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Midbrain dopamine (DA) neurons are key regulators of basal ganglia functions. The axonal domain of these neurons is highly complex, with a large subset of non-synaptic release sites and a smaller subset of synaptic terminals from which in addition to DA, glutamate or GABA are also released. The molecular mechanisms regulating the connectivity of DA neurons and their neurochemical identity are unknown. An emerging literature suggests that neuroligins, trans-synaptic cell adhesion molecules, regulate both DA neuron connectivity and neurotransmission. However, the contribution of their major interaction partners, neurexins (Nrxns), is unexplored. Here, we tested the hypothesis that Nrxns regulate DA neuron neurotransmission. Mice with conditional deletion of all Nrxns in DA neurons (DAT::NrxnsKO) exhibited normal basic motor functions. However, they showed an impaired locomotor response to the psychostimulant amphetamine. In line with an alteration in DA neurotransmission, decreased levels of the membrane DA transporter (DAT) and increased levels of the vesicular monoamine transporter (VMAT2) were detected in the striatum of DAT::NrxnsKO mice, along with reduced activity-dependent DA release. Strikingly, electrophysiological recordings revealed an increase of GABA co-release from DA neuron axons in the striatum of these mice. Together, these findings suggest that Nrxns act as regulators of the functional connectivity of DA neurons.

IntroductionInitiation of tobacco products typically occurs in adolescence. Adolescence is a critical period in development where the maturation of brain neurocircuitry is vulnerable to nicotine. Nicotine-containing products and psychostimulants, such as methamphetamine (METH), are often coabused. Rodent studies have shown that nicotine exposure in early adolescence increases subsequent drug intake and reward. Given the exponential increase in e-cigarette use among adolescents, there is a pressing need to understand whether adolescent nicotine exposure impacts concurrent increased METH use. The objective of this study is to evaluate age, sex, and longitudinal effects of nicotine pretreatment on METH reinforcement.Aims and methodsMale and female Sprague-Dawley rats were pretreated with a subchronic, low-dose nicotine (2×, 30 µg/kg/0.1 mL, intravenous) or saline during early adolescence (postnatal days [PN] 28-31) or adulthood (PN 86-89). Following nicotine pretreatment, on PN 32 or PN 90, animals underwent operant intravenous self-administration for METH (20 µg/kg/inf) over a 2-hour period for five consecutive days.ResultsEarly adolescent nicotine exposure enhances intravenous METH self-administration in male, but not female adolescents. Male adult rats self-administer METH over the 5-day testing period, independent of nicotine exposure. In contrast, nicotine exposure increases METH self-administration in female adults during the later sessions of the 5-day testing period.ConclusionsTaken together, our data highlight age- and sex-dependent effects of low dose, subchronic nicotine pretreatment on subsequent intravenous METH self-administration.ImplicationsA majority of polysubstance users begin smoking before the age of 18. Mounting evidence highlights adolescent susceptibility to nicotine exposure on brain and behavior. With the escalation in nicotine-containing products and stimulant use among adolescents, it is important to identify the consequences from adolescent nicotine use, including polysubstance use. Our study provides evidence that adolescent nicotine exposure enhances subsequent METH use, with important sex- and age-dependent effects.

Methamphetamine (MA) is a highly addictive psychostimulant drug, and the number of MA-related overdose deaths has reached epidemic proportions. Repeated MA exposure induces a robust and persistent neuroinflammatory response, and the evidence supports the potential utility of targeting neuroimmune function using non-selective phosphodiesterase 4 (PDE4) inhibitors as a therapeutic strategy for attenuating addiction-related behavior. Off-target, emetic effects associated with non-selective PDE4 blockade led to the development of isozyme-selective inhibitors, of which the PDE4B-selective inhibitor A33 was demonstrated recently to reduce binge drinking in two genetically related C57BL/6 (B6) substrains (C57BL/6NJ (B6NJ) and C57BL/6J (B6J)) that differ in their innate neuroimmune response. Herein, we determined the efficacy of A33 for reducing MA self-administration and MA-seeking behavior in these two B6 substrains. Female and male mice of both substrains were first trained to nose poke for a 100 mg/L MA solution followed by a characterization of the dose-response function for oral MA reinforcement (20 mg/L-3.2 g/L), the demand-response function for 400 mg/L MA, and cue-elicited MA seeking following a period of forced abstinence. During this substrain comparison of MA self-administration, we also determined the dose-response function for A33 pretreatment (0-1 mg/kg) on the maintenance of MA self-administration and cue-elicited MA seeking. Relative to B6NJ mice, B6J mice earned fewer reinforcers, consumed less MA, and took longer to reach acquisition criterion with males of both substrains exhibiting some signs of lower MA reinforcement than their female counterparts during the acquisition phase of the study. A33 pretreatment reduced MA reinforcement at all doses tested. These findings provide the first evidence that pretreatment with a selective PDE4B inhibitor effectively reduces MA self-administration in both male and female mice of two genetically distinct substrains but does not impact cue-elicited MA seeking following abstinence. If relevant to humans, these results posit the potential clinical utility of A33 or other selective PDE4B inhibitors for curbing active drug-taking in MA use disorder.

The α-pyrrolidino-phenone cathinone stimulants first came to widespread attention because of bizarre behavior consequent to the use of α-pyrrolidinopentiophenone (α-PVP, "flakka") reported in popular press. As with other designer drugs, diversification of cathinones has been driven by desirable subjective effects, but also by attempts to stay ahead of legal controls of specific molecules. The α-pyrrolidinohexiophenone (α-PHP) and α-pyrrolidinopropiophenone (α-PPP) compounds have been relatively under-investigated relative to α-PVP and provide a key opportunity to also investigate structure-activity relationships, i.e., how the extension of the alpha carbon chain may affect potency or efficacy. Female rats were used to contrast the effects of α-PHP and α-PPP with those of α-PVP in altering wheel activity and effects on spontaneous locomotion, temperature and intracranial self-stimulation reward. The α-PPP, α-PHP and α-PVP compounds (5, 10 mg/kg, i.p.) suppressed wheel activity. Inhalation of α-PHP or α-PVP also suppressed wheel activity, but for an abbreviated duration compared with the injection route. Spontaneous activity was increased, and brain reward thresholds decreased, in a dose-dependent manner by all three compounds; only small decrements in body temperature were observed. These data show that all three of the α-pyrrolidino-phenone cathinones exhibit significant stimulant-like activity in female rats. Differences were minor and abuse liability is therefore likely to be equivalent for all three α-pyrrolidino-phenones.

HIV infection and methamphetamine (METH) use are highly comorbid and represent a significant public health problem. Both conditions are known to negatively impact a variety of brain functions. One brain function that may be affected by HIV and METH use is sensorimotor gating, an automatic, pre-conscious filtering of sensory information that is thought to contribute to higher order cognitive processes. Sensorimotor gating is often measured using prepulse inhibition (PPI), a paradigm that can be conducted in both humans and animals, thereby enabling cross-species translational studies. While previous studies suggest HIV and METH may individually impair PPI, little research has been conducted on the effects of combined HIV and METH on PPI. The goal of this cross-species study was to determine the effects of METH on PPI in the inducible Tat (iTat) mouse model of HIV and in people with HIV. PPI was measured in the iTat mouse model before, during, and after chronic METH treatment and after Tat induction. Chronic METH treatment decreased PPI in male but not female mice. PPI normalized with cessation of METH. Inducing Tat expression decreased PPI in male but not in female mice. No interactions between chronic METH treatment and Tat expression were observed in mice. In humans, HIV was associated with decreased PPI in both men and women. Furthermore, PPI was lowest in people with HIV who also had a history of METH dependence. Overall, these results suggest HIV and METH may additively impair early information processing in humans, potentially affecting downstream cognitive function.

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.

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.

We previously identified a 210 kb region on chromosome 11 (50.37-50.58 Mb, mm10) containing two protein-coding genes (Hnrnph1, Rufy1) that was necessary for reduced methamphetamine-induced locomotor activity in C57BL/6J congenic mice harboring DBA/2J polymorphisms. Gene editing of a small deletion in the first coding exon supported Hnrnph1 as a quantitative trait gene. We have since shown that Hnrnph1 mutants also exhibit reduced methamphetamine-induced reward, reinforcement, and dopamine release. However, the quantitative trait variants (QTVs) that modulate Hnrnph1 function at the molecular level are not known. Nine single nucleotide polymorphisms and seven indels distinguish C57BL/6J from DBA/2J within Hnrnph1, including four variants within the 5' untranslated region (UTR). Here, we show that a 114 kb introgressed region containing Hnrnph1 and Rufy1 was sufficient to cause a decrease in MA-induced locomotor activity. Gene-level transcriptome analysis of striatal tissue from 114 kb congenics vs Hnrnph1 mutants identified a nearly perfect correlation of fold-change in expression for those differentially expressed genes that were common to both mouse lines, indicating functionally similar effects on the transcriptome and behavior. Exon-level analysis (including noncoding exons) revealed decreased 5' UTR usage of Hnrnph1 and immunoblot analysis identified a corresponding decrease in hnRNP H protein in 114 kb congenic mice. Molecular cloning of the Hnrnph1 5' UTR containing all four variants (but none of them individually) upstream of a reporter induced a decrease in reporter signal in both HEK293 and N2a cells, thus, identifying a set of QTVs underlying molecular regulation of Hnrnph1.

Caffeine is the most widely consumed psychoactive substance in the world. However, there is controversy about whether becoming addicted to caffeine is possible and a lack of well-established animal models to examine caffeine consumption. The present study sought to establish a model of caffeine consumption in Wistar rats, identify different rat populations based on caffeine preference, and determine whether extended voluntary caffeine consumption produces compulsive-like caffeine intake and withdrawal symptoms. Male Wistar rats were used throughout the experiment. The optimal concentration of caffeine to maximize caffeine consumption and caffeine preference was determined. Rats were then given continuous access to caffeine, followed by intermittent access. Rats were tested for signs of withdrawal-like behavior by measuring mechanical nociception and irritability-like behavior. Rats were further examined for compulsive-like caffeine consumption using quinine adulteration. Dose-response testing indicated an optimal caffeine concentration of 0.3 mg/mL. During intermittent access to caffeine, the rats did not escalate their caffeine intake and instead exhibited a decrease in intake over sessions. Three groups of rats were identified based on caffeine preference (high, medium, and low) across continuous and intermittent access. These three groups of rats matched low (1 cup), medium (2 cups), and high (4 cups) levels of daily coffee consumption in humans. Caffeine-consuming rats did not exhibit differences in mechanical nociception or irritability-like behavior compared with controls. In high caffeine-preferring rats but not in medium or low caffeine-preferring rats, compulsive-like caffeine consumption was observed. The present study established a rodent model of caffeine consumption that resulted in large individual differences in caffeine intake, similar to humans. Compulsive-like caffeine consumption in high caffeine-preferring rats and differences in caffeine preference between groups suggest that caffeine may result in compulsive-like intake in a subpopulation of subjects. Further testing is necessary to determine the factors that contribute to differences in caffeine preference and compulsive-like intake.

The abuse of stimulants, such as methamphetamine (METH), is associated with treatment non-compliance, a greater risk of viral transmission, and the more rapid clinical progression of immunological and central nervous system human immunodeficiency virus (HIV) disease. The behavioral effects of METH in the setting of HIV remain largely uncharacterized. We used a state-of-the-art paradigm of the escalation of voluntary intravenous drug self-administration in HIV transgenic (Tg) and wildtype rats. The rats were first allowed to self-administer METH under short-access (ShA) conditions, which is characterized by a nondependent and more "recreational" pattern of METH use, and then allowed to self-administer METH under long-access (LgA) conditions, which leads to compulsive (dependent) METH intake. HIV Tg and wildtype rats self-administered equal amounts of METH under ShA conditions. HIV Tg rats self-administered METH under LgA conditions following a 4-week enforced abstinence period to model the intermittent pattern of stimulant abuse in humans. These HIV Tg rats developed greater motivation to self-administer METH and self-administered larger amounts of METH. Impairments in function of the medial prefrontal cortex (mPFC) contribute to compulsive drug and alcohol intake. Gene expression profiling of the mPFC in HIV Tg rats with a history of escalated METH self-administration under LgA conditions showed transcriptional evidence of increased inflammation, greater neural injury, and impaired aerobic glucose metabolism than wildtype rats that self-administered METH under LgA conditions. The detrimental effects of the interaction between neuroHIV and escalated METH intake on the mPFC are likely key factors in the greater vulnerability to excessive drug intake in the setting of HIV.

BackgroundPreclinical studies suggest that the non-selective phosphodiesterase inhibitor, Ibudilast (IBUD) may contribute to the treatment of methamphetamine (METH) use disorder through the attenuation of METH-induced inflammatory markers such as adhesion molecules, sICAM-1 and sVCAM-1, and cytokines, IL-6 and TNF-α.ObjectiveThe present study aimed to test whether treatment with IBUD can attenuate peripheral markers of inflammation during a METH challenge in an inpatient clinical trial of 11 patients.MethodsThis trial followed a randomized, within-subjects crossover design where participants received a METH challenge, during which five participants were treated with placebo then with IBUD, while the remaining six participants were treated with IBUD prior to placebo. Mixed effects regression modeled changes in peripheral markers of inflammation-sICAM-1, sVCAM-1, TNF-α, IL-6, MIF, and cathepsin D-by treatment condition, with measurements at baseline, 60 min post-METH infusion, and 360 min post-METH infusion.ResultsWhile on placebo, sICAM-1, sVCAM-1, and cathepsin D significantly increased by 60 min post-METH infusion, while IL-6 significantly increased 360 min post-METH infusion. Treatment with IBUD significantly reduced METH-induced levels of sICAM-1, sVCAM-1, and cathepsin D at 60 min post-METH infusion.ConclusionsOur findings demonstrate that IBUD attenuated acute pro-inflammatory effects of METH administration, which may have implications for treatment of METH use disorder.

Reward-related behavior is complex and its dysfunction correlated with neuropsychiatric illness. Dopamine (DA) neurons of the ventral tegmental area (VTA) have long been associated with different aspects of reward function, but it remains to be disentangled how distinct VTA DA neurons contribute to the full range of behaviors ascribed to the VTA. Here, a recently identified subtype of VTA neurons molecularly defined by NeuroD6 (NEX1M) was addressed. Among all VTA DA neurons, less than 15% were identified as positive for NeuroD6. In addition to dopaminergic markers, sparse NeuroD6 neurons expressed the vesicular glutamate transporter 2 (Vglut2) gene. To achieve manipulation of NeuroD6 VTA neurons, NeuroD6(NEX)-Cre-driven mouse genetics and optogenetics were implemented. First, expression of vesicular monoamine transporter 2 (VMAT2) was ablated to disrupt dopaminergic function in NeuroD6 VTA neurons. Comparing Vmat2lox/lox;NEX-Cre conditional knock-out (cKO) mice with littermate controls, it was evident that baseline locomotion, preference for sugar and ethanol, and place preference upon amphetamine-induced and cocaine-induced conditioning were similar between genotypes. However, locomotion upon repeated psychostimulant administration was significantly elevated above control levels in cKO mice. Second, optogenetic activation of NEX-Cre VTA neurons was shown to induce DA release and glutamatergic postsynaptic currents within the nucleus accumbens. Third, optogenetic stimulation of NEX-Cre VTA neurons in vivo induced significant place preference behavior, while stimulation of VTA neurons defined by Calretinin failed to cause a similar response. The results show that NeuroD6 VTA neurons exert distinct regulation over specific aspects of reward-related behavior, findings that contribute to the current understanding of VTA neurocircuitry.

BackgroundElectronic nicotine delivery systems (ENDS, e-cigarettes) are increasingly used for the self-administration of nicotine by various human populations, including previously nonsmoking adolescents. Studies in preclinical models are necessary to evaluate health impacts of ENDS including the development of nicotine addiction, effects of ENDS vehicles, flavorants and co-administered psychoactive substances such as Δ9-tetrahydrocannabinol (THC). This study was conducted to validate a rat model useful for the study of nicotine effects delivered by inhalation of vapor created by ENDS.MethodsMale Sprague-Dawley rats (N = 8) were prepared with radio telemetry devices for the reporting of temperature and activity. Experiments subjected rats to inhalation of vapor generated by an electronic nicotine delivery system (ENDS) adapted for rodents. Inhalation conditions included vapor generated by the propylene glycol (PG) vehicle, Nicotine (1, 10, 30 mg/mL in the PG) and THC (12.5, 25 mg/mL).ResultsNicotine inhalation increased spontaneous locomotion and decreased body temperature of rats. Pretreatment with the nicotinic cholinergic receptor antagonist mecamylamine (2 mg/kg, i.p.) prevented stimulant effects of nicotine vapor inhalation and attenuated the hypothermic response. Combined inhalation of nicotine and THC resulted in apparently independent effects which were either additive (hypothermia) or opposed (activity).ConclusionsThese studies provide evidence that ENDS delivery of nicotine via inhalation results in nicotine-typical effects on spontaneous locomotion and thermoregulation in male rats. Effects were blocked by a nicotinic antagonist, demonstrating mechanistic specificity. This system will therefore support additional studies of the contribution of atomizer/wick design, vehicle constituents and/or flavorants to the effects of nicotine administered by ENDS.

The broad diversity of synthetic cathinone psychostimulant drugs that are available to users complicates research efforts to provide understanding of health risks. Second generation cathinones pentedrone and pentylone are distinguished from each other by the 3,4-methylenedioxy structural motif (which distinguishes methamphetamine from 3,4-methylenedioxymethamphetamine) and each incorporates the α-alkyl chain motif contained in the transporter-inhibitor cathinones (3,4-methylenedioxypyrovalerone (MDPV), α-pyrrolidinopentiophenone (α-PVP)) but not in the monoamine releasers (mephedrone, methylone). Studies were conducted in male and female Wistar rats to compare locomotor and thermoregulatory effects of pentedrone, pentylone and methylone using an implanted radiotelemetry system. Reinforcing effects were assessed in female Wistar rats trained in the intravenous self-administration (IVSA) procedure and subjected to dose-substitution (0.025-0.3 m/gkg/inf) under a fixed-ratio 1 response contingency. Pentedrone, pentylone and methylone dose-effect curves were contrasted with those for α-PVP and α-pyrrolidinohexiophenone (α-PHP). Dose dependent increases in locomotion were observed after intraperitoneal injection of pentylone (0.5-10.0 mg/kg), pentedrone (0.5-10.0 mg/kg) or mephedrone (0.5-10.0 mg/kg) in male and female rats. The maximum locomotor effect was similar across drugs but lasted longest after pentedrone. Mean body temperature did not vary systematically more than 0.5 °C after pentedrone or pentylone in either sex. A sustained hyperthermia (0.4-0.8 °C) was observed for four hours after 10.0 mg/kg methylone in male rats. More infusions of pentedrone or pentylone were self-administered compared with methylone, but all three were less potent than α-PVP or α-PHP. These studies support the inference that second generation cathinones pentylone and pentedrone have abuse liability greater than that of methylone. This article is part of the Special Issue entitled 'Designer Drugs and Legal Highs.'

We previously reported that acute and selective activation of GABA-releasing parafacial zone (PZVgat) neurons in behaving mice produces slow-wave-sleep (SWS), even in the absence of sleep deficit, suggesting that these neurons may represent, at least in part, a key cellular substrate underlying sleep drive. It remains, however, to be determined if PZVgat neurons actively maintain, as oppose to simply gate, SWS. To begin to experimentally address this knowledge gap, we asked whether activation of PZVgat neurons could attenuate or block the wake-promoting effects of two widely used wake-promoting psychostimulants, armodafinil or caffeine. We found that activation of PZVgat neurons completely blocked the behavioral and electrocortical wake-promoting action of armodafinil. In some contrast, activation of PZVgat neurons inhibited the behavioral, but not electrocortical, arousal response to caffeine. These results suggest that: (1) PZVgat neurons actively maintain, as oppose to simply gate, SWS and cortical slow-wave-activity; (2) armodafinil cannot exert its wake-promoting effects when PZVgat neurons are activated, intimating a possible shared circuit/molecular basis for mechanism of action; (3) caffeine can continue to exert potent cortical desynchronizing, but not behavioral, effects when PZVgat neurons are activated, inferring a shared and divergent circuit/molecular basis for mechanism of action; and 4) PZVgat neurons represent a key cell population for SWS induction and maintenance.

RationaleDrug addiction can be described as aberrant allocation of effort toward acquiring drug, despite associated costs. It is unclear if this behavioral pattern results from an overvaluation of reward or to an altered sensitivity to costs.ObjectivePresent experiments assessed reward sensitivity and effortful choice in rats following 1 week of withdrawal from methamphetamine (mAMPH).MethodsRats were treated with either saline or an escalating dose mAMPH regimen, then tested after a week without the drug. In experiment 1, rats were given a free choice between water and various concentrations of sucrose solution to assess general reward sensitivity. In experiment 2, rats were presented with a choice between lever-pressing for sucrose pellets on a progressive ratio schedule or consuming freely-available chow.ResultsIn experiment 1, we found no differences in sucrose preference between mAMPH- and saline-pretreated rats. In experiment 2, when selecting between two options, mAMPH-pretreated rats engaged in less lever-pressing for sucrose pellets (p

The recreational use of substituted cathinones continues to grow as a public health concern in the United States. Studies have shown that extended access to intravenous (i.v.) self-administration of stimulants, such as cocaine and methamphetamine, results in escalation of drug intake relative to shorter access; however, little is known about the impact of extended access on self-administration of entactogen class stimulants such as methylone and 4-methylmethcathinone (mephedrone). Male Wistar rats were randomly assigned to short-access (ShA, 2- h) and long-access (LgA, 6- h) groups and trained to self-administer methylone or mephedrone (0.5 mg/kg/infusion) using a fixed-ratio 1 response contingency. The methylone-trained groups were evaluated on a progressive-ratio (PR) procedure incorporating dose-substitution of methylone (0.125-2.5 mg/kg/infusion), mephedrone (0.125-2.5 mg/kg/infusion) or methamphetamine (MA; 0.01-0.5 mg/kg/infusion). Mephedrone-trained rats were similarly evaluated on a PR with mephedrone and MA. Rats trained with LgA to methylone and mephedrone earned more infusions during acquisition compared with ShA groups. Mephedrone-trained LgA rats reached significantly higher breakpoints than all other groups in mephedrone and MA PR tests. Methylone-trained LgA rats exhibited a rightward shift of the peak effective dose but no overall efficacy change compared with methylone-trained ShA rats. These findings show that the self-administration of mephedrone escalates under LgA conditions in a manner similar to traditional stimulants whereas escalation of 6 h intakes of methylone is not accompanied by differences in PR performance. Thus mephedrone represents the greater risk for dysregulated drug consumption.

Recreational use of substituted cathinones continues to be an emerging public health problem in the United States; cathinone derivatives α-pyrrolidinopentiophenone (α-PVP) and 3,4-methylenedioxypyrovalerone (MDPV), which have been linked to human fatalities and show high potential for abuse liability in animal models, are of particular concern. The objective of this study was to develop an immunotherapeutic strategy for attenuating the effects of α-PVP and MDPV in rats, using drug-conjugate vaccines created to generate antibodies with neutralizing capacity. Immunoconjugates (α-PVP-KLH and MDPV-KLH) or the control carrier protein, keyhole limpet hemocyanin (KLH), were administered to groups (N = 12) of male Sprague-Dawley rats on Weeks 0, 2 and 4. Groups were administered α-PVP or MDPV (0.0, 0.25, 0.5, 1.0, 5.0 mg/kg, i.p.) in acute drug challenges and tested for changes in wheel activity. Increased wheel activity produced by α-PVP or MDPV in the controls was attenuated in the α-PVP-KLH and MDPV-KLH vaccinated groups, respectively. Rectal temperature decreases produced by MDPV in the controls were reduced in duration in the MDPV-KLH vaccine group. A separate group (N = 19) was trained to intravenously self-administer α-PVP (0.05, 0.1 mg/kg/inf) and vaccinated with KLH or α-PVP-KLH, post-acquisition. Self-administration in α-PVP-KLH rats was initially higher than in the KLH rats but then significantly decreased following a final vaccine booster, unlike the stable intake of KLH rats. The data demonstrate that active vaccination provides functional protection against the effects of α-PVP and MDPV, in vivo, and recommend additional development of vaccines as potential therapeutics for mitigating the effects of designer cathinone derivatives.

The two highly homologous non-visual arrestins, beta-arrestin 1 and 2, are ubiquitously expressed in the central nervous system, yet knowledge of their disparate roles is limited. While beta-arrestin 2 (βarr2) has been implicated in several aspects of reward-related learning and behavior, very little is known about the behavioral function of beta-arrestin 1 (βarr1). Using mice lacking βarr1, we focused on the role of this scaffolding and signal transduction protein in reward-motivated behaviors and in striatal glutamatergic function. We found that βarr1 KO mice were both slower in acquiring cocaine self-administration and in extinguishing this behavior. They also showed deficits in learning tasks supported by a natural food reward, suggesting a general alteration in reward processing. We then examined glutamatergic synaptic strength in WT and KO medium spiny neurons (MSNs) of the Nucleus Accumbens (NAc) shell in naïve animals, and from those that underwent cocaine self-administration. An increase in the AMPA/NMDA (A/N) ratio and a relative lack of GluN2B-enriched NMDARs was found in naïve KO vs WT MSNs. Applying Lim Domain Kinase (LIMK1), the kinase that phosphorylates and inactivates cofilin, to these cells, showed that both βarr1 and LIMK regulate the A/N ratio and GluN2B-NMDARs. Cocaine self-administration increased the A/N ratio and GluN2B-NMDARs in WT MSNs and, although the A/N ratio also increased in KO MSNs, this was accompanied by fewer GluN2B-NMDARs and an appearance of calcium-permeable AMPARs. Finally, to examine the consequences of reduced basal GluN2B-NMDARs in reward-processing seen in KO mice, we chronically infused ifenprodil, a GluN2B antagonist, into the NAc shell of WT mice. This intervention substantially reduced food-motivated behavior. Together these findings identify a previously unknown role of βarr1 in regulating specific reward-motivated behaviors and glutamatergic function.

Although inhaled exposure to drugs is a prevalent route of administration for human substance abusers, preclinical models that incorporate inhaled exposure to psychomotor stimulants are not commonly available. Using a novel method that incorporates electronic cigarette-type technology to facilitate inhalation, male Wistar rats were exposed to vaporized methamphetamine (MA), 3,4-methylenedioxypyrovalerone (MDPV), and mephedrone (4-methylmethcathinone) in propylene glycol vehicle using concentrations ranging from 12.5 to 200 mg/ml. Rats exhibited increases in spontaneous locomotor activity, measured by implanted radiotelemetry, following exposure to methamphetamine (12.5 and 100 mg/ml), MDPV (25, 50, and 100 mg/ml), and mephedrone (200 mg/ml). Locomotor effects were blocked by pretreatment with the dopamine D1-like receptor antagonist SCH23390 (10 μg/kg, intraperitoneal (i.p.)). MA and MDPV vapor inhalation also altered activity on a running wheel in a biphasic manner. An additional group of rats was trained on a discrete trial intracranial self-stimulation (ICSS) procedure interpreted to assess brain reward status. ICSS-trained rats that received vaporized MA, MDPV, or mephedrone exhibited a significant reduction in threshold of ICSS reward compared with vehicle. The effect of vapor inhalation of the stimulants was found comparable to the locomotor and ICSS threshold-reducing effects of i.p. injection of mephedrone (5.0 mg/kg), MA (0.5-1.0 mg/kg), or MDPV (0.5-1.0 mg/kg). These data provide robust validation of e-cigarette-type technology as a model for inhaled delivery of vaporized psychostimulants. Finally, these studies demonstrate the potential for human use of e-cigarettes to facilitate covert use of a range of psychoactive stimulants. Thus, these devices pose health risks beyond their intended application for the delivery of nicotine.

Corticostriatal circuitry supports flexible reward learning and emotional behavior from the critical neurodevelopmental stage of adolescence through adulthood. It is still poorly understood how prescription drug exposure in adolescence may impact these outcomes in the long-term. We studied adolescent methylphenidate (MPH) and fluoxetine (FLX) exposure in rats and their impact on learning and emotion in adulthood. In Experiment 1, male and female rats were administered MPH, FLX, or saline (SAL), and compared with methamphetamine (mAMPH) treatment beginning in postnatal day (PND) 37. The rats were then tested on discrimination and reversal learning in adulthood. In Experiment 2, animals were administered MPH or SAL also beginning in PND 37 and later tested in adulthood for anxiety levels. In Experiment 3, we analyzed striatal dopamine D1 and D2 receptor expression in adulthood following either extensive learning (after Experiment 1) or more brief emotional measures (after Experiment 2). We found sex differences in discrimination learning and attenuated reversal learning after MPH and only sex differences in adulthood anxiety. In learners, there was enhanced striatal D1, but not D2, after either adolescent MPH or mAMPH. Lastly, also in learners, there was a sex x treatment group interaction for D2, but not D1, driven by the MPH-pretreated females, who expressed significantly higher D2 levels compared to SAL. These results show enduring effects of adolescent MPH on reversal learning in rats. Developmental psychostimulant exposure may interact with learning to enhance D1 expression in adulthood, and affect D2 expression in a sex-dependent manner.

Methamphetamine (METH) is an addictive psychostimulant inducing neurotoxicity. Human magnetic resonance imaging and diffusion tensor imaging (DTI) of METH-dependent participants find various structural abnormities. Animal studies demonstrate immunohistochemical changes in multiple cellular pathways after METH exposure. Here, we characterized the long-term effects of METH on brain microstructure in mice exposed to an escalating METH binge regimen using in vivo DTI, a methodology directly translatable across species. Results revealed four patterns of differential fractional anisotropy (FA) and mean diffusivity (MD) response when comparing METH-exposed (n=14) to saline-treated mice (n=13). Compared to the saline group, METH-exposed mice demonstrated: 1) decreased FA with no change in MD [corpus callosum (posterior forceps), internal capsule (left), thalamus (medial aspects), midbrain], 2) increased MD with no change in FA [posterior isocortical regions, caudate-putamen, hypothalamus, cerebral peduncle, internal capsule (right)], 3) increased FA with decreased MD [frontal isocortex, corpus callosum (genu)], and 4) increased FA with no change or increased MD [hippocampi, amygdala, lateral thalamus]. MD was negatively associated with calbindin-1 in hippocampi and positively with dopamine transporter in caudate-putamen. These findings highlight distributed and differential METH effects within the brain suggesting several distinct mechanisms. Such mechanisms likely change brain tissue differentially dependent upon neural location.

Methamphetamine (MA) is a widely misused, highly addictive psychostimulant that elicits pronounced deficits in neurocognitive function related to hypo-functioning of the prefrontal cortex (PFC). Our understanding of how repeated MA impacts excitatory glutamatergic transmission within the PFC is limited, as is information about the relationship between PFC glutamate and addiction vulnerability/resiliency. In vivo microdialysis and immunoblotting studies characterized the effects of MA (ten injections of 2 mg/kg, i.p.) upon extracellular glutamate in C57BL/6J mice and upon glutamate receptor and transporter expression, within the medial PFC. Glutamatergic correlates of both genetic and idiopathic variance in MA preference/intake were determined through studies of high vs. low MA-drinking selectively bred mouse lines (MAHDR vs. MALDR, respectively) and inbred C57BL/6J mice exhibiting spontaneously divergent place-conditioning phenotypes. Repeated MA sensitized drug-induced glutamate release and lowered indices of N-methyl-d-aspartate receptor expression in C57BL/6J mice, but did not alter basal extracellular glutamate content or total protein expression of Homer proteins, or metabotropic or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors. Elevated basal glutamate, blunted MA-induced glutamate release and ERK activation, as well as reduced protein expression of mGlu2/3 and Homer2a/b were all correlated biochemical traits of selection for high vs. low MA drinking, and Homer2a/b levels were inversely correlated with the motivational valence of MA in C57BL/6J mice. These data provide novel evidence that repeated, low-dose MA is sufficient to perturb pre- and post-synaptic aspects of glutamate transmission within the medial PFC and that glutamate anomalies within this region may contribute to both genetic and idiopathic variance in MA addiction vulnerability/resiliency.

RationaleCross-species quantification of physiological behavior enables a better understanding of the biological systems underlying neuropsychiatric diseases such as bipolar disorder (BD). Cardinal symptoms of manic BD include increased motor activity and goal-directed behavior, thought to be related to increased catecholamine activity, potentially selective to dopamine homeostatic dysregulation.ObjectivesThe objective of this study was to test whether acute administration of amphetamine, a norepinephrine/dopamine transporter inhibitor and dopamine releaser, would replicate the profile of activity and exploration observed in both humans with manic BD and mouse models of mania.MethodsHealthy volunteers with no psychiatric history were randomized to a one-time dose of placebo (n = 25), 10 mg d-amphetamine (n = 18), or 20 mg amphetamine (n = 23). Eighty mice were administered one of four doses of d-amphetamine or vehicle. Humans and mice were tested in the behavioral pattern monitor (BPM), which quantifies motor activity, exploratory behavior, and spatial patterns of behavior.ResultsIn humans, the 20-mg dose of amphetamine increased motor activity as measured by acceleration without marked effects on exploration or spatial patterns of activity. In mice, amphetamine increased activity, decreased specific exploration, and caused straighter, one-dimensional movements in a dose-dependent manner.ConclusionsConsistent with mice, amphetamine increased motoric activity in humans without increasing exploration. Given that BD patients exhibit heightened exploration, these data further emphasize the limitation of amphetamine-induced hyperactivity as a suitable model for BD. Further, these studies highlight the utility of cross-species physiological paradigms in validating biological mechanisms of psychiatric diseases.

Psychostimulant addiction is a heritable substance use disorder; however its genetic basis is almost entirely unknown. Quantitative trait locus (QTL) mapping in mice offers a complementary approach to human genome-wide association studies and can facilitate environment control, statistical power, novel gene discovery, and neurobiological mechanisms. We used interval-specific congenic mouse lines carrying various segments of chromosome 11 from the DBA/2J strain on an isogenic C57BL/6J background to positionally clone a 206 kb QTL (50,185,512-50,391,845 bp) that was causally associated with a reduction in the locomotor stimulant response to methamphetamine (2 mg/kg, i.p.; DBA/2J < C57BL/6J)-a non-contingent, drug-induced behavior that is associated with stimulation of the dopaminergic reward circuitry. This chromosomal region contained only two protein coding genes-heterogeneous nuclear ribonucleoprotein, H1 (Hnrnph1) and RUN and FYVE domain-containing 1 (Rufy1). Transcriptome analysis via mRNA sequencing in the striatum implicated a neurobiological mechanism involving a reduction in mesolimbic innervation and striatal neurotransmission. For instance, Nr4a2 (nuclear receptor subfamily 4, group A, member 2), a transcription factor crucial for midbrain dopaminergic neuron development, exhibited a 2.1-fold decrease in expression (DBA/2J < C57BL/6J; p 4.2 x 10-15). Transcription activator-like effector nucleases (TALENs)-mediated introduction of frameshift deletions in the first coding exon of Hnrnph1, but not Rufy1, recapitulated the reduced methamphetamine behavioral response, thus identifying Hnrnph1 as a quantitative trait gene for methamphetamine sensitivity. These results define a novel contribution of Hnrnph1 to neurobehavioral dysfunction associated with dopaminergic neurotransmission. These findings could have implications for understanding the genetic basis of methamphetamine addiction in humans and the development of novel therapeutics for prevention and treatment of substance abuse and possibly other psychiatric disorders.

Despite aggressive efforts to contain it, methamphetamine use disorder continues to be major public health problem; and with generic behavioral therapies still the mainstay of treatment for methamphetamine abuse, rates of attrition and relapse remain high. This review summarizes the findings of structural, molecular, and functional neuroimaging studies of methamphetamine abusers, focusing on cortical and striatal abnormalities and their potential contributions to cognitive and behavioral phenotypes that can serve to promote compulsive drug use. These studies indicate that individuals with a history of chronic methamphetamine abuse often display several signs of corticostriatal dysfunction, including abnormal gray- and white-matter integrity, monoamine neurotransmitter system deficiencies, neuroinflammation, poor neuronal integrity, and aberrant patterns of brain connectivity and function, both when engaged in cognitive tasks and at rest. More importantly, many of these neural abnormalities were found to be linked with certain addiction-related phenotypes that may influence treatment response (e.g., poor self-control, cognitive inflexibility, maladaptive decision-making), raising the possibility that they may represent novel therapeutic targets.

Exposure to drugs of abuse can produce many neurobiological changes which may lead to increased valuation of rewards and decreased sensitivity to their costs. Many of these behavioral alterations are associated with activity of D2-expressing medium spiny neurons in the striatum. Additionally, Bdnf in the striatum has been shown to play a role in flexible reward-seeking behavior. Given that voluntary aerobic exercise can affect the expression of these proteins in healthy subjects, and that exercise has shown promise as an anti-addictive therapy, we set out to quantify changes in D2 and Bdnf expression in methamphetamine-exposed rats given access to running wheels. Sixty-four rats were treated for two weeks with an escalating dose of methamphetamine or saline, then either sacrificed, housed in standard cages, or given free access to a running wheel for 6 weeks prior to sacrifice. Rats treated with methamphetamine ran significantly greater distances than saline-treated rats, suggesting an augmentation in the reinforcement value of voluntary wheel running. Transcription of Drd2 and Bdnf was assessed via RT-qPCR. Protein expression levels of D2 and phosphorylation of the TrkB receptor were measured via western blot. Drd2 and Bdnf mRNA levels were impacted independently by exercise and methamphetamine, but exposure to methamphetamine prior to the initiation of exercise blocked the exercise-induced changes seen in rats treated with saline. Expression levels of both proteins were elevated immediately after methamphetamine, but returned to baseline after six weeks, regardless of exercise status.

Despite aggressive efforts to contain it, methamphetamine use disorder continues to be major public health problem; and with generic behavioral therapies still the mainstay of treatment for methamphetamine abuse, rates of attrition and relapse remain high. This review summarizes the findings of structural, molecular, and functional neuroimaging studies of methamphetamine abusers, focusing on cortical and striatal abnormalities and their potential contributions to cognitive and behavioral phenotypes that can serve to promote compulsive drug use. These studies indicate that individuals with a history of chronic methamphetamine abuse often display several signs of corticostriatal dysfunction, including abnormal gray- and white-matter integrity, monoamine neurotransmitter system deficiencies, neuroinflammation, poor neuronal integrity, and aberrant patterns of brain connectivity and function, both when engaged in cognitive tasks and at rest. More importantly, many of these neural abnormalities were found to be linked with certain addiction-related phenotypes that may influence treatment response (e.g., poor self-control, cognitive inflexibility, maladaptive decision-making), raising the possibility that they may represent novel therapeutic targets.

The intravenous self-administration (IVSA) of 3,4-methylenedioxymethamphetamine (MDMA) is inconsistent in rats, with up to half of subjects failing to acquire reliable drug intake. It is unknown if this changes under long-access conditions (6 h sessions) under which the IVSA of cocaine and methamphetamine escalates. The entactogen class cathinone stimulants which exhibit MDMA-like monoamine effects in the nucleus accumbens, mephedrone (4-methylmethcathinone) and methylone (3,4-methylenedioxymethcathinone), may support more reliable IVSA but results have been mixed. This study was designed to directly compare the IVSA of these three compounds. Groups of male Wistar rats were trained to self-administer mephedrone, methylone or MDMA (0.5 mg/kg/inf) under a Fixed-Ratio (FR) 1 schedule of reinforcement for 14 sessions. Following the acquisition interval, animals were evaluated in FR (0.0, 0.125, 0.25, 0.5, 1.0, 2.5 mg/kg/inf) and Progressive Ratio (PR; 0.125, 1.0 mg/kg/inf) dose-substitution procedures. Long access conditions escalated MDMA intake over the 6 h session but not in the first 2 h. In short access, drug intake was significantly higher in mephedrone-trained rats compared with either the methylone-trained or MDMA-trained groups during acquisition. Mephedrone resulted in the highest intakes during FR and PR dose-substitution in MDMA- and mephedrone-trained groups. Overall it was found that mephedrone is a more effective reinforcer than methylone or MDMA and represents a higher risk for compulsive use.

This study reviews the findings from the Infant Development, Environment, and Lifestyle (IDEAL) study, a multisite, longitudinal, prospective study designed to determine maternal outcome and child growth and developmental findings following prenatal methamphetamine exposure from birth up to age 7.5 years. These findings are presented in the context of the home environment and caregiver characteristics to determine how the drug and the environment interact to affect the outcome of these children. No neonatal abstinence syndrome requiring pharmacologic intervention was observed but heavy drug exposure was associated with increased stress responses in the neonatal period. Poorer inhibitory control was also observed in heavy methamphetamine exposed children placing them at high risk for impaired executive function. Independent of methamphetamine exposure, children with more responsive home environments to developmental and emotional needs demonstrated lower risks for internalizing and externalizing behavior.

Methamphetamine dependence is a common comorbid condition among people living with HIV, and may exacerbate HIV-associated neurocognitive disorders. Animal models of neuroAIDS suggest that the gp120 protein may also cause cognitive impairment. The present work evaluated the separate and combined effects of HIV/gp120 and methamphetamine on learning and executive functions in both humans and transgenic mice. Human participants were grouped by HIV serostatus (HIV+ or HIV-) and lifetime methamphetamine dependence (METH+ or METH-). A neurocognitive test battery included domain-specific assessments of learning and executive functions. Mice (gp120+ and gp120-) were exposed to either a methamphetamine binge (METH+) or saline (METH-), then tested in the attentional-set-shifting task to assess learning and executive functions. In humans, HIV status was associated with significant impairments in learning, but less so for executive functions. The frequency of learning impairments varied between groups, with the greatest impairment observed in the HIV+/METH+ group. In mice, gp120 expression was associated with impairments in learning but not reversal learning (executive component). The greatest proportion of mice that failed to complete the task was observed in the gp120+/METH+ group, suggesting greater learning impairments. Our cross-species study demonstrated that HIV in humans and gp120 in mice impaired learning, and that a history of methamphetamine exposure increased the susceptibility to HIV-associated neurocognitive deficits in both species. Finally, the similar pattern of results in both species suggest that the gp120 protein may contribute to HIV-associated learning deficits in humans.

BackgroundExercise influences drug craving and consumption in humans and drug self-administration in laboratory animals, but the effects can be variable. Improved understanding of how exercise affects drug intake or craving would enhance applications of exercise programs to human drug users attempting cessation.MethodsRats were trained in the intravenous self-administration (IVSA) of D-methamphetamine (METH; 0.05 mg/kg/inf), 3,4-methylenedioxymethamphetamine (MDMA; 0.5 mg/kg/inf) or methylone (0.5 mg/kg/inf). Once IVSA was established, the effect of ∼ 22 h of wheel access in the home cage on subsequent drug taking was assessed in a two cohort crossover design.ResultsProvision of home cage wheel access during the day prior to IVSA sessions significantly decreased the self-administration of METH, MDMA and methylone. At the individual level, there was no correlation between the amount a rat used the wheel and the size of the individual's decrease in drug intake.ConclusionsWheel access can reduce self-administration of a variety of psychomotor stimulants. It does so immediately, i.e., without a need for weeks of exercise prior to drug access. This study therefore indicates that future mechanistic investigations should focus on acute effects of exercise. In sum, the results predict that exercise programs can be used to decrease stimulant drug use in individuals even with no exercise history and an established drug taking pattern.

RationaleIndividuals with fetal alcohol spectrum disorder (FASD) are at increased risk for substance use disorders (SUD). In typically developing individuals, susceptibility to SUD is associated with alterations in dopamine and hypothalamic-pituitary-adrenal (HPA) systems, and their interactions. Prenatal alcohol exposure (PAE) alters dopamine and HPA systems, yet effects of PAE on dopamine-HPA interactions are unknown. Amphetamine-stress cross-sensitization paradigms were utilized to investigate sensitivity of dopamine and stress (HPA) systems, and their interactions following PAE.MethodsAdult Sprague-Dawley offspring from PAE, pair-fed, and ad libitum-fed control groups were assigned to amphetamine-(1-2 mg/kg) or saline-treated conditions, with injections every other day for 15 days. Fourteen days later, all animals received an amphetamine challenge (1 mg/kg) and 5 days later, hormones were measured under basal or acute stress conditions. Amphetamine sensitization (augmented locomotion, days 1-29) and cross-sensitization with acute restraint stress (increased stress hormones, day 34) were assessed.ResultsPAE rats exhibited a lower threshold for amphetamine sensitization compared to controls, suggesting enhanced sensitivity of dopaminergic systems to stimulant-induced changes. Cross-sensitization between amphetamine (dopamine) and stress (HPA hormone) systems was evident in PAE, but not in control rats. PAE males exhibited increased dopamine receptor expression (medial prefrontal cortex (mPFC)) compared to controls.ConclusionsPAE alters induction and expression of sensitization/cross-sensitization, as reflected in locomotor, neural, and endocrine changes, in a manner consistent with increased sensitivity of dopamine and stress systems. These results provide insight into possible mechanisms that could underlie increased prevalence of SUD, as well as the impact of widely prescribed stimulant medications among adolescents with FASD.

Male rats will intravenously self-administer (IVSA) the substituted cathinone stimulants ("bath salts") mephedrone (4-methylmethcathione) and methylone (3,4-methylenedioxymethcathinone) robustly, whereas the IVSA of 3,4-methylenedioxymethamphetamine (MDMA) is inconsistent in many rat models. There are no data available on the self-administration of these drugs in female rats, thus a study was undertaken to contrast them directly. Groups of female Wistar rats were trained to self-administer mephedrone, methylone or MDMA (0.5 mg/kg/inf) under a Fixed-Ratio (FR) 1 schedule of reinforcement for 14 sessions. Following the acquisition interval, animals were evaluated in FR (0.0, 0.125, 0.25, 0.5, 1.0, 2.5 mg/kg/inf) and PR (0.125, 1.0 mg/kg/inf) dose-substitution procedures. The results show that female rats acquired the self-administration of all three compounds with intakes in mephedrone-trained rats that were significantly higher than that of methylone-trained or MDMA-trained rats. In dose-substitution under either FR or PR contingencies, however, the potencies of all three drugs were similar within the original training groups. The mephedrone-trained animals exhibited higher intakes of all drugs during dose-substitution, indicating lasting consequences of the training drug. Abuse liability of these three compounds is therefore predicted to be similar in established stimulant users but may differ in liability if they are primary drugs of initiation.

Alterations in reward valuation are thought to have a central role at all stages of the addiction process. We previously reported work aversion in an effortful T-maze task following a binge exposure to methamphetamine, and no such changes in effort following escalating doses. Limitations of the T-maze task include its two available options, with an effort requirement, in the form of increasing barrier height, varying incrementally as a function of time, and reward magnitudes held constant. Reward preferences and choices, however, are likely affected by the number of options available and the manner in which alternatives are presented. In the present experiment, we investigated the long-lasting, off-drug effects of methamphetamine on reward choices in a novel effortful maze task with three possible courses of action, each associated with different effort requirements and reward magnitudes. Neuroinflammatory responses associated with drug exposure, proposed as one of the mechanisms contributing to suboptimal choices on effort-based tasks, were also examined. We investigated region-specific changes in pro- and anti-inflammatory markers in the mesocorticolimbic pathway after methamphetamine, and their relationship with animals' reward choices. We observed long-lasting, increased sensitivity to differences in reward magnitude in the methamphetamine group: animals were more likely to overcome greater effort costs to obtain larger rewards on our novel effortful maze task. These behavioral changes were strongly predicted by pronounced decreases in frontocortical cytokines, but not amygdalar or striatal markers. The present results provide the first evidence that neuroinflammatory processes are associated with alterations in reward valuation during protracted drug withdrawal.

Most animal studies using methylphenidate (MP) do not administer it the same way it is administered clinically (orally), but rather by injection, resulting in an altered pharmacokinetic profile (quicker and higher peak concentrations). We evaluated several oral-dosing regimens in rats, including dual-dose drinking, to mimic clinical drug delivery. Using an 8-hour-limited-access-drinking-paradigm, MP solutions were delivered at different doses (20, 30, or 60mg/kg/day; as well as dual-dosages of 4 and 10mg/kg/day, 20 and 30mg/kg/day, or 30 and 60mg/kg/day, in which the low dose was administered in the first hour of drinking followed by 7 h of drinking the high dose). Plasma was assayed for MP levels at many time points. Results showed that an 8-hour limited drinking of a dual-dosage 30/60mg/kg MP solution achieved a pharmacokinetic profile similar to clinically administered doses of MP at the high end of the spectrum (peaking at ~30ng/mL), while the 4/10mg/kg MP dual-dosage produced plasma levels in the range produced by typically prescribed clinical doses of MP (peaking at ~8ng/mL). Treatment with the higher dual-dosage (HD: 30/60mg/kg) resulted in hyperactivity, while the lower (LD: 4/10mg/kg) had no effect. Chronic effects of these dual-dosages were assessed throughout three months of treatment and one month of abstinence, beginning in adolescence. MP dose-dependently decreased body weight, which remained attenuated throughout abstinence. MP decreased food intake during early treatment, suggesting that MP may be an appetite suppressant and may also speed metabolism and/or suppress growth. Chronic HD MP resulted in hyperactivity limited during the dark cycle, decreased exploratory behavior, and increased anxiolytic behavior. Findings suggest that these dual-dosage-drinking-paradigms can be used to examine the effects of clinically relevant pharmacokinetic doses of MP and that chronic treatment with such dosages can result in long-lasting developmental and behavioral changes.

Given that the κ opioid receptor (KOR) system has been implicated in psychostimulant abuse, we evaluated whether the selective KOR antagonist norbinaltorphimine dihydrochloride (nor-BNI) would attenuate the escalation of methamphetamine (METH) intake in an extended-access self-administration model. Systemic nor-BNI decreased the escalation of intake of long-access (LgA) but not short-access (ShA) self-administration. nor-BNI also decreased elevated progressive-ratio (PR) breakpoints in rats in the LgA condition and continued to decrease intake after 17 d of abstinence, demonstrating that the effects of a nor-BNI injection are long lasting. Rats with an ShA history showed an increase in prodynorphin immunoreactivity in both the nucleus accumbens (NAc) core and shell, but LgA animals showed a selective increase in the NAc shell. Other cohorts of rats received nor-BNI directly into the NAc shell or core and entered into ShA or LgA. nor-BNI infusion in the NAc shell, but not NAc core, attenuated escalation of intake and PR responding for METH in LgA rats. These data indicate that the development and/or expression of compulsive-like responding for METH under LgA conditions depends on activation of the KOR system in the NAc shell and suggest that the dynorphin-KOR system is a central component of the neuroplasticity associated with negative reinforcement systems that drive the dark side of addiction.

Methamphetamine (METH) abuse is frequent in individuals infected with human immunodeficiency virus type-1 (HIV-1) and is suspected to aggravate HIV-associated neurocognitive disorders (HAND). METH is a psychostimulant that compromises several neurotransmitter systems and HIV proteins trigger neuronal injury but the combined effects of viral infection and METH abuse are incompletely understood. In this study we treated transgenic mice expressing the HIV envelope protein gp120 in the brain (HIV-1 gp120tg) at 3-4 months of age with an escalating-dose, multiple-binge METH regimen. The long-term effects were analyzed after 6-7 months of drug abstinence employing behavioral tests and analysis of neuropathology, electrophysiology and gene expression. Behavioral testing showed that both HIV-1 gp120tg and WT animals treated with METH displayed impaired learning and memory. Neuropathological analysis revealed that METH similar to HIV-1 gp120 caused a significant loss of neuronal dendrites and pre-synaptic terminals in hippocampus and cerebral cortex of WT animals. Electrophysiological studies in hippocampal slices showed that METH exposed HIV-1 gp120tg animals displayed reduced post-tetanic potentiation, whereas both gp120 expression and METH lead to reduced long-term potentiation. A quantitative reverse transcription-polymerase chain reaction array showed that gp120 expression, METH and their combination each caused a significant dysregulation of specific components of GABAergic and glutamatergic neurotransmission systems, providing a possible mechanism for synaptic dysfunction and behavioral impairment. In conclusion, both HIV-1 gp120 and METH caused lasting behavioral impairment in association with neuropathology and altered gene expression. However, combined METH exposure and HIV-1 gp120 expression resulted in the most pronounced, long lasting pre- and post-synaptic alterations coinciding with impaired learning and memory.

Methamphetamine abuse is common among individuals infected by human immunodeficiency virus (HIV). Neurocognitive outcomes tend to be worse in methamphetamine users with HIV. However, it is unclear whether discrete cognitive domains are susceptible to impairment after combined HIV infection and methamphetamine abuse. The expression of HIV/gp120 protein induces neuropathology in mice similar to HIV-induced pathology in humans. We investigated the separate and combined effects of methamphetamine exposure and gp120 expression on cognitive function in transgenic (gp120-tg) and control mice. The mice underwent an escalating methamphetamine binge regimen and were tested in novel object/location recognition, object-in-place recognition, and Barnes maze tests. gp120 expression disrupted performance in the object-in-place test (i.e. similar time spent with all objects, regardless of location), indicating deficits in associative recognition memory. gp120 expression also altered reversal learning in the Barnes maze, suggesting impairments in executive function. Methamphetamine exposure impaired spatial strategy in the Barnes maze, indicating deficits in spatial learning. Methamphetamine-exposed gp120-tg mice had the lowest spatial strategy scores in the final acquisition trials in the Barnes maze, suggesting greater deficits in spatial learning than all of the other groups. Although HIV infection involves interactions between multiple proteins and processes, in addition to gp120, our findings in gp120-tg mice suggest that humans with the dual insult of HIV infection and methamphetamine abuse may exhibit a broader spectrum of cognitive deficits than those with either factor alone. Depending on the cognitive domain, the combination of both insults may exacerbate deficits in cognitive performance compared with each individual insult.

Methamphetamine is a highly addictive psychostimulant that causes profound damage to the brain and other body organs. Post mortem studies of human tissues have linked the use of this drug to diseases associated with aging, such as coronary atherosclerosis and pulmonary fibrosis, but the molecular mechanism underlying these findings remains unknown. Here we used functional lipidomics and transcriptomics experiments to study abnormalities in lipid metabolism in select regions of the brain and, to a greater extent, peripheral organs and tissues of rats that self-administered methamphetamine. Experiments in various cellular models (primary mouse fibroblasts and myotubes) allowed us to investigate the molecular mechanisms of systemic inflammation and cellular aging related to methamphetamine abuse. We report now that methamphetamine accelerates cellular senescence and activates transcription of genes involved in cell-cycle control and inflammation by stimulating production of the sphingolipid messenger ceramide. This pathogenic cascade is triggered by reactive oxygen species, likely generated through methamphetamine metabolism via cytochrome P450, and involves the recruitment of nuclear factor-κB (NF-κB) to induce expression of enzymes in the de novo pathway of ceramide biosynthesis. Inhibitors of NF-κB signaling and ceramide formation prevent methamphetamine-induced senescence and systemic inflammation in rats self-administering the drug, attenuating their health deterioration. The results suggest new therapeutic strategies to reduce the adverse consequences of methamphetamine abuse and improve effectiveness of abstinence treatments.

Circulating triglycerides (TGs) normally increase after a meal but are altered in pathophysiological conditions, such as obesity. Although TG metabolism in the brain remains poorly understood, several brain structures express enzymes that process TG-enriched particles, including mesolimbic structures. For this reason, and because consumption of high-fat diet alters dopamine signaling, we tested the hypothesis that TG might directly target mesolimbic reward circuits to control reward-seeking behaviors. We found that the delivery of small amounts of TG to the brain through the carotid artery rapidly reduced both spontaneous and amphetamine-induced locomotion, abolished preference for palatable food and reduced the motivation to engage in food-seeking behavior. Conversely, targeted disruption of the TG-hydrolyzing enzyme lipoprotein lipase specifically in the nucleus accumbens increased palatable food preference and food-seeking behavior. Finally, prolonged TG perfusion resulted in a return to normal palatable food preference despite continued locomotor suppression, suggesting that adaptive mechanisms occur. These findings reveal new mechanisms by which dietary fat may alter mesolimbic circuit function and reward seeking.

Learning from mistakes and prospectively adjusting behavior in response to reward feedback is an important facet of performance monitoring. Dopamine (DA) pathways play an important role in feedback learning and a growing literature has also emerged on the importance of serotonin (5HT) in reward learning, particularly during punishment or reward omission (negative feedback). Cognitive impairments resulting from psychostimulant exposure may arise from altered patterns in feedback learning, which in turn may be modulated by DA and 5HT transmission. We analyzed long-term, off-drug changes in learning from positive and negative feedback and associated striatal DA transporter (DAT) and frontocortical 5HT transporter (SERT) binding in rats pretreated with methamphetamine (mAMPH). Specifically, we assessed the reversal phase of pairwise visual discrimination learning in rats receiving single dose- (mAMPHsingle) vs. escalating-dose exposure (mAMPHescal). Using fine-grained trial-by-trial analyses, we found increased sensitivity to and reliance on positive feedback in mAMPH-pretreated animals, with the mAMPHsingle group showing more pronounced use of this type of feedback. In contrast, overall negative feedback sensitivity was not altered following any mAMPH treatment. In addition to validating the enduring effects of mAMPH on early reversal learning, we found more consecutive error commissions before the first correct response in mAMPH-pretreated rats. This behavioral rigidity was negatively correlated with subregional frontocortical SERT whereas positive feedback sensitivity negatively correlated with striatal DAT binding. These results provide new evidence for the overlapping, yet dissociable roles of DA and 5HT systems in overcoming perseveration and in learning new reward rules.

AMPA-type glutamate receptors mediate fast, excitatory neurotransmission in the brain, and their concentrations at synapses are important determinants of synaptic strength. We investigated the post-transcriptional regulation of GluA2, the calcium-impermeable AMPA receptor subunit, by examining the subcellular distribution of its mRNA and evaluating its translational regulation by microRNA in cultured mouse hippocampal neurons. Using computational approaches, we identified a conserved microRNA-124 (miR-124) binding site in the 3'UTR of GluA2 and demonstrated that miR-124 regulated the translation of GluA2 mRNA reporters in a sequence-specific manner in luciferase assays. While we hypothesized that this regulation might occur in dendrites, our biochemical and fluorescent in situ hybridization (FISH) data indicate that GluA2 mRNA does not localize to dendrites or synapses of mouse hippocampal neurons. In contrast, we detected significant concentrations of miR-124 in dendrites. Overexpression of miR-124 in dissociated neurons results in a 30% knockdown of GluA2 protein, as measured by immunoblot and quantitative immunocytochemistry, without producing any changes in GluA2 mRNA concentrations. While total GluA2 concentrations are reduced, we did not detect any changes in the concentration of synaptic GluA2. We conclude from these results that miR-124 interacts with GluA2 mRNA in the cell body to downregulate translation. Our data support a model in which GluA2 is translated in the cell body and subsequently transported to neuronal dendrites and synapses, and suggest that synaptic GluA2 concentrations are modified primarily by regulated protein trafficking rather than by regulated local translation.

Chronic increases in myofilament Ca(2+)-sensitivity in the heart are known to alter gene expression potentially modifying Ca(2+)-homeostasis and inducing arrhythmias. We tested age-dependent effects of a chronic increase in myofilament Ca(2+)-sensitivity on induction of altered alter gene expression and activity of Ca(2+) transport systems in cardiac myocytes. Our approach was to determine the relative contributions of the major mechanisms responsible for restoring Ca(2+) to basal levels in field stimulated ventricular myocytes. Comparisons were made from ventricular myocytes isolated from non-transgenic (NTG) controls and transgenic mice expressing the fetal, slow skeletal troponin I (TG-ssTnI) in place of cardiac TnI (cTnI). Replacement of cTnI by ssTnI induces an increase in myofilament Ca(2+)-sensitivity. Comparisons included myocytes from relatively young (5-7months) and older mice (11-13months). Employing application of caffeine in normal Tyrode and in 0Na(+) 0Ca(2+) solution, we were able to dissect the contribution of the sarcoplasmic reticulum Ca(2+) pump (SR Ca(2+)-ATPase), the Na(+)/Ca(2+) exchanger (NCX), and "slow mechanisms" representing the activity of the sarcolemmal Ca(2+) pump and the mitochondrial Ca(2+) uniporter. The relative contribution of the SR Ca(2+)-ATPase to restoration of basal Ca(2+) levels in younger TG-ssTnI myocytes was lower than in NTG (81.12±2.8% vs 92.70±1.02%), but the same in the older myocytes. Younger and older NTG myocytes demonstrated similar contributions from the SR Ca(2+)-ATPase and NCX to restoration of basal Ca(2+). However, the slow mechanisms for Ca(2+) removal were increased in the older NTG (3.4±0.3%) vs the younger NTG myocytes (1.4±0.1%). Compared to NTG, younger TG-ssTnI myocytes demonstrated a significantly bigger contribution of the NCX (16±2.7% in TG vs 6.9±0.9% in NTG) and slow mechanisms (3.3±0.4% in TG vs 1.4±0.1% in NTG). In older TG-ssTnI myocytes the contributions were not significantly different from NTG (NCX: 4.9±0.6% in TG vs 5.5±0.7% in NTG; slow mechanisms: 2.5±0.3% in TG vs 3.4±0.3% in NTG). Our data indicate that constitutive increases in myofilament Ca(2+)-sensitivity alter the relative significance of the NCX transport system involved in Ca(2+)-homeostasis only in a younger group of mice. This modification may be of significance in early changes in altered gene expression and electrical stability hearts with increased myofilament Ca-sensitivity.

Psychostimulants containing methylphenidate (MPH) are increasingly being used both on and off-label to enhance learning and memory. Still, almost no studies have investigated MPH's ability to specifically improve spatial or long-term memory. Here we examined the effect of training with 1 or 10mg/kg MPH on hidden platform learning in the Morris water maze. 10mg/kg MPH improved memory acquisition and retention, while 1mg/kg MPH improved memory retention. Taken together with prior evidence that low, clinically relevant, doses of MPH (0.01-1mg/kg MPH) enhance fear memory we conclude that MPH broadly enhances memory.

HIV infection is frequently comorbid with methamphetamine (METH) dependence. Both factors are associated with impairment in inhibitory function that continues even after abstinence from the drug. Deficits in prepulse inhibition (PPI), a measure of sensorimotor gating, are induced by acute stimulant administration, but the combined effect of HIV and chronic METH exposure on PPI is not well characterized. We quantified baseline acoustic startle and PPI in mice expressing the HIV-1 gp120 envelope protein (gp120tg) and in wild-type (WT) littermates; thereafter, we administered a chronic regimen of METH or vehicle and tested startle and PPI after 7 days of drug withdrawal. We hypothesized that METH-treated gp120tg mice would exhibit PPI deficits compared with vehicle-treated WT or gp120tg animals. Before METH administration, drug-naive female gp120tg mice exhibited decreased PPI compared with female WT mice, whereas male gp120tg mice exhibited increased startle compared with other groups. After drug withdrawal, no consistent genotype effect was observed, but METH-treated mice exhibited increased PPI compared with vehicle, in contrast to previous reports of acute METH-induced PPI deficits. In summary, PPI impairment in HIV could depend on factors such as sex, whereas changes in PPI following METH withdrawal may depend on the quantity and duration of drug exposure.

Methylphenidate (MPH), introduced more than 60 years ago, accounts for two-thirds of current prescriptions for attention deficit hyperactivity disorder (ADHD). Although many studies have modeled MPH's effect on executive function, almost none have directly modeled its effect on long-term memory (LTM), even though improvement in LTM is a critical target of therapeutic intervention in ADHD. We examined the effects of a wide range of doses of MPH (0.01-10 mg/kg, i.p.) on Pavlovian fear learning, a leading model of memory. MPH's effects were then compared to those of atomoxetine (0.1-10 mg/kg, i.p.), bupropion (0.5-20 mg/kg, i.p.), and citalopram (0.01-10 mg/kg, i.p.). At low, clinically relevant doses, MPH enhanced fear memory; at high doses it impaired memory. MPH's memory-enhancing effects were not confounded by its effects on locomotion or anxiety. Further, MPH-induced memory enhancement seemed to require both dopamine and norepinephrine transporter inhibition. Finally, the addictive potential of MPH (1 mg/kg and 10 mg/kg) was compared to those of two other psychostimulants, amphetamine (0.005 mg/kg and 1.5 mg/kg) and cocaine (0.15 mg/kg and 15 mg/kg), using a conditioned place preference and behavioral sensitization paradigm. We found that memory-enhancing effects of psychostimulants observed at low doses are readily dissociable from their reinforcing and locomotor activating effects at high doses. Together, our data suggest that fear conditioning will be an especially fruitful platform for modeling the effects of psychostimulants on LTM in drug development.

Psychostimulants such as cocaine have been used as performance enhancers throughout recorded history. Although psychostimulants are commonly prescribed to improve attention and cognition, a great deal of literature has described their ability to induce cognitive deficits, as well as addiction. How can a single drug class be known to produce both cognitive enhancement and impairment? Properties of the particular stimulant drug itself and individual differences between users have both been suggested to dictate the outcome of stimulant use. A more parsimonious alternative, which we endorse, is that dose is the critical determining factor in cognitive effects of stimulant drugs. Herein, we review several popular stimulants (cocaine, amphetamine, methylphenidate, modafinil, and caffeine), outlining their history of use, mechanism of action, and use and abuse today. One common graphic depiction of the cognitive effects of psychostimulants is an inverted U-shaped dose-effect curve. Moderate arousal is beneficial to cognition, whereas too much activation leads to cognitive impairment. In parallel to this schematic, we propose a continuum of psychostimulant activation that covers the transition from one drug effect to another as stimulant intake is increased. Low doses of stimulants effect increased arousal, attention, and cognitive enhancement; moderate doses can lead to feelings of euphoria and power, as well as addiction and cognitive impairment; and very high doses lead to psychosis and circulatory collapse. This continuum helps account for the seemingly disparate effects of stimulant drugs, with the same drug being associated with cognitive enhancement and impairment.

Bath salts are designer drugs with stimulant properties that are a growing medical and psychiatric concern due to their widespread availability and use. Although the chemical compounds in the mixtures referred to as "bath salts" vary, many are derivatives of cathinone, a monoamine alkaloid. Cathinones have an affinity for dopamine, serotonin, and norepinephrine synapses in the brain. Because of the strong selection for these neurotransmitters, these drugs induce stimulating effects similar to those of methamphetamines, cocaine, and 3,4-methylenedioxy-N-methylamphetamine (MDMA). Much of the emerging information about bath salts is from emergency department evaluation and treatment of severe medical and neuropsychiatric adverse outcomes. This review consists of a compilation of case reports and describes the emergent literature that illustrates the chemical composition of bath salts, patterns of use, administration methods, medical and neuropsychiatric effects, and treatments of patients with bath salt toxicity.

Recently, the notion that memory and addiction share similar neural substrates has become widely accepted. N-methyl-d-aspartate receptors (NMDAR) are the cornerstones of synaptic models of memory. The present study examined the effect of the competitive NMDAR antagonist CPP on the induction of behavioral sensitization and conditioned place preference to cocaine. Conditioned place preference is an associative memory model of drug seeking, while sensitization is a non-associative model of the transition from casual to compulsive use. There were three principal findings: (1) co-administration of CPP and cocaine altered the acute response to cocaine, suggesting a direct interaction between the two drugs; (2) NMDAR antagonism had no effect on behavioral sensitization; and (3) NMDAR antagonism abolished conditioned place preference. A review of prior evidence supporting a role for NMDARs in sensitization suggests that NMDAR antagonists directly interfere with cocaine's psychostimulant effects, and this interaction could be misinterpreted as a disruption of sensitization. Finally, we suggest that addiction recruits multiple kinds of plasticity, with sensitization recruiting NMDAR-independent mechanisms.