Long-term ethanol and corticosterone co-exposure sensitize the hippocampal ca1 region pyramidal cells to insult during ethanol withdrawal in an NMDA GluN2B subunit-dependent manner.

ETHANOL WITHDRAWAL (EWD) is associated with NMDA-mediated excitotoxicity in vitro. Clinically, multiple withdrawals have been correlated with greater long-term cognitive deficits (Duka et al., 2003). Currently, treatment of EWD reduces acute symptoms; however, normalizing dysfunction of the hypothalamic–pituitary–adrenal (HPA) axis is a viable target for alleviation of neuronal excitability (and perhaps neurodegeneration), EWD behaviors, craving, and relapse prevention (Kiefer et al., 2006; Mulholland et al., 2006; Sinha et al., 2011). In particular, the polyamine-sensitive site on the NMDA GluN2B subunit has been investigated as a potential pharmacotherapeutic target for relieving EtOH-related neurodegeneration and alcohol-dependent behaviors (Barron et al., 2012). Effects of the glucocorticoid corticosterone (CORT) and ethanol (EtOH) exposure have been studied extensively in the hippocampus, as the hippocampus is particularly vulnerable to neurodegeneration by prolonged exposure to CORT and EtOH exposure/EWD(Packan and Sapolsky, 1990). The glucocorticoid CORT is the primary stress hormone in rodents that is released upon HPA axis activation, and it modulates neuronal function via canonical intracellular hormone receptors and membrane-bound receptors. CORT levels are increased during EtOH exposure and further potentiated during EWD in rodents and humans (Adinoff et al., 1991; Borlikova et al., 2006; Little et al., 2008). CORT levels are elevated in both plasma and brain samples during EWD, but CORT levels in brain tissue may remain elevated long after CORT levels in plasma return to basal levels (Little et al., 2008). Chronic EtOH exposure in various experimental regimens results in up-regulation of NMDA receptor subunits in vitro and in vivo, making the brain more vulnerable to damage by concurrent CORT exposure. In vitro, 10-day continuous EtOH exposure (100 mM) in organotypic hippocampal slices significantly increases GluN1 and GluN2B subunits and NMDA-dependent toxicity during EWD (Harris et al., 2003; Prendergast et al., 2000, 2004). Prolonged exposure to CORT has also been shown to increase abundance of GluN2A and GluN2B mRNA and subunit protein (Meyer et al., 2004; Weiland et al., 1997); increase calcium (Ca2+) influx in hippocampal neurons (Takahashi et al., 2002); potentiate glutamate-induced excitotoxicity in hippocampal neurons in vitro (Goodman et al., 1996); and increase extracellular glutamate levels in vivo (Stein-Behrens et al., 1994). GluN2B-containing receptors confer greater open channel time and Ca2+ influx (Chen et al., 1999), and the GluN2B subunit contains an allosteric binding site for the endogenous polyamines spermidine, spermine, and putrescine. Upon binding, polyamines potentiate NMDA receptor open channel time and Ca2+ influx (Williams et al., 1991, 1994), and polyamine concentrations increase during excitotoxic events (Adibhatla et al., 2002; Davidson and Wilce, 1998). Further, CORT has been shown to increase expression of the rate-limiting enzyme in polyamine synthesis, ornithine decarboxylase (ODC; Cousin et al., 1982). During EWD from long-term EtOH exposure, in which CORT levels are increased and GluN2B subunit protein and/or function may be up-regulated, polyamine potentiation at the GluN2B subunit may contribute to pathological levels of intracellular Ca2+ and excitotoxicity. As both long-term EtOH exposure and CORT exposure have been shown to increase glutamatergic neurotransmission and NMDA receptor function and/or abundance, vulnerability to excitotoxicity during EWD coupled with increased CORT exposure likely promote neurotoxicity. In fact, previous data from our laboratory have shown that CORT administered during EtOH exposure and EWD in vitro potentiates Ca2+ influx and hippocampal cell death, and this damage is blocked by antagonism of the NMDA receptor or glucocorticoid receptor (GR) (Mulholland et al., 2005). The current studies investigated the effect of CORT and EtOH co-exposure on toxicity during EWD with a specific focus on the NMDA GluN2B subunit. We hypothesized that: (i) toxicity would be greater in EtOH and CORT co-treated cultures compared to cultures exposed to CORT or EtOH only; (ii) the greatest damage would be observed within the CA1 region, as CA1 pyramidal neurons are most vulnerable to cell death in models of excitotoxicity, including EWD (Butler et al., 2010; Prendergast et al., 2004), and as such, we focused exclusively on this region; (iii) antagonism of the polyamine-sensitive GluN2B subunit with ifenprodil would reduce the hypothesized toxicity; and (iv) GluN2B protein expression may be increased following EtOH and CORT co-exposure, as measured using Western blots.