Negative modulation of AMPA receptors bound to transmembrane AMPA receptor regulatory protein γ-8 blunts the positive reinforcing properties of alcohol and sucrose in a brain region-dependent manner in male mice

Abstract

Rationale: The development and progression of alcohol use disorder (AUD) are widely viewed as maladaptive neuroplasticity. The transmembrane alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) regulatory protein γ8 (TARP γ-8) is a molecular mechanism of neuroplasticity that has not been evaluated in AUD or other addictions.

Objective: To address this gap in knowledge, we evaluated the mechanistic role of TARP γ-8 bound AMPAR activity in the basolateral amygdala (BLA) and ventral hippocampus (vHPC) in the positive reinforcing effects of alcohol, which drive repetitive alcohol use throughout the course of AUD, in male C57BL/6 J mice. These brain regions were selected because they exhibit high levels of TARP γ-8 expression and send glutamate projections to the nucleus accumbens (NAc), which is a key nucleus in the brain reward pathway.

Methods and results: Site-specific pharmacological inhibition of AMPARs bound to TARP γ-8 in the BLA via bilateral infusion of the selective negative modulator JNJ-55511118 (0-2 µg/µl/side) significantly decreased operant alcohol self-administration with no effect on sucrose self-administration in behavior-matched controls. Temporal analysis showed that reductions in alcohol-reinforced response rate occurred > 25 min after the onset of responding, consistent with a blunting of the positive reinforcing effects of alcohol in the absence of nonspecific behavioral effects. In contrast, inhibition of TARP γ-8 bound AMPARs in the vHPC selectively decreased sucrose self-administration with no effect on alcohol.

Conclusions: This study reveals a novel brain region-specific role of TARP γ-8 bound AMPARs as a molecular mechanism of the positive reinforcing effects of alcohol and non-drug rewards.

Keywords: AMPA receptor; Alcohol use; Basolateral amygdala; JNJ-55511118; TARP γ-8.

Fig. 1

Sequence of experimental procedures. Mice were first trained to self-administer sweetened alcohol or sucrose only in operant conditioning chambers. Training was followed by bilateral cannulation surgery and recovery and then a return to operant responding. Mice were then microinjected with JNJ-5 (0, .3, 1, 2 μg/μl) into the BLA or vHPC prior to operant self-administration to determine the effects of inhibiting TARP γ-8 bound AMPAR on alcohol or sucrose self-administration. The JNJ-5 microinjections were repeated prior to an open-field test to account for any nonspecific locomotor or anxiety-like (thigmotaxis) effects. Finally, brains were removed for histological verification of cannula placement

Fig. 2

Inhibition of TARP γ-8 bound AMPARs in the BLA selectively decreases EtOH-reinforced responding. A Schematic showing mouse operant conditioning chamber and cannula placement for intra-BLA microinjections of JNJ-55511118 prior to self-administration of sweetened alcohol (n = 8) and sucrose only (n = 9). B Coronal sections of adult (P56) male C57BL/6 J mouse brain showing the expression pattern of the TARP γ-8 gene (Cacng8) by in situ hybridization (Allen Mouse Brain Atlas) and cresyl violet stained section showing representative injector placement in the BLA. C-D Bar graphs (top rows) showing MEAN ± SEM parameters of operant ethanol (C) and sucrose (D) self-administration plotted as a function of dosage of JNJ-5 infused in the BLA. Line graphs (bottom rows) show MEAN ± SEM rate of ethanol (C) and sucrose (D) reinforced responding (cumulative responses/5-min interval) as compared to vehicle control for each dose of JNJ-5 tested. Asterisk denotes statistically significant difference as compared to vehicle, p < .05

Fig. 3

TARP γ-8 bound AMPAR inhibition in vHPC selectively decreases sucrose-reinforced responding. A Schematic showing mouse operant conditioning chamber and cannular placement for intra-vHPC microinjections of JNJ-55511118 prior to operant self-administration of sweetened alcohol (n = 8) and sucrose only (n = 9). B Coronal sections of adult (P56) male C57BL/6 J mouse brain showing the expression pattern of the TARP γ-8 gene (Cacng8) by in situ hybridization (Allen Mouse Brain Atlas) and cresyl violet stained section showing representative injector placement in the vHPC. C-D Bar graphs (top rows) showing MEAN ± SEM parameters of operant ethanol (C) and sucrose (D) self-administration plotted as a function of dosage of JNJ-5 infused in the vHPC. Line graphs (bottom rows) show MEAN ± SEM rate of ethanol (C) and sucrose (D) reinforced responding (cumulative responses/5-min interval) as compared to vehicle control for each dose of JNJ-5 tested. Asterisk denotes statistically significant difference as compared to vehicle, p < .05

Fig. 4

Site-specific inhibition of TARP γ-8 bound AMPAR has no effect on locomotor activity or anxiety-like behavior. A Experimental schematic showing open field with center zone and intra-BLA microinjections prior to the open-field test. B Motor activity is shown as MEAN ± SEM distance traveled (cm) during each 60-min session for EtOH (left, blue bars, n = 9) and sucrose (right, gray bars, n = 10) exposed mice plotted as a function of JNJ-5 dosage in the BLA. C Thigmotaxis shown as MEAN ± SEM distance traveled (cm) in the center of the open field for EtOH (left, blue bars) and sucrose (right, gray bars) exposed mice plotted as a function of JNJ-5 dosage in the BLA. D Experimental schematic of the apparatus and intra-vHPC microinjections prior to the open-field test. E Motor activity plotted as MEAN ± SEM distance traveled (cm) during 60-min sessions for EtOH (left, blue bars, n = 9) and sucrose (right, gray bars, n = 9) exposed mice plotted as a function of JNJ-5 dosage in the VHPC. F Thigmotaxis shown as MEAN ± SEM distance traveled (cm) in the center of the open field for EtOH (left, blue bars) and sucrose (right, gray bars) exposed mice plotted as a function of JNJ-5 dosage in the vHPC