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 including Alzheimer's, Parkinson's, schizophrenia, and bipolar disorder. Evidence indicates that alcohol exposure increases GSK-3β (ser9) phosphorylation (pGSK-3β); however, few studies have investigated whether GSK-3 regulates the positive reinforcing effects of alcohol, which drive repetitive drug use. This study aimed to address the potential role of GSK-3 in alcohol self-administration by investigating whether direct pharmacological inhibition of GSK-3 alters the positive reinforcing effect of alcohol in mice. 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 trihydrochloride (0-10 mg/kg, ip) was injected 45-min prior to self-administration sessions in a counterbalanced design. 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, ip) was evaluated on pGSK-3β, GSK-3β PICK1, and AMPA receptor GluA2 subunit protein expression in amygdala, nucleus accumbens (NAcb), and frontal cortex in the absence of alcohol. 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-3β 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-3β and PICK1, and increased GluA2 protein expression. Given prior results showing that AMPA receptor activity regulates alcohol selfadministration, 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.

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.

Background: A prominent therapeutic indication for alcohol use disorder (AUD) is reduction in chronic repetitive alcohol use. Glutamate α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (AMPARs) regulate chronic alcohol selfadministration in preclinical models. Recent evidence indicates that the expression and function of AMPARs require the transmembrane AMPAR regulatory protein γ-8 (TARP γ-8). This study evaluated the preclinical efficacy of JNJ-55511118, a novel, selective, high-affinity inhibitor of TARP γ-8-bound AMPARs, in reducing chronic operant alcohol self-administration.Methods: Separate groups of male and female C57BL/6J mice (n = 8/sex/group) were trained to lever press for sweetened alcohol (9% v/v + sucrose 2% w/v) or sucrose only (2% w/v) in operant conditioning chambers using an FR-4 schedule of reinforcement. After a 40-day baseline, JNJ-55511118 (0, 1, and 10 mg/kg, p.o.) was administered in randomized order 1 h before self-administration sessions. Parameters of operant behavior including response rate, total reinforcers, and head entries in the drinking troughs were computer recorded.Results: During baseline, responding to alcohol, but not sucrose, was greater in female than male mice. In male mice, both doses of JNJ-55511118 decreased multiple parameters of alcohol self-administration but did not reduce behavior-matched sucrose-only self-administration. JNJ-55511118 had no effect on sweetened alcohol or sucrose self-administration in female mice. Subsequent tests of motor function showed that the lowest effective dose of JNJ-55511118 (1 mg/kg) had no effect on open-field activity in male mice. Conclusions:This study shows for the first time that TARP γ-8-bound AMPARs regulate a behavioral pathology associated with addiction. The preclinical efficacy of JNJ-55511118 in reducing alcohol self-administration in male mice suggests that inhibition of TARP γ-8-bound AMPARs is a novel and highly significant neural target for developing medications to treat AUD and other forms of addiction.

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.