration and motivation for nicotine seeking in rats and mice. The reduction in nicotine self-administration was blocked by AM630, a selective CB2 receptor antagonist, but not by AM251, a selective CB1 receptor antagonist, suggesting involvement of a CB2 receptor mechanism. Genetic deletion of CB2 receptors in mice blocked the reduction in nicotine self-administration produced only by low doses, but not by high doses, of BCP, suggesting involvement of both CB2 and non-CB2 receptor mechanisms. Furthermore, in the intracranial self-stimulation paradigm, BCP attenuated electrical brain-stimulation reward and nicotine-enhanced brain-stimulation reward in rats. Lastly, BCP also attenuated brain-stimulation reward maintained by optogenetic stimulation of dopaminergic neurons in the ventral tegmental area in DAT-cre mice, suggesting the involvement of a dopamine-dependent mechanism in BCP's action. CONCLUSIONS AND IMPLICATIONS: The present findings suggest that BCP has significant anti-nicotine effects via both CB2 and non-CB2 receptor mechanisms and, therefore, deserves further study as a potential new pharmacotherapy for cigarette smoking cessation.

Different receptor mechanisms underlying phytocannabinoid- versus synthetic cannabinoid-induced tetrad effects: Opposite roles of CB1 /CB2 versus GPR55 receptors.

BACKGROUND AND PURPOSE: Cannabis or cannabinoids produce characteristic tetrad effects-analgesia, hypothermia, catalepsy and suppressed locomotion, which are believed to be mediated by the activation of cannabinoid CB1 receptors. Given recent findings of CB2 and GPR55 receptors in the brain, we examined whether these receptors are also involved in cannabinoid action. EXPERIMENTAL APPROACH: We compared Δ9 -tetrahydrocannabinol (Δ9 -THC)-, WIN55212-2-, or XLR11-induced tetrad effects between wild-type (WT) and each genotype of CB1 -, CB2 - or GPR55-knockout (KO) mice and then observed the effects of antagonists of these receptors on these tetrad effects in WT mice. KEY RESULTS: Systemic administration of Δ9 -THC, WIN55212-2 or XLR11 produced dose-dependent tetrad effects in WT mice. Genetic deletion or pharmacological blockade of CB1 receptors abolished the tetrad effects produced by all three cannabinoids. Unexpectedly, genetic deletion of CB2 receptor abolished analgesia and catalepsy produced by Δ9 -THC or WIN55212-2, but not by XLR11. Microinjections of Δ9 -THC into the lateral ventricles also produced tetrad effects in WT, but not in CB1 -KO mice. CB2 -KO mice displayed a reduction in intraventricular Δ9 -THC-induced analgesia and catalepsy. In contrast to CB1 and CB2 receptors, genetic deletion of GPR55 receptors caused enhanced responses to Δ9 -THC or WIN55212-2. Antagonisim of CB1 , CB2 or GPR55 receptors produced alterations similar to those observed in each genotype mouse line. CONCLUSIONS AND IMPLICATIONS: These findings suggest that in addition to CB1 , both CB2 and GPR55 receptors are also involved in some pharmacological effects produced by cannabinoids. CB1 /CB2 , in contrast to GPR55, receptors appears to play opposite roles in cannabinoid action.

Δ8 -Tetrahydrocannabivarin has potent anti-nicotine effects in several rodent models of nicotine dependence.

BACKGROUND AND PURPOSE: Both types of cannabinoid receptors-CB1 and CB2 -regulate brain functions relating to addictive drug-induced reward and relapse. CB1 receptor antagonists and CB2 receptor agonists have anti-addiction efficacy, in animal models, against a broad range of addictive drugs. Δ9 -Tetrahydrocannabivarin (Δ9 -THCV)-a cannabis constituent-acts as a CB1 antagonist and a CB2 agonist. Δ8 -Tetrahydrocannabivarin (Δ8 -THCV) is a Δ9 -THCV analogue with similar combined CB1 antagonist/CB2 agonist properties. EXPERIMENTAL APPROACH: We tested Δ8 -THCV in seven different rodent models relevant to nicotine dependence-nicotine self-administration, cue-triggered nicotine-seeking behaviour following forced abstinence, nicotine-triggered reinstatement of nicotine-seeking behaviour, acquisition of nicotine-induced conditioned place preference, anxiety-like behaviour induced by nicotine withdrawal, somatic withdrawal signs induced by nicotine withdrawal, and hyperalgesia induced by nicotine withdrawal. KEY RESULTS: Δ8 -THCV significantly attenuated intravenous nicotine self-administration and both cue-induced and nicotine-induced relapse to nicotine-seeking behaviour in rats. Δ8 -THCV also significantly attenuated nicotine-induced conditioned place preference and nicotine withdrawal in mice. CONCLUSIONS AND IMPLICATIONS: We conclude that Δ8 -THCV may have therapeutic potential for the treatment of nicotine dependence. We also suggest that tetrahydrocannabivarins should be tested for possible anti-addiction efficacy in a broader range of preclinical animal models, against other addictive drugs, and eventually in humans.

Cannabinoid CB1 and CB2 receptor mechanisms underlie cannabis reward and aversion in rats.

BACKGROUND AND PURPOSE: Endocannabinoids are critically involved in brain reward functions, mediated by activation of CB1 receptors, reflecting their high density in the brain. However, the recent discovery of CB2 receptors in the brain, particularly in the midbrain dopamine neurons, has challenged this view and inspired us to re-examine the roles of both CB1 and CB2 receptors in the effects of cannabis. EXPERIMENTAL APPROACH: In the present study, we used the electrical intracranial self-stimulation paradigm to evaluate the effects of various cannabinoid drugs on brain reward in laboratory rats and the roles of CB1 and CB2 receptors activation in brain reward function(s). KEY RESULTS: Two mixed CB1 / CB2 receptor agonists, Δ9 -tetrahydrocannabinol (Δ9 -THC) and WIN55,212-2, produced biphasic effects-mild enhancement of brain-stimulation reward (BSR) at low doses but inhibition at higher doses. Pretreatment with a CB1 receptor antagonist (AM251) attenuated the low dose-enhanced BSR, while a CB2 receptor antagonist (AM630) attenuated high dose-inhibited BSR. To confirm these opposing effects, rats were treated with selective CB1 and CB2 receptor agonists. These compounds produced significant BSR enhancement and inhibition, respectively. CONCLUSIONS AND IMPLICATIONS: CB1 receptor activation produced reinforcing effects, whereas CB2 receptor activation was aversive. The subjective effects of cannabis depend on the balance of these opposing effects. These findings not only explain previous conflicting results in animal models of addiction but also explain why cannabis can be either rewarding or aversive in humans, as expression of CB1 and CB2 receptors may differ in the brains of different subjects.

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.