Prefrontal cortex glutamatergic adaptations in a mouse model of alcohol use disorder

Abstract

Alcohol use disorder (AUD) produces cognitive deficits, indicating a shift in prefrontal cortex (PFC) function. PFC glutamate neurotransmission is mostly mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type ionotropic receptors (AMPARs); however preclinical studies have mostly focused on other receptor subtypes. Here we examined the impact of early withdrawal from chronic ethanol on AMPAR function in the mouse medial PFC (mPFC). Dependent male C57BL/6J mice were generated using the chronic intermittent ethanol vapor-two bottle choice (CIE-2BC) paradigm. Non-dependent mice had access to water and ethanol bottles but did not receive ethanol vapor. Naïve mice had no ethanol exposure. We used patch-clamp electrophysiology to measure glutamate neurotransmission in layer 2/3 prelimbic mPFC pyramidal neurons. Since AMPAR function can be impacted by subunit composition or plasticity-related proteins, we probed their mPFC expression levels. Dependent mice had higher spontaneous excitatory postsynaptic current (sEPSC) amplitude and kinetics compared to the Naïve/Non-dependent mice. These effects were seen during intoxication and after 3-8 days withdrawal, and were action potential-independent, suggesting direct enhancement of AMPAR function. Surprisingly, 3 days withdrawal decreased expression of genes encoding AMPAR subunits (Gria1/2) and synaptic plasticity proteins (Dlg4 and Grip1) in Dependent mice. Further analysis within the Dependent group revealed a negative correlation between Gria1 mRNA levels and ethanol intake. Collectively, these data establish a role for mPFC AMPAR adaptations in the glutamatergic dysfunction associated with ethanol dependence. Future studies on the underlying AMPAR plasticity mechanisms that promote alcohol reinforcement, seeking, drinking and relapse behavior may help identify new targets for AUD treatment.

Keywords: AMPA; Addiction; Excitatory transmission; GluR1; Neuroplasticity.

Fig. 1.

Ethanol intake escalated in Dependent mice. A. Schematic of the CIE-2BC protocol used to generate ethanol dependence, with mice experiencing alternating weeks of chronic intermittent ethanol vapor (CIE) and two bottle choice ethanol drinking (2BC). Non-dependent mice experienced 2BC but not CIE. Naïve mice did not receive any ethanol exposure (not illustrated). B. There was an escalation of ethanol intake in the 2BC drinking sessions during Weeks 4–6 in the Dependent vs. Non-dependent mice. C-D. (C) 13 out of 22 Non-dependent mice increased their weekly ethanol intake from their last baseline week to their final drinking week (Week 1 to Week 6), (D) while the majority of Dependent mice escalated their ethanol drinking during this time period (35/40 mice). E-F. Dependent mice had a higher ethanol intake (E) during the last week of 2BC (Week 6), and (F) totaled across all 2BC sessions vs. the Non-dependent group. N = 22–40 mice per group. **p < 0.01, ***p < 0.001 by unpaired t-test.

Fig. 2.

Ethanol dependence increased glutamate receptor function after 3–8 days withdrawal. A. Schematic of a coronal brain slice illustrating layer 2/3 of the prelimbic mPFC (adapted from [47]), and a 40x micrograph of a representative pyramidal neuron. B. Representative sEPSC traces from Naïve, Non-dependent and Dependent neurons. C. There was no significant difference in sEPSC frequency across mice groups. D–F. The sEPSC (D) amplitude, (E) rise time and (F) decay time were higher in Dependent vs. Naïve and Non-dependent mice, n = 9–25 cells from N = 7–11 mice per group. **p < 0.01; ***p < 0.001 by one-way ANOVA and Tukey’s post hoc test.

Fig. 3.

Ethanol dependence increased glutamate receptor function via an action potential-independent mechanism. A. Representative mEPSC traces from Naïve and Dependent neurons. B. There was no significant difference in mEPSC frequency of mPFC prelimbic layer 2/3 pyramidal neurons between mice groups. C–E. The mEPSC (C) amplitude and (D) rise time were higher in Dependent vs. Naïve mice, while there was a trend approaching significance in the (E) decay time, n = 8–12 cells from N = 5–6 mice per group. *p < 0.05 by unpaired t-test.

Fig. 4.

Ethanol dependence decreased mPFC AMPAR subunit gene expression. A. Schematic of a coronal brain slice illustrating the mPFC micropunch site (adapted from [47]). B and C. Gene expression levels for (B) Gria1 and (C) Gria2 were lower in Dependent vs. Naïve and Non-dependent mice. D and E. Total ethanol intake within the Dependent group (D) negatively correlated with Gria1 transcript levels, while there was a trend for a negative correlation in (E) Gria2. F. There was no difference in Gria3 mRNA levels across all three mice groups. N = 9–12 mice per group. *p < 0.05; **p < 0.01 by one-way ANOVA and Tukey’s post hoc test.

Fig. 5.

Ethanol dependence decreased mPFC AMPAR subunit protein expression. A. GluA1 levels were lower in Dependent vs. Naïve mice. B and C. There was no difference in the phosphorylation ratios of (B) GluA1-Ser831 and (C) GluA1-Ser841 across both mice groups. N 12 mice per group. *p < 0.05 by unpaired t-test.

Fig. 6.

Ethanol dependence decreased mPFC plasticity gene expression. A and B. mPFC mRNA levels for (A) Grip1 and (B) Dlg4 were lower in Dependent vs. Naïve and Non-dependent mice. C and D. There were no significant correlations between the total ethanol intake of the Dependent group and (C) Grip1 and (D) Dlg4 transcript levels. N = 10–11 mice per group. *p < 0.05; **p < 0.01; ***p < 0.001 by one-way ANOVA and Tukey’s post hoc test.