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.

Neuropharmacological Evidence Implicating Drug-Induced Glutamate Receptor Dysfunction in Affective and Cognitive Sequelae of Subchronic Methamphetamine Self-Administration in Mice.

Methamphetamine (MA) is a highly addictive drug, and MA use disorder is often comorbid with anxiety and cognitive impairment. These comorbid conditions are theorized to reflect glutamate-related neurotoxicity within the frontal cortical regions. However, our prior studies of MA-sensitized mice indicate that subchronic, behaviorally non-contingent MA treatment is sufficient to dysregulate glutamate transmission in mouse brain. Here, we extend this prior work to a mouse model of high-dose oral MA self-administration (0.8, 1.6, or 3.2 g/L; 1 h sessions × 7 days) and show that while female C57BL/6J mice consumed more MA than males, MA-experienced mice of both sexes exhibited some signs of anxiety-like behavior in a behavioral test battery, although not all effects were concentration-dependent. No MA effects were detected for our measures of visually cued spatial navigation, spatial learning, or memory in the Morris water maze; however, females with a history of 3.2 g/L MA exhibited reversal-learning deficits in this task, and mice with a history of 1.6 g/L MA committed more working-memory incorrect errors and relied upon a non-spatial navigation strategy during the radial-arm maze testing. Relative to naïve controls, MA-experienced mice exhibited several changes in the expression of certain glutamate receptor-related proteins and their downstream effectors within the ventral and dorsal areas of the prefrontal cortex, the hippocampus, and the amygdala, many of which were sex-selective. Systemic pretreatment with the mGlu1-negative allosteric modulator JNJ 162596858 reversed the anxiety-like behavior expressed by MA-experienced mice in the marble-burying test, while systemic pretreatment with NMDA or the NMDA antagonist MK-801 bi-directionally affected the MA-induced reversal-learning deficit. Taken together, these data indicate that a relatively brief history of oral MA is sufficient to induce some signs of anxiety-like behavior and cognitive dysfunction during early withdrawal that reflect, at least in part, MA-induced changes in the corticolimbic expression of certain glutamate receptor subtypes of potential relevance to treating symptoms of MA use disorder.

A subchronic history of binge-drinking elicits mild, age- and sex-selective, affective, and cognitive anomalies in C57BL/6J mice.

Introduction: Alcohol abuse is a risk factor for affective and cognitive disorders, with evidence indicating that adolescent-onset excessive drinking can result in long-term deficiencies in emotional regulation and cognition, with females more susceptible to the negative emotional and cognitive consequences of excessive alcohol consumption. However, our prior examination of the interactions between sex and the age of drinking-onset indicated minimal signs of anxiety-like behavior during alcohol withdrawal, which may have related to the concurrent anxiety testing of male and female subjects. Methods: The present study addressed this potential confound by assaying for alcohol withdrawal-induced negative affect separately in males and females and expanded our investigation to include measures of spatial and working memory. Results: Following 14 days of drinking under modified Drinking-in-the-Dark procedures (10, 20, and 40% alcohol v/v; 2 h/day), adolescent and adult binge-drinking mice of both sexes exhibited, respectively, fewer and more signs of negative affect in the light-dark shuttle-box and forced swim tests than their water-drinking counterparts. Adolescent-onset binge-drinking mice also exhibited signs of impaired working memory early during radial arm maze training during early alcohol withdrawal. When tested in late (30 days) withdrawal, only adult female binge-drinking mice buried more marbles than their water-drinking counterparts. However, adolescent-onset binge-drinking mice exhibited poorer spatial memory recall in a Morris water maze. Discussion: These findings indicate that a subchronic (14-day) binge-drinking history induces mild, age- and sex-selective, changes in negative affect and cognition of potential relevance to understanding individual variability in the etiology and treatment of alcohol abuse and alcohol use disorder.

Alcohol-drinking during later life by C57BL/6J mice induces sex- and age-dependent changes in hippocampal and prefrontal cortex expression of glutamate receptors and neuropathology markers.

Heavy drinking can induce early-onset dementia and increase the likelihood of the progression and severity of Alzheimer's Disease and related dementias (ADRD). Recently, we showed that alcohol-drinking by mature adult C57BL/6J mice induces more signs of cognitive impairment in females versus males without worsening age-related cognitive decline in aged mice. Here, we immunoblotted for glutamate receptors and protein markers of ADRD-related neuropathology within the hippocampus and prefrontal cortex (PFC) of these mice after three weeks of alcohol withdrawal to determine protein correlates of alcohol-induced cognitive decline. Irrespective of alcohol history, age-related changes in protein expression included a male-specific decline in hippocampal glutamate receptors and an increase in the expression of a beta-site amyloid precursor protein cleaving enzyme (BACE) isoform in the PFC as well as a sex-independent increase in hippocampal amyloid precursor protein. Alcohol-drinking was associated with altered expression of glutamate receptors in the hippocampus in a sex-dependent manner, while all glutamate receptor proteins exhibited significant alcohol-related increases in the PFC of both sexes. Expression of BACE isoforms and phosphorylated tau varied in the PFC and hippocampus based on age, sex, and drinking history. The results of this study indicate that withdrawal from a history of alcohol-drinking during later life induces sex- and age-selective effects on glutamate receptor expression and protein markers of ADRD-related neuropathology within the hippocampus and PFC of potential relevance to the etiology, treatment and prevention of alcohol-induced dementia and Alzheimer's Disease.

The effect of MK-801 on stress-ethanol cross-sensitization is dissociable from its effects on nNOS activity.

Locomotor behavioral sensitization represents an animal model for understanding neuroadaptive processes related to repeated drug exposure. Repeated stress can elicit a cross-sensitization to the stimulant response of ethanol, which involves neuronal nitric oxide synthase (nNOS). Activation of N-methyl d-aspartate (NMDA) glutamate receptors triggers nNOS and the synthesis of nitric oxide (NO). In this study, we investigated the effects of blocking NMDA receptors using the NMDA receptor antagonist MK-801 on the cross-sensitization between restraint stress and ethanol. We also evaluated the nNOS activity in the prefrontal cortex (PFC) and hippocampus. Mice were pretreated with saline or MK-801 30 min before an injection of saline or stress exposure for 14 days. On the following day, they were challenged with either saline or 1.8 g/kg ethanol. Swiss male mice pretreated with 0.25 mg/kg MK-801 exhibited a sensitized response to ethanol. Moreover, MK-801 potentiated the cross-sensitization between stress and ethanol. However, MK-801 prevented the enhanced nNOS activity in stress-exposed groups (challenged with saline or ethanol) in the PFC; the antagonist also prevented the ethanol-induced increase in nNOS activity and reduced this enzyme activity in mice exposed to stress in the hippocampus. These data indicate that systemic treatment with the NMDA antagonist potentiated, rather than blocked, ethanol-induced behavioral sensitization and that this effect is dissociable from the capacity of NMDA antagonists to reduce ethanol/stress-induced NOS stimulation in the PFC and hippocampus.

Evidence for Phosphorylation-Dependent, Dynamic, Regulation of mGlu5 and Homer2 in Expression of Cocaine Aversion in Mice.

Cocaine-induced changes in the expression of the glutamate-related scaffolding protein Homer2 influence this drug's psychostimulant and rewarding properties. In response to neuronal activity, Homer2 is phosphorylated on S117/S216 by calcium-calmodulin kinase IIα (CaMKIIα), which induces a rapid dissociation of mGlu5-Homer2 scaffolds. Herein, we examined the requirement for Homer2 phosphorylation in cocaine-induced changes in mGlu5-Homer2 coupling, to include behavioral sensitivity to cocaine. For this, mice with alanine point mutations at (S117/216)-Homer2 (Homer2AA/AA ) were generated, and we determined their affective, cognitive and sensorimotor phenotypes, as well as cocaine-induced changes in conditioned reward and motor hyperactivity. The Homer2AA/AA mutation prevented activity-dependent phosphorylation of S216 Homer2 in cortical neurons, but Homer2AA/AA mice did not differ from wild-type (WT) controls with respect to Morris maze performance, acoustic startle, spontaneous or cocaine-induced locomotion. Homer2AA/AA mice exhibited signs of hypoanxiety similar to the phenotype of transgenic mice with a deficit in signal-regulated mGluR5 phosphorylation (Grm5AA/AA ). However, opposite of Grm5AA/AA mice, Homer2AA/AA mice were less sensitive to the aversive properties of high-dose cocaine under both place-conditioning and taste-conditioning procedures. Acute injection with cocaine caused dissociation of mGluR5 and Homer2 in striatal lysates from WT, but not Homer2AA/AA mice, suggesting a molecular basis for the deficit in cocaine aversion. These findings indicate that CaMKIIα-dependent phosphorylation of Homer2 gates the negative motivational valence of high-dose cocaine via regulation of mGlu5 binding, furthering an important role for dynamic changes in mGlu5-Homer interactions in addiction vulnerability.

Alcohol-Drinking Under Limited-Access Procedures During Mature Adulthood Accelerates the Onset of Cognitive Impairment in Mice.

A history of heavy drinking increases vulnerability to, and the severity of, Alzheimer's disease (AD) and related dementias, with alcohol use disorder identified as the strongest modifiable risk factor for early-onset dementia. Heavy drinking has increased markedly in women over the past 10 years, particularly in mature adult women during the coronavirus (COVID-19) pandemic. This is concerning as women are more sensitive to many alcohol-related disease states, including AD and related dementias. Herein, we conducted two studies to determine if a 1-month period of binge drinking during mature adulthood (i.e., 5-9 months of age) impairs spatial and working memory to a greater extent in female vs. male C57BL/6J (B6J) mice. The anxiogenic and cognitive-impairing effects of binge drinking were also compared between mature adult and old B6J mice (18 months of age) in a third study. Throughout, females consumed more alcohol than males, indicating that a sex difference in binge drinking persists into old age. Despite the sex difference in intake, we detected no consistent sex difference in our measures of alcohol withdrawal-induced anxiety during a behavioral test battery. Although mature adult females exhibited more cognitive deficits than males, the precise outcome exhibiting a female-selective effect varied across studies. Old mice drank lower amounts of alcohol than mature adult mice, yet their blood ethanol concentrations (BECs) were within error of the 80 mg/dl criterion for binge drinking, indicative of an age-related slowing of alcohol metabolism. As expected, 18-month-old controls exhibited more signs of cognitive impairment than their 6-month-old counterparts, and binge drinking history impaired the Morris water maze performance of mice of both ages. In contrast, binge drinking history impaired the radial arm maze performance of 6-month-old mice only, and the extent of the impairment was comparable to the behavior exhibited by the older mice. We conclude from our studies that: (1) both biological sex and the age of drinking onset are subject factors that impact voluntary alcohol consumption by mice into old age; (2) binge drinking during later life elicits a negative affective state that is relatively sex-independent; (3) binge drinking during both mature adulthood and old age impairs spatial learning and memory; (4) binge drinking during mature adulthood accelerates deficits in working memory; and (5) mature adult females tend to exhibit more alcohol-induced cognitive impairments than males. If relevant to humans, these findings suggest that binge-like drinking by older adult men and women induces a negative affective state and cognitive decline, but that mature adult women, in particular, may be more sensitive to both the immediate and persistent cognitive-impairing effects of heavy drinking.

Selective Inhibition of PDE4B Reduces Methamphetamine Reinforcement in Two C57BL/6 Substrains.

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.

Profiling prefrontal cortex protein expression in rats exhibiting an incubation of cocaine craving following short-access self-administration procedures.

Introduction: Incubation of drug-craving refers to a time-dependent increase in drug cue-elicited craving that occurs during protracted withdrawal. Historically, rat models of incubated cocaine craving employed extended-access (typically 6 h/day) intravenous drug self-administration (IV-SA) procedures, although incubated cocaine craving is reported to occur following shorter-access IV-SA paradigms. The notoriously low-throughput of extended-access IV-SA prompted us to determine whether two different short-access IV-SA procedures akin to those in the literature result in qualitatively similar changes in glutamate receptor expression and the activation of downstream signaling molecules within prefrontal cortex (PFC) subregions as those reported previously by our group under 6h-access conditions. Methods: For this, adult, male Sprague-Dawley rats were trained to intravenously self-administer cocaine for 2 h/day for 10 consecutive days (2-h model) or for 6 h on day 1 and 2 h/day for the remaining 9 days of training (Mixed model). A sham control group was also included that did not self-administer cocaine. Results: On withdrawal day 3 or 30, rats were subjected to a 2-h test of cue-reinforced responding in the absence of cocaine and a time-dependent increase in drug-seeking was observed under both IV-SA procedures. Immunoblotting of brain tissue collected immediately following the cue test session indicated elevated phospho-Akt1, phospho-CaMKII and Homer2a/b expression within the prelimbic subregion of the PFC of cocaine-incubated rats. However, we failed to detect incubation-related changes in Group 1 metabotropic glutamate receptor or ionotropic glutamate receptor subunit expression in either subregion. Discussion: These results highlight further a role for Akt1-related signaling within the prelimbic cortex in driving incubated cocaine craving, and provide novel evidence supporting a potential role also for CaMKII-dependent signaling through glutamate receptors in this behavioral phenomenon.

Selective Inhibition of PDE4B Reduces Binge Drinking in Two C57BL/6 Substrains.

Cyclic AMP (cAMP)-dependent signaling is highly implicated in the pathophysiology of alcohol use disorder (AUD), with evidence supporting the efficacy of inhibiting the cAMP hydrolyzing enzyme phosphodiesterase 4 (PDE4) as a therapeutic strategy for drinking reduction. Off-target emetic effects associated with non-selective PDE4 inhibitors has prompted the development of selective PDE4 isozyme inhibitors for treating neuropsychiatric conditions. Herein, we examined the effect of a selective PDE4B inhibitor A33 (0-1.0 mg/kg) on alcohol drinking in both female and male mice from two genetically distinct C57BL/6 substrains. Under two different binge-drinking procedures, A33 pretreatment reduced alcohol intake in male and female mice of both substrains. In both drinking studies, there was no evidence for carry-over effects the next day; however, we did observe some sign of tolerance to A33's effect on alcohol intake upon repeated, intermittent, treatment (5 injections of 1.0 mg/kg, every other day). Pretreatment with 1.0 mg/kg of A33 augmented sucrose intake by C57BL/6NJ, but not C57BL/6J, mice. In mice with a prior history of A33 pretreatment during alcohol-drinking, A33 (1.0 mg/kg) did not alter spontaneous locomotor activity or basal motor coordination, nor did it alter alcohol's effects on motor activity, coordination or sedation. In a distinct cohort of alcohol-naïve mice, acute pretreatment with 1.0 mg/kg of A33 did not alter motor performance on a rotarod and reduced sensitivity to the acute intoxicating effects of alcohol. These data provide the first evidence that selective PDE4B inhibition is an effective strategy for reducing excessive alcohol intake in murine models of binge drinking, with minimal off-target effects. Despite reducing sensitivity to acute alcohol intoxication, PDE4B inhibition reduces binge alcohol drinking, without influencing behavioral sensitivity to alcohol in alcohol-experienced mice. Furthermore, A33 is equally effective in males and females and exerts a quantitatively similar reduction in alcohol intake in mice with a genetic predisposition for high versus moderate alcohol preference. Such findings further support the safety and potential clinical utility of targeting PDE4 for treating AUD.

Preclinical evidence to support repurposing everolimus for craving reduction during protracted drug withdrawal.

Cue-elicited drug-craving is a cardinal feature of addiction that intensifies (incubates) during protracted withdrawal. In a rat model, these addiction-related behavioral pathologies are mediated, respectively, by time-dependent increases in PI3K/Akt1 signaling and reduced Group 1 metabotropic glutamate receptor (mGlu) expression, within the ventromedial prefrontal cortex (vmPFC). Herein, we examined the capacity of single oral dosing with everolimus, an FDA-approved inhibitor of the PI3K/Akt effector mTOR, to reduce incubated cocaine-craving and reverse incubation-associated changes in vmPFC kinase activity and mGlu expression. Rats were trained to lever-press for intravenous infusions of cocaine or delivery of sucrose pellets and then subjected to tests for cue-reinforced responding during early (3 days) or late (30-46 days) withdrawal. Rats were gavage-infused with everolimus (0-1.0 mg/kg), either prior to testing to examine for effects upon reinforcer-seeking behavior, or immediately following testing to probe effects upon the consolidation of extinction learning. Single oral dosing with everolimus dose-dependently blocked cocaine-seeking during late withdrawal and the effect lasted at least 24 h. No everolimus effects were observed for cue-elicited sucrose-seeking or cocaine-seeking in early withdrawal. In addition, everolimus treatment, following initial cue-testing, reduced subsequent cue hyper-responsivity exhibited observed during late withdrawal, arguing a facilitation of extinction memory consolidation. everolimus' "anti-incubation" effect was associated with a reversal of withdrawal-induced changes in indices of PI3K/Akt1/mTOR activity, as well as Homer protein and mGlu1/5 expression, within the prelimbic (PL) subregion of the prefrontal cortex. Our results indicate mTOR inhibition as a viable strategy for interrupting heightened cocaine-craving and facilitating addiction recovery during protracted withdrawal.

Targeting mGlu5 for Methamphetamine Use Disorder.

Methamphetamine abuse leads to devastating consequences, including addiction, crime, and death. Despite decades of research, no medication has been approved by the U.S. Food and Drug Administration for the treatment of Methamphetamine Use Disorder. Thus, there is a need for new therapeutic approaches. Animal studies demonstrate that methamphetamine exposure dysregulates forebrain function involving the Group-I metabotropic glutamate receptor subtype 5 (mGlu5), which is predominantly localized to postsynaptic sites. Allosteric modulators of mGlu5 offer a unique opportunity to modulate glutamatergic neurotransmission selectively, thereby potentially ameliorating methamphetamine-induced disruptions. Negative allosteric modulators of mGlu5 attenuate the effects of methamphetamine, including rewarding/reinforcing properties of the drug across animal models, and have shown promising effects in clinical trials for Anxiety Disorder and Major Depressive Disorder. Preclinical studies have also sparked great interest in mGlu5 positive allosteric modulators, which exhibit antipsychotic and anxiolytic properties, and facilitate extinction learning when access to methamphetamine is removed, possibly via the amelioration of methamphetamine-induced cognitive deficits. Clinical research is now needed to elucidate the mechanisms underlying the mGlu5 receptor-related effects of methamphetamine and the contributions of these effects to addictive behaviors. The growing array of mGlu5 allosteric modulators provides excellent tools for this purpose and may offer the prospect of developing tailored and effective medications for Methamphetamine Use Disorder.

Intracranial self-stimulation and concomitant behaviors following systemic methamphetamine administration in Hnrnph1 mutant mice.

RATIONALE: Methamphetamine (MA) addiction is a major public health issue in the USA, with a poorly understood genetic component. We previously identified heterogeneous nuclear ribonucleoprotein H1 (Hnrnph1; H1) as a quantitative trait gene underlying sensitivity to MA-induced behavioral sensitivity. Mice heterozygous for a frameshift deletion in the first coding exon of H1 (H1+/-) showed reduced MA phenotypes including oral self-administration, locomotor activity, dopamine release, and dose-dependent differences in MA conditioned place preference. However, the effects of H1+/- on innate and MA-modulated reward sensitivity are not known. OBJECTIVES: We examined innate reward sensitivity and facilitation by MA in H1+/- mice via intracranial self-stimulation (ICSS). METHODS: We used intracranial self-stimulation (ICSS) of the medial forebrain bundle to assess shifts in reward sensitivity following acute, ascending doses of MA (0.5-4.0 mg/kg, i.p.) using a within-subjects design. We also assessed video-recorded behaviors during ICSS testing sessions. RESULTS: H1+/- mice displayed reduced normalized maximum response rates in response to MA. H1+/- females had lower normalized M50 values compared to wild-type females, suggesting enhanced reward facilitation by MA. Finally, regardless of genotype, there was a dose-dependent reduction in distance to the response wheel following MA administration, providing an additional measure of MA-induced reward-driven behavior. CONCLUSIONS: H1+/- mice displayed a complex ICSS phenotype following MA, displaying indications of both blunted reward magnitude (lower normalized maximum response rates) and enhanced reward sensitivity specific to H1+/- females (lower normalized M50 values).

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.

Persistently Elevated mTOR Complex 1-S6 Kinase 1 Disrupts DARPP-32-Dependent D1 Dopamine Receptor Signaling and Behaviors.

BACKGROUND: The serine-threonine kinase mTORC1 (mechanistic target of rapamycin complex 1) is essential for normal cell function but is aberrantly activated in the brain in both genetic-developmental and sporadic diseases and is associated with a spectrum of neuropsychiatric symptoms. The underlying molecular mechanisms of cognitive and neuropsychiatric symptoms remain controversial. METHODS: The present study examines behaviors in transgenic models that express Rheb, the most proximal known activator of mTORC1, and profiles striatal phosphoproteomics in a model with persistently elevated mTORC1 signaling. Biochemistry, immunohistochemistry, electrophysiology, and behavior approaches are used to examine the impact of persistently elevated mTORC1 on D1 dopamine receptor (D1R) signaling. The effect of persistently elevated mTORC1 was confirmed using D1-Cre to elevate mTORC1 activity in D1R neurons. RESULTS: We report that persistently elevated mTORC1 signaling blocks canonical D1R signaling that is dependent on DARPP-32 (dopamine- and cAMP-regulated neuronal phosphoprotein). The immediate downstream effector of mTORC1, ribosomal S6 kinase 1 (S6K1), phosphorylates and activates DARPP-32. Persistent elevation of mTORC1-S6K1 occludes dynamic D1R signaling downstream of DARPP-32 and blocks multiple D1R responses, including dynamic gene expression, D1R-dependent corticostriatal plasticity, and D1R behavioral responses including sociability. Candidate biomarkers of mTORC1-DARPP-32 occlusion are increased in the brain of human disease subjects in association with elevated mTORC1-S6K1, supporting a role for this mechanism in cognitive disease. CONCLUSIONS: The mTORC1-S6K1 intersection with D1R signaling provides a molecular framework to understand the effects of pathological mTORC1 activation on behavioral symptoms in neuropsychiatric disease.

Sex-dependent effects of an Hnrnph1 mutation on fentanyl addiction-relevant behaviors but not antinociception in mice.

Opioid Use Disorder (OUD) and opioid-related deaths remain a major public health concern in the United States. Both environmental and genetic factors influence risk for OUD. We previously identified Hnrnph1 as a quantitative trait gene underlying the stimulant, rewarding, and reinforcing properties of methamphetamine. Prior work shows that hnRNP H1, the RNA-binding protein encoded by Hnrnph1, post-transcriptionally regulates Oprm1 (mu opioid receptor gene)-the primary molecular target for the therapeutic and addictive properties of opioids. Because genetic variants can exert pleiotropic effects on behaviors induced by multiple drugs of abuse, in the current study, we tested the hypothesis that Hnrnph1 mutants would show reduced behavioral sensitivity to the mu opioid receptor agonist fentanyl. Hnrnph1 mutants showed reduced sensitivity to fentanyl-induced locomotor activity, along with a female-specific reduction in, and a male-specific induction of, locomotor sensitization following three, daily injections (0.2 mg/kg, i.p.). Hnrnph1 mutants also required a higher dose of fentanyl to exhibit opioid reward as measured via conditioned place preference (CPP). Male Hnrnph1 mutants showed reduced fentanyl reinforcement. Hnrnph1 mutants also showed reduced sucrose motivation, suggesting a reward deficit. No genotypic differences were observed in baseline thermal nociception, fentanyl-induced antinociception, physical or negative affective signs of opioid dependence, or in sensorimotor gating. In the context of our prior work, these findings suggest that Hnrnph1 dysfunction exerts a selective role in reducing the addiction liability to drugs of abuse (opioids and psychostimulants), which could provide new biological pathways to improve their therapeutic profiles.

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.

Enduring dysregulation of nucleus accumbens catecholamine and glutamate transmission by developmental exposure to phenylpropanolamine.

For over 50 years, the sympathomimetic phenylpropanolamine (PPA; ±-norephedrine) was a primary active ingredient in over-the-counter nasal decongestants for both children and adults and continues to be prevalent in the vast majority of countries today. Previously, we reported that juvenile PPA exposure alters the developmental trajectory of catecholamine and amino acid neurotransmitter systems in the nucleus accumbens (NAC), impacting the motivational valence of cocaine in later life. The present study employed a combination of in vivo microdialysis and immunoblotting approaches to better understand how juvenile PPA exposure impacts catecholamine and glutamate function within the NAC. For this, C57BL/6J mice were pretreated repeatedly with PPA (0 or 40 mg/kg) during postnatal days 21-33. Starting at 70 days of age, the function and expression of receptors and transporters regulating extracellular dopamine and glutamate were determined. Juvenile PPA pretreatment completely abolished the capacity of selective dopamine and epinephrine reuptake inhibitors to increase NAC levels of both catecholamines, without impacting D2 or α2 receptor regulation of catecholamine release. Juvenile PPA pretreatment facilitated the rise in NAC glutamate elicited by dopamine, norepinephrine and glutamate transporter inhibitors and blunted mGlu2/3 inhibition of glutamate release in this region. These data confirm that juvenile exposure to PPA produces protracted perturbations in the regulation of extracellular catecholamine and glutamate levels within the NAC and further the hypothesis that early exposure to sympathomimetic drugs found in cough, cold and allergy medicines, have long-lasting effects upon neurotransmission within brain regions gating motivation.