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

Abstract

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-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. 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-3β, GSK-3β, 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-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 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.

Figure 1.

Effect of the GSK-3 inhibitor CHIR 99021 on lever press responding reinforced by sweetened alcohol. (A) Dose-effect curve for CHIR 99021 on alcohol-reinforced response rate expressed as cumulative responses per 5-min interval on the active lever. Symbols represent means for all doses and vertical error bars represent SEM. Open circles represent responding 45 min after an i.p. injection of NaCl. Ascending doses of the inhibitor are represented by squares (1 mg/kg), upright triangle (3 mg/kg) and upside-down triangle (10 mg/kg), respectively. Asterisks indicate significant difference from vehicle control at corresponding time point, p≤0.05, 2-way RM ANOVA, Dunnett’s multiple comparison test. (B – D) Total number of alcohol reinforcers (B), head pokes per reinforcer (C), and alcohol dosage consumed (D) plotted as a function of CHIR 99021 dosage. White bars represent means for NaCl vehicle and black bars represent means of doses of CHIR 99021. Vertical error bars represent SEM. Asterisks indicates significance from NaCl, P≤0.05, 1-way RM ANOVA followed by Dunnett’s multiple comparison test.

Figure 2.

Effect of the GSK-3 inhibitor CHIR 99021 on sucrose reinforced responding. (A) Sucrose reinforced response rate plotted as a function of session time. Symbols represent individual doses of CHIR 99021 (0 – 10 mg/kg). (B – C) Total number of reinforcers (B) and head pokes per reinforcer (C) plotted as a function of CHIR 99021 dosage. White bars represent means for NaCl vehicle and black bars represent means of doses of CHIR 99021. Vertical error bars represent SEM.

Figure 3.

Effect of the GSK-3 inhibitor, CHIR 99021, on open field activity in alcohol- and sucrose-trained mice. Spontaneous locomotor activity of (A) alcohol-trained and (B) sucrose-trained mice. Distance traveled was measured in an open field 45 min after an i.p. injection of NaCl (white bars) or 10 mg/kg CHIR 99021 (black bars). Vertical error bars represent SEM.

Figure 4.

Effect of the GSK-3 inhibitor CHIR 99021 on pGSK-3β (ser9) and tGSK-3β protein expression in reward-related corticolimbic brain regions. Representative immunoblots (pGSK-3β (ser9), tGSK-3β, pGSK-3β/tGSK-3β, left, center, right panels, respectively) and quantitative analyses (bar graphs) showing systemic administration of the 10 mg/kg CHIR 99021 (black bars), on pGSK-3β (left graphs), tGSK-3β (center graphs), pGSK-3β/tGSK-3β (right graphs) immunoreactivity relative to NaCl (white bars) in the (A) prefrontal cortex, (B) nucleus accumbens and (C) amygdala. All data represent group MEAN±SEM and are plotted relative to GAPDH. Asterisks indicate statistical significance, p≤0.05, t-test.

Figure 5.

Effect of the GSK-3 inhibitor CHIR 99021 on GluA2 and PICK1 protein expression in the nucleus accumbens. Representative immunoblots (GluA2 and PICK1, left and right panels, respectively) from nucleus accumbens mouse tissue and quantitative analyses (bar graphs) showing systemic administration of 10 mg/kg CHIR 99021 (black bars), on (A) GluA2 and (B) PICK1 immunoreactivity relative to NaCl (white bars). All data represent group MEAN±SEM and are plotted relative to GAPDH. Asterisks indicate statistical significance, p≤0.05, t-test.