Calcium-permeable AMPA receptor activity and GluA1 trafficking in the basolateral amygdala regulate operant alcohol self-administration.

Addiction is viewed as maladaptive glutamate-mediated neuroplasticity that is regulated, in part, by calcium-permeable AMPA receptor (CP-AMPAR) activity. However, the contribution of CP-AMPARs to alcohol-seeking behavior remains to be elucidated. We evaluated CP-AMPAR activity in the basolateral amygdala (BLA) as a potential target of alcohol that also regulates alcohol self-administration in C57BL/6J mice. Operant self-administration of sweetened alcohol increased spontaneous EPSC frequency in BLA neurons that project to the nucleus accumbens as compared with behavior-matched sucrose controls indicating an alcohol-specific upregulation of synaptic activity. Bath application of the CP-AMPAR antagonist NASPM decreased evoked EPSC amplitude only in alcohol self-administering mice indicating alcohol-induced synaptic insertion of CP-AMPARs in BLA projection neurons. Moreover, NASPM infusion in the BLA dose-dependently decreased the rate of operant alcohol self-administration providing direct evidence for CP-AMPAR regulation of alcohol reinforcement. As most CP-AMPARs are GluA1-containing, we asked if alcohol alters the activation state of GluA1-containing AMPARs. Immunocytochemistry results showed elevated GluA1-S831 phosphorylation in the BLA of alcohol as compared with sucrose mice. To investigate mechanistic regulation of alcohol self-administration by GluA1-containing AMPARs, we evaluated the necessity of GluA1 trafficking using a TET-ON AAV encoding a dominant-negative GluA1 c-terminus (GluA1ct) that blocks activity-dependent synaptic delivery of native GluA1-containing AMPARs. GluA1ct expression in the BLA reduced alcohol self-administration with no effect on sucrose controls. These results show that CP-AMPAR activity and GluA1 trafficking in the BLA mechanistically regulate the reinforcing effects of sweetened alcohol. Pharmacotherapeutic targeting these mechanisms of maladaptive neuroplasticity may aid medical management of alcohol use disorder.

Pharmacological inhibition of glycogen synthase kinase 3 increases operant alcohol self-administration in a manner associated with altered pGSK-3ß, protein interacting with C kinase and GluA2 protein expression in the reward pathway of male C57BL/6J mice.

Glycogen synthase kinase 3 (GSK-3) is a constitutively active serine-threonine kinase that regulates numerous signaling pathways and has been implicated in neurodegenerative and neuropsychiatric diseases. Alcohol exposure increases GSK-3ß (ser9) phosphorylation (pGSK-3ß); however, few studies have investigated whether GSK-3 regulates the positive reinforcing effects of alcohol, which drive repetitive drug use. To address this goal, male C57BL/6J mice were trained to lever press on a fixed-ratio 4 schedule of sweetened alcohol or sucrose-only reinforcement in operant conditioning chambers. The GSK-3 inhibitor CHIR 99021 (0-10 mg/kg, i.p.) was injected 45 minutes prior to self-administration sessions. After completion of the self-administration dose-effect curve, potential locomotor effects of the GSK-3 inhibitor were assessed. To determine molecular efficacy, CHIR 99021 (10 mg/kg, i.p.) was evaluated on pGSK-3ß, GSK-3ß, protein interacting with C kinase (PICK1), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluA2 subunit protein expression in amygdala, nucleus accumbens (NAcb), and frontal cortex. Results showed that CHIR 99021 (10 mg/kg) dose-dependently increased alcohol reinforced responding with no effect on sucrose self-administration or locomotor activity. CHIR 99021 (10 mg/kg) significantly decreased pGSK-3ß expression in all brain regions tested, reduced PICK1 and increased GluA2 total expression only in the NAcb. We conclude that GSK-3 inhibition increased the reinforcing effects of alcohol in mice. This was associated with reduced pGSK-3ß and PICK1, and increased GluA2 expression. Given prior results showing that AMPA receptor activity regulates alcohol self-administration, we propose that signaling through the GSK-3/PICK1/GluA2 molecular pathway drives the positive reinforcing effects of the drug, which are required for abuse liability.

Mining the nucleus accumbens proteome for novel targets of alcohol self-administration in male C57BL/6J mice.

RATIONALE: There is a clear need for discovery of effective medications to treat behavioral pathologies associated with alcohol addiction, such as chronic drinking. OBJECTIVE: The goal of this preclinical study was to assess effects of chronic alcohol drinking on the nucleus accumbens (NAcb) proteome to identify and validate novel targets for medications development. MATERIALS AND METHODS: Two-dimensional difference in-gel electrophoresis (2D-DIGE) with matrix-assisted laser desorption ionization tandem time-of-flight (MALDI-TOF/TOF) was used to assess effects of chronic voluntary home-cage (24-h access) alcohol drinking on the NAcb proteome of C57BL/6J mice. To extend these findings to a model of alcohol self-administration and reinforcement, we investigated potential regulation of the positive reinforcing effects of alcohol by the target protein glutathione S-transferase Pi 1 (GSTP1) using a pharmacological inhibition strategy in mice trained to self-administer alcohol or sucrose. RESULTS: Expression of 52 unique proteins in the NAcb was changed by chronic alcohol drinking relative to water control (23 upregulated, 29 downregulated). Ingenuity Pathway Analysis showed that alcohol drinking altered an array of protein networks associated with neurological and psychological disorders, molecular and cellular functions, and physiological systems and development. DAVID functional annotation analysis identified 9 proteins (SNCA, GSTP1, PRDX3, PPP3R1, EIF5A, PHB, PEBP1/RKIP, GAPDH, AND SOD1) that were significantly overrepresented in a functional cluster that included the Gene Ontology categories "response to alcohol" and "aging." Immunoblots confirmed changes in Pebp1 (RKIP) and GSTP1 in NAcb with no change in amygdala or frontal cortex, suggesting anatomical specificity. Systemic inhibition of GSTP1 with Ezatiostat (0-30 mg/kg, i.p.) dose-dependently reduced the reinforcing effects of alcohol as measured by operant self-administration, in the absence of motor effects. Sucrose self-administration was also reduced but in a manner associated with nonspecific motor inhibition. CONCLUSIONS: Protein expression profiling identified an array of proteins and networks in the NAcb, including GSTP1, that are novel molecular targets of chronic alcohol drinking. Pharmacological inhibition of GSTP1 significantly reduced the positive reinforcing effects of alcohol, which regulate repetitive use and abuse liability. The observation that this protein was both upregulated after chronic drinking and that its inhibition could modulate the reinforcing properties of alcohol suggests that it is a key target for alcohol-related pathologies. Proteomic strategies combined with specific preclinical models has potential to identify and validate novel targets of alcohol that may be useful in the medical management of alcohol addiction.

Cue-induced reinstatement of alcohol-seeking behavior is associated with increased CaMKII T286 phosphorylation in the reward pathway of mice.

Cue-induced reinstatement of alcohol-seeking is a hallmark behavioral pathology of addiction. Evidence suggests that reinstatement (e.g., relapse), may be regulated by cell signaling systems that underlie neuroplasticity. A variety of plasticity events require activation of calcium calmodulin-dependent protein kinase II (CaMKII) in components of the reward pathway, such as the nucleus accumbens and amygdala. We sought to determine if cue-induced reinstatement of alcohol-seeking behavior is associated with changes in the activation state (e.g., phosphorylation) of CaMKII-T286. Male C57BL/6J mice (n=14) were trained to lever press on a fixed-ratio-4 schedule of sweetened alcohol (2% sucrose+9% EtOH) reinforcement. After 14-d of extinction (no cues or reinforcers), mice underwent a response-contingent reinstatement (n=7) vs. an additional day of extinction (n=7). Brains were removed immediately after the test and processed for evaluation of pCaMKII-T286 immunoreactivity (IR). Number of pCaMKII-T286 positive cells/mm2 was quantified from coronal brain sections using Bioquant Image Analysis software. Mice emitted significantly more responses on the alcohol vs. the inactive lever throughout the baseline phase with average alcohol intake of 1.1±0.03g/kg/1-h. During extinction, responses on the alcohol lever decreased to inactive lever levels by day 7. Significant cue-induced reinstatement of alcohol-seeking was observed during a single test with no effects on the inactive lever. Reinstatement was associated with increased pCaMKII-T286 IR specifically in amygdala (LA and BLA), nucleus accumbens (AcbSh), lateral septum, mediodorsal thalamus, and piriform cortex as compared to extinction control. Brain regions showing no change included the dorsal striatum, medial septum, cingulate cortex, habenula, paraventricular thalamus, and ventral hypothalamus. These results show response contingent cue-induced reinstatement of alcohol-seeking behavior is associated with selective increases in pCaMKII-T286 in specific reward- and memory-related brain regions of male C57BL/6J mice. Primary molecular mechanisms of associative learning and memory may regulate relapse in alcohol addiction.

Cannabinoid CB1 receptor inhibition blunts adolescent-typical increased binge alcohol and sucrose consumption in male C57BL/6J mice.

Increased binge alcohol consumption has been reported among adolescents as compared to adults in both humans and rodent models, and has been associated with serious long-term health consequences. However, the neurochemical mechanism for age differences in binge drinking between adolescents and adults has not been established. The present study was designed to evaluate the mechanistic role of the cannabinoid CB1 receptor in adolescent and adult binge drinking. Binge consumption was established in adolescent and adult male C57BL/6J mice by providing access to 20% alcohol or 1% sucrose for 4h every other day. Pretreatment with the CB1 antagonist/inverse agonist AM-251 (0, 1, 3, and 10mg/kg) in a Latin square design dose-dependently reduced adolescent alcohol consumption to adult levels without altering adult intake. AM-251 (3mg/kg) also reduced adolescent but not adult sucrose consumption. Adolescent reductions in alcohol and sucrose were not associated with alterations in open-field locomotor activity or thigmotaxis. These findings point to age differences in CB1 receptor activity as a functional mediator of adolescent-typical increased binge drinking as compared to adults. Developmental alterations in endocannabinoid signaling in the adolescent brain may therefore be responsible for the drinking phenotype seen in this age group.