Effects of systemic pretreatment with the NAALADase inhibitor 2-PMPA on oral methamphetamine reinforcement in C57BL/6J mice.

Introduction: Repeated exposure to methamphetamine (MA) in laboratory rodents induces a sensitization of glutamate release within the corticoaccumbens pathway that drives both the rewarding and reinforcing properties of this highly addictive drug. Such findings argue the potential for pharmaceutical agents inhibiting glutamate release or its postsynaptic actions at glutamate receptors as treatment strategies for MA use disorder. One compound that may accomplish both of these pharmacological actions is the N-acetylated-alpha-linked-acidic dipeptidase (NAALADase) inhibitor 2-(phosphonomethyl)pentanedioic acid (2-PMPA). 2-PMPA elevates brain levels of the endogenous agonist of glutamate mGluR3 autoreceptors, N-acetyl-aspartatylglutamate (NAAG), while potentially acting as an NMDA glutamate receptor antagonist. Of relevance to treating psychomotor stimulant use disorders, 2-PMPA is reported to reduce indices of both cocaine and synthetic cathinone reward, as well as cocaine reinforcement in preclinical rodent studies. Method: Herein, we conducted three experiments to pilot the effects of systemic pretreatment with 2-PMPA (0-100 mg/kg, IP) on oral MA self-administration in C57BL/6J mice. The first experiment employed female mice with a prolonged history of MA exposure, while the mice in the second (females) and third (males and females) experiment were MA-naïve prior to study. In all experiments, mice were trained daily to nose-poke for delivery of unadulterated MA solutions until responding stabilized. Then, mice were pretreated with 2-PMPA prior to operant-conditioning sessions in which nose-poking behavior was reinforced by delivery of 120 mg/L or 200 mg/L MA (respectively, in Experiments 1 and 2/3). Results: Contrary to our expectations, 30 mg/kg 2-PMPA pretreatment altered neither appetitive nor consummatory measures related to MA self-administration. In Experiment 3, 100 mg/kg 2-PMPA reduced responding in the MA-reinforced hole, as well as the number of reinforcers earned, but did not significantly lower drug intake. Discussion: These results provide mixed evidenced related to the efficacy of this NAALADase inhibitor for reducing oral MA reinforcement in female mice.

ERK-Directed Phosphorylation of mGlu5 Gates Methamphetamine Reward and Reinforcement in Mouse.

Methamphetamine (MA) is a highly addictive psychomotor stimulant drug. In recent years, MA use has increased exponentially on a global scale, with the number of MA-involved deaths reaching epidemic proportions. There is no approved pharmacotherapy for treating MA use disorder, and we know relatively little regarding the neurobiological determinants of vulnerability to this disease. Extracellular signal-regulated kinase (ERK) is an important signaling molecule implicated in the long-lasting neuroadaptations purported to underlie the development of substance use disorders, but the role for this kinase in the propensity to develop addiction, particularly MA use disorder, is uncharacterized. In a previous MA-induced place-conditioning study of C57BL/6J mice, we characterized mice as MA-preferring, -neutral, or -avoiding and collected tissue from the medial prefrontal cortex (mPFC). Using immunoblotting, we determined that elevated phosphorylated ERK expression within the medial prefrontal cortex (mPFC) is a biochemical correlate of the affective valence of MA in a population of C57BL/6J mice. We confirmed the functional relevance for mPFC ERK activation for MA-induced place-preference via site-directed infusion of the MEK inhibitor U0126. By contrast, ERK inhibition did not have any effect upon MA-induced locomotion or its sensitization upon repeated MA treatment. Through studies of transgenic mice with alanine point mutations on T1123/S1126 of mGlu5 that disrupt ERK-dependent phosphorylation of the receptor, we discovered that ERK-dependent mGlu5 phosphorylation normally suppresses MA-induced conditioned place-preference (MA-CPP), but is necessary for this drug's reinforcing properties. If relevant to humans, the present results implicate individual differences in the capacity of MA-associated cues/contexts to hyper-activate ERK signaling within mPFC in MA Use Disorder vulnerability and pose mGlu5 as one ERK-directed target contributing to the propensity to seek out and take MA.

Hnrnph1 is a novel regulator of alcohol reward.

BACKGROUND: Hnrnph1 is a validated quantitative trait gene for methamphetamine behavioral sensitivity that encodes for heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1). This RNA-binding protein is involved in all stages of RNA metabolism that impacts mesocorticolimbic dopamine neurotransmission to influence addiction-related behavior. METHODS: We characterized the alcohol behavioral phenotypes of mice heterozygous for a deletion in the first coding exon of Hnrnph1 (Hnrnph1+/-). We examined alcohol intake under both continuous- and limited-access procedures, as well as alcohol-induced place-conditioning. Follow-up studies examined genotypic differences in the psychomotor-activating and sedative-hypnotic effects of acute and repeated alcohol, and a behavioral test battery was employed to determine the effects of Hnrnph1 deletion on the manifestation of negative affect during alcohol withdrawal. RESULTS: Relative to wild-type (WT) controls, Hnrnph1+/- males exhibited blunted intake of high alcohol concentrations under both drinking procedures. Hnrnph1 deletion did not impact the conditioned rewarding properties of low-dose alcohol, but reversed the conditioned place-aversion elicited by higher alcohol doses (2 and 4 g/kg), with more robust effects in male versus female mice. No genotypic differences were observed for alcohol-induced locomotor activity. Hnrnph1+/- mice exhibited a modest increase in sensitivity to alcohol's sedative-hypnotic effects, but did not differ from WT mice with regard to tolerance to alcohol's sedative-hypnotic effects or alcohol metabolism, Inconsistent effects of Hnrnph1 deletion were observed in models for withdrawal-induced negative affect. CONCLUSIONS: These data identify Hnrnph1 as a novel, male-selective, driver of alcohol consumption and high-dose alcohol aversion that is potentially relevant to the neurobiology of alcohol abuse and alcoholism.

The motivational valence of methamphetamine relates inversely to subsequent methamphetamine self-administration in female C57BL/6J mice.

Understanding the mechanisms underpinning individual variance in addiction vulnerability requires the development of validated, high-throughput screens. In a prior study of a large sample of male isogenic C57BL/6J mice, the direction and magnitude of methamphetamine (MA)-induced place-conditioning predicts the propensity to acquire oral MA self-administration, as well as the efficacy of MA to serve as a reinforcer. The present study examined whether or not such a predictive relationship also exists in females. Adult C57BL/6J females underwent a 4-day MA place-conditioning paradigm (once daily injections of 2 mg/kg) and were then trained to nose-poke for delivery of a 20 mg/L MA solution under increasing schedules of reinforcement, followed by dose-response testing (5-400 mg/L MA). Akin to males, 53 % of the females exhibited a conditioned place-preference, while 32 % of the mice were MA-neutral and 15 % exhibited a conditioned place-aversion. However, unlike males, the place-conditioning phenotype did not transfer to MA-reinforced nose-poking behavior under operant-conditioning procedures, with 400 mg/L MA intake being inversely correlated place-conditioning. While only one MA-conditioning dose has been assayed to date, these data indicate that sex does not significantly shift the proportion of C57BL/6J mice that perceive MA's interoceptive effects as positive, neutral or aversive. However, a sex difference appears to exist regarding the predictive relationship between the motivational valence of MA and subsequent drug-taking behavior; females exhibit MA-taking behavior and reinforcement, despite their initial perception of the stimulant interoceptive effects as positive, neutral or negative.

Transgenic Analyses of Homer2 Function Within Nucleus Accumbens Subregions in the Regulation of Methamphetamine Reward and Reinforcement in Mice.

Problems associated with the abuse of amphetamine-type stimulants, including methamphetamine (MA), pose serious health and socioeconomic issues world-wide. While it is well-established that MA's psychopharmacological effects involve interactions with monoamine neurotransmission, accumulating evidence from animal models implicates dysregulated glutamate in MA addiction vulnerability and use disorder. Recently, we discovered an association between genetic vulnerability to MA-taking and increased expression of the glutamate receptor scaffolding protein Homer2 within both the shell and core subregions of the nucleus accumbens (NAC) and demonstrated a necessary role for Homer2 within the shell subregion in MA reward and reinforcement in mice. This report extends our earlier work by interrogating the functional relevance of Homer2 within the NAC core for the conditioned rewarding and reinforcing properties of MA. C57BL/6J mice with a virus-mediated knockdown of Homer2b expression within the NAC core were first tested for the development and expression of a MA-induced conditioned place-preference/CPP (four pairings of 2 mg/kg MA) and then were trained to self-administer oral MA under operant-conditioning procedures (5-80 mg/L). Homer2b knockdown in the NAC core augmented a MA-CPP and shifted the dose-response function for MA-reinforced responding, above control levels. To determine whether Homer2b within NAC subregions played an active role in regulating MA reward and reinforcement, we characterized the MA phenotype of constitutive Homer2 knockout (KO) mice and then assayed the effects of virus-mediated overexpression of Homer2b within the NAC shell and core of wild-type and KO mice. In line with the results of NAC core knockdown, Homer2 deletion potentiated MA-induced CPP, MA-reinforced responding and intake, as well as both cue- and MA-primed reinstatement of MA-seeking following extinction. However, there was no effect of Homer2b overexpression within the NAC core or the shell on the KO phenotype. These data provide new evidence indicating a globally suppressive role for Homer2 in MA-seeking and MA-taking but argue against specific NAC subregions as the neural loci through which Homer2 actively regulates MA addiction-related behaviors.

A prior history of binge-drinking increases sensitivity to the motivational valence of methamphetamine in female C57BL/6J mice.

Methamphetamine (MA) and alcohol use disorders exhibit a high degree of co-morbidity and sequential alcohol-MA mixing increases risk for co-abuse. Recently, we reported greater MA-conditioned reward in male C57BL/6J mice with a prior history of binge alcohol-drinking (14 days of 2-hour access to 5, 10, 20 and 40% alcohol). As female mice tend to binge-drink more alcohol than males and females tend to be more sensitive than males to the psychomotor-activating properties of MA, we first characterized the effects of binge-drinking upon MA-induced place-conditioning (four pairings of 0.25, 0.5, 1, 2, or 4 mg/kg IP) in females and then incorporated our prior data to analyze for sex differences in MA-conditioned reward. Prior binge-drinking history did not significantly affect locomotor hyperactivity or its sensitization in female mice. However, the dose-response function for place-conditioning was shifted to the left of water-drinking controls, indicating an increase in sensitivity to MA-conditioned reward. The examination of sex differences revealed no sex differences in alcohol intake, although females exhibited greater MA-induced locomotor stimulation than males, irrespective of their prior drinking history. No statistically significant sex difference was apparent for the potentiation of MA-conditioned reward produced by prior binge-drinking history. If relevant to humans, these data argue that both males and females with a prior binge-drinking history are similarly vulnerable to MA abuse and it remains to be determined whether or not the neural substrates underpinning this increased vulnerability reflect common or sex-specific adaptations in reward-related brain regions.

A Mutation in Hnrnph1 That Decreases Methamphetamine-Induced Reinforcement, Reward, and Dopamine Release and Increases Synaptosomal hnRNP H and Mitochondrial Proteins.

Individual variation in the addiction liability of amphetamines has a heritable genetic component. We previously identified Hnrnph1 (heterogeneous nuclear ribonucleoprotein H1) as a quantitative trait gene underlying decreased methamphetamine-induced locomotor activity in mice. Here, we showed that mice (both females and males) with a heterozygous mutation in the first coding exon of Hnrnph1 (H1+/-) showed reduced methamphetamine reinforcement and intake and dose-dependent changes in methamphetamine reward as measured via conditioned place preference. Furthermore, H1+/- mice showed a robust decrease in methamphetamine-induced dopamine release in the NAc with no change in baseline extracellular dopamine, striatal whole-tissue dopamine, dopamine transporter protein, dopamine uptake, or striatal methamphetamine and amphetamine metabolite levels. Immunohistochemical and immunoblot staining of midbrain dopaminergic neurons and their forebrain projections for TH did not reveal any major changes in staining intensity, cell number, or forebrain puncta counts. Surprisingly, there was a twofold increase in hnRNP H protein in the striatal synaptosome of H1+/- mice with no change in whole-tissue levels. To gain insight into the mechanisms linking increased synaptic hnRNP H with decreased methamphetamine-induced dopamine release and behaviors, synaptosomal proteomic analysis identified an increased baseline abundance of several mitochondrial complex I and V proteins that rapidly decreased at 30 min after methamphetamine administration in H1+/- mice. In contrast, the much lower level of basal synaptosomal mitochondrial proteins in WT mice showed a rapid increase. We conclude that H1+/- decreases methamphetamine-induced dopamine release, reward, and reinforcement and induces dynamic changes in basal and methamphetamine-induced synaptic mitochondrial function.SIGNIFICANCE STATEMENT Methamphetamine dependence is a significant public health concern with no FDA-approved treatment. We discovered a role for the RNA binding protein hnRNP H in methamphetamine reward and reinforcement. Hnrnph1 mutation also blunted methamphetamine-induced dopamine release in the NAc, a key neurochemical event contributing to methamphetamine addiction liability. Finally, Hnrnph1 mutants showed a marked increase in basal level of synaptosomal hnRNP H and mitochondrial proteins that decreased in response to methamphetamine, whereas WT mice showed a methamphetamine-induced increase in synaptosomal mitochondrial proteins. Thus, we identified a potential role for hnRNP H in basal and dynamic mitochondrial function that informs methamphetamine-induced cellular adaptations associated with reduced addiction liability.

Prior binge-drinking history promotes the positive affective valence of methamphetamine in mice.

An alcohol use disorder is a major predisposing factor for methamphetamine (MA) abuse. Further, MA-alcohol co-abuse is a risk factor for treatment discontinuation and non-compliance in MA-dependent individuals. No effective treatment exists for MA addiction, let alone treatments directed at those suffering from MA-alcohol addiction co-morbidity. Thus, it is imperative that we develop high-throughput animal models to study the biobehavioral interactions between MA and alcohol of relevance to the etiology and treatment of co-abuse. To this end, we reported that a history of binge alcohol-drinking [5,10, 20 and 40% (v/v); 2 h/day for 10-14 days] reduces MA reinforcement and intake, but it augments MA-preference and intake when drug availability is behaviorally non-contingent. To reconcile this apparent discrepancy in findings, we employed a comparable 2-week binge-drinking paradigm as that employed in our previous studies followed by place-conditioning procedures (4 pairings of 0.25, 0.5, 1, 2 or 4 mg/kg MA, i.p.). This was meant to determine how a prior binge-drinking history impacts the affective valence of MA and sensitivity to MA-induced psychomotor-activation/sensitization. Prior binge-drinking history blunted spontaneous locomotor activity and shifted the MA dose-place-preference function upwards of water drinking controls. The potentiation of MA-conditioned reward by prior binge-drinking history was independent of any alcohol effects upon the locomotor-activating or -sensitizing effects of MA. Based on these results we propose that the reduced MA reinforcement reported previously by our group likely reflects a compensatory response to an increased sensitivity to MA's positive subjective effects rather than increased sensitivity to the drug's psychomotor-activating effects.

Methamphetamine-alcohol interactions in murine models of sequential and simultaneous oral drug-taking.

BACKGROUND: A high degree of co-morbidity exists between methamphetamine (MA) addiction and alcohol use disorders and both sequential and simultaneous MA-alcohol mixing increases risk for co-abuse. As little preclinical work has focused on the biobehavioral interactions between MA and alcohol within the context of drug-taking behavior, we employed simple murine models of voluntary oral drug consumption to examine how prior histories of either MA- or alcohol-taking influence the intake of the other drug. METHODS: In one study, mice with a 10-day history of binge alcohol-drinking [5,10, 20 and 40% (v/v); 2h/day] were trained to self-administer oral MA in an operant-conditioning paradigm (10-40mg/L). In a second study, mice with a 10-day history of limited-access oral MA-drinking (5, 10, 20 and 40mg/L; 2h/day) were presented with alcohol (5-40% v/v; 2h/day) and then a choice between solutions of 20% alcohol, 10mg/L MA or their mix. RESULTS: Under operant-conditioning procedures, alcohol-drinking mice exhibited less MA reinforcement overall, than water controls. However, when drug availability was not behaviorally-contingent, alcohol-drinking mice consumed more MA and exhibited greater preference for the 10mg/L MA solution than drug-naïve and combination drug-experienced mice. Conversely, prior MA-drinking history increased alcohol intake across a range of alcohol concentrations. DISCUSSION: These exploratory studies indicate the feasibility of employing procedurally simple murine models of sequential and simultaneous oral MA-alcohol mixing of relevance to advancing our biobehavioral understanding of MA-alcohol co-abuse.

Methamphetamine Addiction Vulnerability: The Glutamate, the Bad, and the Ugly.

BACKGROUND: The high prevalence and severity of methamphetamine (MA) abuse demands greater neurobiological understanding of its etiology. METHODS: We conducted immunoblotting and in vivo microdialysis procedures in MA high/low drinking mice, as well as in isogenic C57BL/6J mice that varied in their MA preference/taking, to examine the glutamate underpinnings of MA abuse vulnerability. Neuropharmacological and Homer2 knockdown approaches were also used in C57BL/6J mice to confirm the role for nucleus accumbens (NAC) glutamate/Homer2 expression in MA preference/aversion. RESULTS: We identified a hyperglutamatergic state within the NAC as a biochemical trait corresponding with both genetic and idiopathic vulnerability for high MA preference and taking. We also confirmed that subchronic subtoxic MA experience elicits a hyperglutamatergic state within the NAC during protracted withdrawal, characterized by elevated metabotropic glutamate 1/5 receptor function and Homer2 receptor-scaffolding protein expression. A high MA-preferring phenotype was recapitulated by elevating endogenous glutamate within the NAC shell of mice and we reversed MA preference/taking by lowering endogenous glutamate and/or Homer2 expression within this subregion. CONCLUSIONS: Our data point to an idiopathic, genetic, or drug-induced hyperglutamatergic state within the NAC as a mediator of MA addiction vulnerability.