Ethanol dose-dependently elicits opposing regulatory effects on hippocampal AMPA receptor GluA2 subunits through a zeta inhibitory peptide-sensitive kinase in adolescent and adult Sprague-Dawley rats

Abstract

AMPA receptor GluA2 subunits are strongly implicated in cognition, and prior work suggests that these subunits may be regulated by atypical protein kinase C (aPKC) isoforms. The present study assessed whether hippocampal and cortical AMPA receptor GluA2 subunit regulation may be an underlying factor in known age-related differences to cognitive-impairing doses of ethanol, and if aPKC isoforms modulate such responses. Hippocampal AMPA receptor GluA2 subunit, protein kinase Mζ (PKMζ), and PKCι/λ expression were elevated during adolescence compared to adults. 1 h following a low-dose (1.0-g/kg) ethanol exposure, hippocampal AMPA receptor GluA2 subunit serine 880 phosphorylation was decreased in adolescents, but was increased in adults. Age-dependent changes in GluA2 subunit phosphorylation were paralleled by alterations in aPKC isoforms, and zeta inhibitory peptide (ZIP) administration prevented ethanol-induced increases in both in adults. Ethanol-induced changes in GluA2 subunit phosphorylation were associated with delayed regulation in synaptosomal GluA2 subunit expression 24 h later. A higher ethanol dose (3.5-g/kg) failed to elicit changes in most measures in the hippocampus at either age. Similar to the hippocampus, analysis of cerebral cortical tissue also revealed age-related declines. However, no demonstrable effects were found following a low-dose ethanol exposure at either age. High-dose ethanol exposure reduced adolescent GluA2 subunit phosphorylation and aPKC isoform expression that were again accompanied by delayed reductions in synaptosomal GluA2 subunit expression. Together, these results suggest that GluA2-containing AMPA receptor modulation by aPKC isoforms is age-, region- and dose-dependently regulated, and may potentially be involved in developmentally regulated ethanol-induced cognitive impairment and other ethanol behaviors.

Keywords: AMPA receptors GluA2 subunits; PKCι/λ; PKMζ; adolescence; ethanol; protein kinase C (PKC).

Figure 1

Hippocampal AMPA receptor GluA2 subunit and aPKC isoform synaptosomal expression decreases across adolescence. Graphs depict: (A) GluA2, (B) PKMζ, and (C) PKCι/λ expression. Representative immunoblot images are shown for each. Data is presented as mean ± SEM. *p < 0.05 compared to P75, n = 7–8/group.

Figure 2

Effects of low dose (1.0 g/kg) ethanol exposure on hippocampal synaptosomal AMPA receptor GluA2 subunit expression and phosphorylation, as well as aPKC isoform expression 1 hour following ethanol administration. Graphs represent adolescents (A) and adults (B). Representative immunoblot images are shown for AMPA receptor GluA2 subunit expression, pGluA2 (serine 880), and PKMζ, PKCı/λ. Data is presented as mean ± SEM. *p < 0.05 compared with age-matched saline controls, n = 8/group.

Figure 3

Effects of zeta inhibitory peptide (ZIP) on synaptosomal AMPA receptor GluA2 subunit ser880 phosphorylation and aPKC isoform expression 1 hour following low dose (1.0 g/kg) ethanol exposure. (A) Ethanol exposure increased GluA2 phosphorylation that was prevented by zeta inhibitory peptide (ZIP, 10 nmol) administration (*, p < 0.05, compared to vehicle; #, p < 0.05, compared to ZIP + ethanol). (B) PKMζ expression was reduced overall following ZIP administration (p < 0.01). (C) PKCι/λ expression did not differ following ZIP administration. Representative immunoblot images are shown. Data are presented as mean ± SEM, n = 6–8/group.

Figure 4

Hippocampal AMPA receptor GluA2 subunit synaptosomal expression 24 hours following low dose (1.0 g/kg) ethanol exposure. Representative immunoblot images are shown. Data are presented as mean ± SEM. *p < 0.05 compared with age-matched saline controls, n = 8/group.

Figure 5

Cortical AMPA receptor GluA2 subunit and aPKC isoform synaptosomal expression decreases across adolescence. Graphs depict: (A) GluA2, and (B) PKMζ expression. Cortical PKCı/λ was previously published elsewhere (Santerre et al., 2013). Representative immunoblot images are shown. Data are presented as mean ± SEM. *p < 0.05 compared to P75, n = 7 – 8/group for GluA2 and 14–16/group for PKMζ.

Figure 6

Effects of high dose (3.5 g/kg) ethanol exposure on cortical synaptosomal AMPA receptor GluA2 subunit expression phosphorylation expression as well as PKMζ isoform expression. Analysis of adolescent (A) and adult (B) AMPA receptor GluA2 subunit expression, serine 880 phosphorylation and aPKC isoform expression at 1 hour following ethanol exposure. (C) Adolescent and adult AMPA receptor GluA2 subunit expression 24 hours following ethanol exposure. Adolescent and adult PKCι/λ results were previously reported in Santerre et al., 2013, but are shown again for completeness. Representative blots are shown for each. Data represent mean ± SEM. *p < 0.05 compared with age-matched saline controls, n = 8/group.