Melissa G. Wroten

Nucleus accumbens mGluR5-associated signaling regulates binge alcohol drinking under drinking-in-the-dark procedures.

BACKGROUND Alcohol increases the expression of Group 1 metabotropic glutamate receptors (mGluRs) and their associated scaffolding protein Homer2 and stimulates phosphatidylinositol 3-kinase (PI3K) within the nucleus accumbens (NAC). Moreover, functional studies suggest that NAC Group 1 mGluR/Homer2/PI3K signaling may be a potential target for pharmacotherapeutic intervention in alcoholism. METHODS Immunoblotting was conducted to examine the effects of alcohol consumption under drinking-in-the-dark (DID) procedures on Group 1 mGluR-associated proteins in C57BL/6J (B6) mice. Follow-up behavioral studies examined the importance of Group 1 mGluR/Homer2/PI3K signaling within the NAC shell for limited-access alcohol drinking. Finally, immunoblotting examined whether the NAC expression of Group 1 mGluR-associated proteins is a genetic correlate of high alcohol drinking using a selectively bred high DID (HDID-1) mouse line. RESULTS Limited-access alcohol drinking under DID procedures up-regulated NAC shell Homer2 levels, concomitant with increases in mGluR5 and NR2B. Intra-NAC shell blockade of mGluR5, Homer2, or PI3K signaling, as well as transgenic disruption of the Homer binding site on mGluR5, decreased alcohol consumption in B6 mice. Moreover, transgenic disruption of the Homer binding site on mGluR5 and Homer2 deletion both prevented the attenuating effect of mGluR5 and PI3K blockade upon intake. Finally, the basal NAC shell protein expression of mGluR1 and Homer2 was increased in offspring of HDID-1 animals. CONCLUSIONS Taken together, these data further implicate Group 1 mGluR signaling through Homer2 within the NAC in excessive alcohol consumption.

Binge Alcohol Drinking by Mice Requires Intact Group1 Metabotropic Glutamate Receptor Signaling Within the Central Nucleus of the Amygdale

Despite the fact that binge alcohol drinking (intake resulting in blood alcohol concentrations (BACs) ⩾80 mg% within a 2-h period) is the most prevalent form of alcohol-use disorders (AUD), a large knowledge gap exists regarding how this form of AUD influences neural circuits mediating alcohol reinforcement. The present study employed integrative approaches to examine the functional relevance of binge drinking-induced changes in glutamate receptors, their associated scaffolding proteins and certain signaling molecules within the central nucleus of the amygdala (CeA). A 30-day history of binge alcohol drinking (for example, 4–5 g kg−1 per 2 h−1) elevated CeA levels of mGluR1, GluN2B, Homer2a/b and phospholipase C (PLC) β3, without significantly altering protein expression within the adjacent basolateral amygdala. An intra-CeA infusion of mGluR1, mGluR5 and PLC inhibitors all dose-dependently reduced binge intake, without influencing sucrose drinking. The effects of co-infusing mGluR1 and PLC inhibitors were additive, whereas those of coinhibiting mGluR5 and PLC were not, indicating that the efficacy of mGluR1 blockade to lower binge intake involves a pathway independent of PLC activation. The efficacy of mGluR1, mGluR5 and PLC inhibitors to reduce binge intake depended upon intact Homer2 expression as revealed through neuropharmacological studies of Homer2 null mutant mice. Collectively, these data indicate binge alcohol-induced increases in Group1 mGluR signaling within the CeA as a neuroadaptation maintaining excessive alcohol intake, which may contribute to the propensity to binge drink.

Deficits in Ventromedial Prefrontal Cortex Group 1 Metabotropic Glutamate Receptor Function Mediate Resistance to Extinction during Protracted Withdrawal from an Extensive History of Cocaine Self-Administration

Anomalies in prefrontal cortex (PFC) function are posited to underpin difficulties in learning to suppress drug-seeking behavior during abstinence. Because group 1 metabotropic glutamate receptors (mGluRs) regulate drug-related learning, we assayed the consequences of extended access to intravenous cocaine (6 h/d; 0.25 mg/infusion for 10 d) on the PFC expression of group 1 mGluRs and the relevance of observed changes for cocaine seeking. After protracted withdrawal, cocaine-experienced animals exhibited a time-dependent intensification of cue-induced cocaine-seeking behavior and an impaired extinction of this behavior. These behavioral phenomena were associated with a time-dependent reduction in mGluR1/5 expression within ventromedial PFC (vmPFC) of cocaine-experienced animals exposed to extinction testing but not in untested ones. Interestingly, pharmacological manipulations of vmPFC mGluR1/5 produced no immediate effects on cue-induced cocaine-seeking behavior but produced residual effects on a subsequent test for cocaine seeking. At 3 d withdrawal, cocaine-experienced rats infused intra-vmPFC with mGluR1/5 antagonists, either before or after an initial test for cocaine seeking, persisted in their cocaine seeking akin to cocaine-experienced rats in protracted withdrawal. Conversely, cocaine-experienced rats infused with an mGluR1/5 agonist before the initial test for cocaine-seeking at 30 d withdrawal exhibited a facilitation of extinction learning. These data indicate that cue-elicited deficits in vmPFC group 1 mGluR function mediate resistance to extinction during protracted withdrawal from a history of extensive cocaine self-administration and pose pharmacological stimulation of these receptors as a potential approach to facilitate learned suppression of drug-seeking behavior that may aid drug abstinence.

Imbalances in Prefrontal Cortex CC-Homer1 versus CC-Homer2 Expression Promote Cocaine Preference

Homer postsynaptic scaffolding proteins regulate forebrain glutamate transmission and thus, are likely molecular candidates mediating hypofrontality in addiction. Protracted withdrawal from cocaine experience increases the relative expression of Homer2 versus Homer1 isoforms within medial prefrontal cortex (mPFC). Thus, this study used virus-mediated gene transfer strategies to investigate the functional relevance of an imbalance in mPFC Homer1/2 expression as it relates to various measures of sensorimotor, cognitive, emotional and motivational processing, as well as accompanying alterations in extracellular glutamate in C57BL/6J mice. mPFC Homer2b overexpression elevated basal glutamate content and blunted cocaine-induced glutamate release within the mPFC, whereas Homer2b knockdown produced the opposite effects. Despite altering mPFC glutamate, Homer2b knockdown failed to influence cocaine-elicited conditioned place preferences, nor did it produce consistent effects on any other behavioral measures. In contrast, elevating the relative expression of Homer2b versus Homer1 within mPFC, by overexpressing Homer2b or knocking down Homer1c, shifted the dose-response function for cocaine-conditioned reward to the left, without affecting cocaine locomotion or sensitization. Intriguingly, both these transgenic manipulations produced glutamate anomalies within the nucleus accumbens (NAC) of cocaine-naive animals that are reminiscent of those observed in cocaine experienced animals, including reduced basal extracellular glutamate content, reduced Homer1/2 and glutamate receptor expression, and augmented cocaine-elicited glutamate release. Together, these data provide novel evidence in support of opposing roles for constitutively expressed Homer1 and Homer2 isoforms in regulating mPFC glutamate transmission in vivo and support the hypothesis that cocaine-elicited increases in the relative amount of mPFC Homer2 versus Homer1 signaling produces abnormalities in NAC glutamate transmission that enhance vulnerability to cocaine reward.

Protein Kinase C Epsilon Activity in the Nucleus Accumbens and Central Nucleus of the Amygdala Mediates Binge Alcohol Consumption.

BACKGROUND Protein kinase C epsilon (PKCε) is emerging as a potential target for the development of pharmacotherapies to treat alcohol use disorders, yet little is known regarding how a history of a highly prevalent form of drinking, binge alcohol intake, influences enzyme priming or the functional relevance of kinase activity for excessive alcohol intake. METHODS Immunoblotting was employed on tissue from subregions of the nucleus accumbens (NAc) and the amygdala to examine both idiopathic and binge drinking-induced changes in constitutive PKCε priming. The functional relevance of PKCε translocation for binge drinking and determination of potential upstream signaling pathways involved were investigated using neuropharmacologic approaches within the context of two distinct binge drinking procedures, drinking in the dark and scheduled high alcohol consumption. RESULTS Binge alcohol drinking elevated p(Ser729)-PKCε levels in both the NAc and the central nucleus of the amygdala (CeA). Moreover, immunoblotting studies of selectively bred and transgenic mouse lines revealed a positive correlation between the propensity to binge drink alcohol and constitutive p(Ser729)-PKCε levels in the NAc and CeA. Finally, neuropharmacologic inhibition of PKCε translocation within both regions reduced binge alcohol consumption in a manner requiring intact group 1 metabotropic glutamate receptors, Homer2, phospholipase C, and/or phosphotidylinositide-3 kinase function. CONCLUSIONS Taken together, these data indicate that PKCε signaling in both the NAc and CeA is a major contributor to binge alcohol drinking and to the genetic propensity to consume excessive amounts of alcohol.

Mesocorticolimbic monoamine correlates of methamphetamine sensitization and motivation

Methamphetamine (MA) is a highly addictive psychomotor stimulant, with life-time prevalence rates of abuse ranging from 5–10% world-wide. Yet, a paucity of research exists regarding MA addiction vulnerability/resiliency and neurobiological mediators of the transition to addiction that might occur upon repeated low-dose MA exposure, more characteristic of early drug use. As stimulant-elicited neuroplasticity within dopamine neurons innervating the nucleus accumbens (NAC) and prefrontal cortex (PFC) is theorized as central for addiction-related behavioral anomalies, we used a multi-disciplinary research approach in mice to examine the interactions between sub-toxic MA dosing, motivation for MA and mesocorticolimbic monoamines. Biochemical studies of C57BL/6J (B6) mice revealed short- (1 day), as well as longer-term (21 days), changes in extracellular dopamine, DAT and/or D2 receptors during withdrawal from 10, once daily, 2 mg/kg MA injections. Follow-up biochemical studies conducted in mice selectively bred for high vs. low MA drinking (respectively, MAHDR vs. MALDR mice), provided novel support for anomalies in mesocorticolimbic dopamine as a correlate of genetic vulnerability to high MA intake. Finally, neuropharmacological targeting of NAC dopamine in MA-treated B6 mice demonstrated a bi-directional regulation of MA-induced place-conditioning. These results extend extant literature for MA neurotoxicity by demonstrating that even subchronic exposure to relatively low MA doses are sufficient to elicit relatively long-lasting changes in mesocorticolimbic dopamine and that drug-induced or idiopathic anomalies in mesocorticolimbic dopamine may underpin vulnerability/resiliency to MA addiction.

Homer2 regulates alcohol and stress cross-sensitization

An interaction exists between stress and alcohol in the etiology and chronicity of alcohol use disorders, yet a knowledge gap exists regarding the neurobiological underpinnings of this interaction. In this regard, we employed an 11‐day unpredictable, chronic, mild stress (UCMS) procedure to examine for stress–alcohol cross‐sensitization of motor activity as well as alcohol consumption/preference and intoxication. We also employed immunoblotting to relate the expression of glutamate receptor‐related proteins within subregions of the nucleus accumbens (NAC) to the manifestation of behavioral cross‐sensitization. UCMS mice exhibited a greater locomotor response to an acute injection of 2 g/kg alcohol than unstressed controls and this cross‐sensitization extended to alcohol intake (0–20 percent), as well as to the intoxicating and sedative properties of 3 and 5 g/kg alcohol, respectively. Regardless of prior alcohol injection (2 g/kg), UCMS mice exhibited elevated NAC shell levels of mGlu1α, GluN2b and Homer2, as well as lower phospholipase Cβ within this subregion. GluN2b levels were also lower within the NAC core of UCMS mice. The expression of stress–alcohol locomotor cross‐sensitization was associated with lower mGlu1α within the NAC core and lower extracellular signal‐regulated kinase activity within both NAC subregions. As Homer2 regulates alcohol sensitization, we assayed also for locomotor cross‐sensitization in Homer2 wild‐type (WT) and knock‐out (KO) mice. WT mice exhibited a very robust cross‐sensitization that was absent in KO animals. These results indicate that a history of mild stress renders an animal more sensitive to the psychomotor and rewarding properties of alcohol, which may depend on neuroplasticity within NAC glutamate transmission.

Prefrontal glutamate correlates of methamphetamine sensitization and preference

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.

Cocaine-elicited imbalances in ventromedial prefrontal cortex Homer1 versus Homer2 expression: implications for relapse.

Withdrawal from a history of extended access to self‐administered cocaine produces a time‐dependent intensification of drug seeking, which might relate to a cocaine‐induced imbalance in the relative expression of constitutively expressed Homer1 versus Homer2 isoforms within the ventromedial aspect of the prefrontal cortex (vmPFC). Thus, we employed immunoblotting to examine the relation between cue‐reinforced lever pressing at 3‐ versus 30‐day withdrawal from a 10‐day history of extended access (6 hours/day) to intravenous cocaine (0.25 mg/infusion) or saline (Sal6h), and the expression of Homer1b/c and Homer2a/b within the vmPFC versus the more dorsomedial aspect of this structure (dmPFC). Behavioral studies employed adeno‐associated virus (AAV) vectors to reverse cocaine‐elicited changes in the relative expression of Homer1 versus Homer2 isoforms and tested animals for cocaine prime‐, and cue‐induced responding following extinction training. Cocaine self‐administration elevated both Homer1b/c and Homer2a/b levels within the vmPFC at 3‐day withdrawal, and the rise in Homer2a/b persisted for at least 30 days. dmPFC Homer levels did not change as a function of self‐administration history. Reversing the relative increase in Homer2 versus Homer1 expression via Homer1c overexpression or Homer2b knockdown failed to influence cue‐reinforced lever pressing when animals were tested in a drug‐free state, but both AAV treatments prevented cocaine‐primed reinstatement of lever‐pressing behavior. These data suggest that a cocaine‐elicited imbalance in the relative expression of constitutively expressed Homer2 versus Homer1 within the vmPFC is necessary for the capacity of cocaine to reinstate drug‐seeking behavior, posing drug‐induced changes in vmPFC Homer expression as a molecular trigger contributing to drug‐elicited relapse.

Cocaine craving during protracted withdrawal requires PKCε priming within vmPFC

In individuals with a history of drug taking, the capacity of drug‐associated cues to elicit indices of drug craving intensifies or incubates with the passage of time during drug abstinence. This incubation of cocaine craving, as well as difficulties with learning to suppress drug‐seeking behavior during protracted withdrawal, are associated with a time‐dependent deregulation of ventromedial prefrontal cortex (vmPFC) function. As the molecular bases for cocaine‐related vmPFC deregulation remain elusive, the present study assayed the consequences of extended access to intravenous cocaine (6 hours/day; 0.25 mg/infusion for 10 day) on the activational state of protein kinase C epsilon (PKCε), an enzyme highly implicated in drug‐induced neuroplasticity. The opportunity to engage in cocaine seeking during cocaine abstinence time‐dependently altered PKCε phosphorylation within vmPFC, with reduced and increased p‐PKCε expression observed in early (3 days) and protracted (30 days) withdrawal, respectively. This effect was more robust within the ventromedial versus dorsomedial PFC, was not observed in comparable cocaine‐experienced rats not tested for drug‐seeking behavior and was distinct from the rise in phosphorylated extracellular signal‐regulated kinase observed in cocaine‐seeking rats. Further, the impact of inhibiting PKCε translocation within the vmPFC using TAT infusion proteins upon cue‐elicited responding was determined and inhibition coinciding with the period of testing attenuated cocaine‐seeking behavior, with an effect also apparent the next day. In contrast, inhibitor pretreatment prior to testing during early withdrawal was without effect. Thus, a history of excessive cocaine taking influences the cue reactivity of important intracellular signaling molecules within the vmPFC, with PKCε playing a critical role in the manifestation of cue‐elicited cocaine seeking during protracted drug withdrawal.