GluN2B subunit deletion reveals key role in acute and chronic ethanol sensitivity of glutamate synapses in bed nucleus of the stria terminalis

Abstract

The bed nucleus of the stria terminalis (BNST) is a critical region for alcohol/drug-induced negative affect and stress-induced reinstatement. NMDA receptor (NMDAR)-dependent plasticity, such as longterm potentiation (LTP), has been postulated to play key roles in alcohol and drug addiction; yet, to date, little is understood regarding the mechanisms underlying LTP of the BNST, or its regulation by ethanol. Acute and chronic exposure to ethanol modulates glutamate transmission via actions on NMDARs. Despite intense investigation, tests of subunit specificity of ethanol actions on NMDARs using pharmacological approaches have produced mixed results. Thus, we use a conditional GluN2B KO mouse line to assess both basal and ethanol-dependent function of this subunit at glutamate synapses in the BNST. Deletion of GluN2B eliminated LTP, as well as actions of ethanol on NMDAR function. Further, we show that chronic ethanol exposure enhances LTP formation in the BNST. Using KO-validated pharmacological approaches with Ro25-6981 and memantine, we provide evidence suggesting that chronic ethanol exposure enhances LTP in the BNST via paradoxical extrasynaptic NMDAR involvement. These findings demonstrate that GluN2B is a key point of regulation for ethanol's actions and suggest a unique role of extrasynaptic GluN2B-containing receptors in facilitating LTP.extended amygdala | synaptic plasticity | excitatory transmission T he bed nucleus of the stria terminalis (BNST) is an area of the brain that underlies the negative reinforcing properties associated with drug/alcohol dependence, and has been shown in numerous studies to be critical for expression of stress-induced reinstatement of drug-seeking behavior (1-7). Although the molecular and physiological regulation of drug-related behaviors is not completely understood, numerous studies suggest a key role for synaptic plasticity in the long-term actions of alcohol and drugs of abuse. Thus, understanding how plasticity is regulated by ethanol and drugs of abuse in the BNST will be vital to the therapeutic development of treatments for alcohol and drug use disorders.A primary action of ethanol is inhibition of NMDA receptor (NMDAR) function (8-10). The NMDAR is a heterotetrameric complex composed of two obligatory GluN1 (formerly NR1) subunits and two GluN2 (formerly NR2) and/or GluN3 subunits (11). Numerous subunit combinations are possible, with eight different splice variants of GluN1 subunit and four distinct GluN2 subunit isoforms (A, B, C, and D). The predominant GluN2 subunits in adult forebrain are GluN2A and GluN2B, which dictate many channel properties, such as decay time, localization, intracellular signaling, and conductance (11,12).Exact mechanisms by which ethanol inhibits NMDAR function are not well-defined but fall into two categories: one involving direct interactions of ethanol with the NMDAR and/or the NMDAR-membrane interface, and another involving ethanoldriven posttranslational modifications of the NMDAR (13-17). It remains unclear whether there is subunit ...

Ethanol Reduces Neuronal Excitability of Lateral Orbitofrontal Cortex Neurons Via a Glycine Receptor Dependent Mechanism

“…However, these data are mixed with some reports showing enhanced sensitivity of GluN2B receptors (Anders et al, 1999;Blevins et al, 1997;Lovinger, 1995), others reporting no difference (Chu et al, 1995;Kuner et al, 1993;Otton et al, 2009) and one showing that GluN1 splice variants also influences overall sensitivity (Jin and Woodward, 2006). Support for a role of EtOH-sensitive GluN2B subunits in neurons comes from brain slice studies showing that EtOH has little effect on NMDA EPSCs recorded in the presence of selective GluN2B antagonists (Kash et al, 2008;Roberto et al, 2004) or from GluN2B conditional knockout mice (Wills et al, 2012). However, unlike NMDA currents in those reports that were recorded from central amygdala and BNST, NMDA EPSCs in lOFC neurons and hippocampus (Suvarna et al, 2005) are significantly inhibited by EtOH even in the presence of Ro-25-6981 that by itself reduces the amplitude of NMDA evoked responses.…”

Viral-Mediated Knockdown of mGluR7 in the Nucleus Accumbens Mediates Excessive Alcohol Drinking and Increased Ethanol-Elicited Conditioned Place Preference in Rats

“…It is currently believed that the nucleus accumbens (NAcc) is critically involved in many drug-related behaviors (eg, (Badanich et al, 2006;Dackis and O'Brien, 2001;Di Chiara et al, 1993;Li and Kauer, 2004;Wenger et al, 2003)). It is well established that the central glutamate system is involved in significant neuro-adaptive changes in response to ethanol exposure (Flatscher-Bader et al, 2008;Kemppainen et al, 2010;Wills et al, 2012;Xiao et al, 2009). Thus, targeting the glutamatergic transmission may provide important therapeutic targets for investigation of exposure to alcohol (De Witte et al, 2005;Pelc, 1997).…”

Ethanol Effects on N-Methyl-D-Aspartate Receptors in the Bed Nucleus of the Stria Terminalis

“…Additionally, the efficacy of these GluN2B-selective compounds can be modulated by zinc occupancy of the receptor, pH, and whether the receptor contains di-versus triheteromeric subunit composition (Kash and Winder 2007;Paoletti and Neyton 2007). As will be discussed below, recent genetic deletion studies now provide additional evidence for a key role of GluN2B in NMDAR ethanol sensitivity (Wills et al 2012). …”

“…Collectively, this evidence defines the GluN2B subunit as a key determinant of acute ethanol sensitivity within the BNST (Fig. 1A,C) (Wills et al 2012).…”

“…The extended amygdala is a series of interconnected, embryologically similar nuclei in the brain that are thought to play key roles in aspects of alcohol dependence, specifically in stress-induced increases in alcohol-seeking behaviors (reviewed in McCool 2011 andWills et al 2012). Plasticity of excitatory transmission in these and other brain regions is currently an intense area of scrutiny as a mechanism underlying aspects of addiction.…”