Hippocampal N‐Methyl‐d‐Aspartate Receptor Subunit Expression Profiles in a Mouse Model of Prenatal Alcohol Exposure

Abstract

Background Although several reports have been published showing prenatal ethanol exposure is associated with alterations in N-methyl-D-aspartate (NMDA) receptor subunit levels and, in a few cases, subcellular distribution, results of these studies are conflicting. Methods We used semi-quantitative immunoblotting techniques to analyze NMDA receptor NR1, NR2A, and NR2B subunit levels in the adult mouse hippocampal formation isolated from offspring of dams who consumed moderate amounts of ethanol throughout pregnancy. We employed subcellular fractionation and immunoprecipitation techniques to isolate synaptosomal membrane- and postsynaptic density protein-95 (PSD-95)-associated pools of receptor subunits. Results We found that, compared to control animals, fetal alcohol-exposed (FAE) adult mice had: (i) increased synaptosomal membrane NR1 levels with no change in association of this subunit with PSD-95 and no difference in total NR1 expression in tissue homogenates; (ii) decreased NR2A subunit levels in hippocampal homogenates, but no alterations in synaptosomal membrane NR2A levels and no change in NR2A-PSD-95 association; and (iii) no change in tissue homogenate or synaptosomal membrane NR2B levels but a reduction in PSD-95-associated NR2B subunits. No alterations were found in mRNA levels of NMDA receptor subunits suggesting that prenatal alcohol-associated differences in subunit protein levels are the result of differences in post-transcriptional regulation of subunit localization. Conclusions Our results demonstrate that prenatal alcohol exposure induces selective changes in NMDA receptor subunit levels in specific subcellular locations in the adult mouse hippocampal formation. Of particular interest is the finding of decreased PSD-95-associated NR2B levels, suggesting that synaptic NR2B-containing NMDA receptor concentrations are reduced in FAE animals. This result is consistent with various biochemical, physiological, and behavioral findings that have been linked with prenatal alcohol exposure.

Neonatal Ethanol Exposure Impairs Trace Fear Conditioning and Alters NMDA Receptor Subunit Expression in Adult Male and Female Rats

“…5A). Adult mice exposed to EtOH before birth also had significant reductions in GluN2B (though not PSD-95) in SMFs (Samudio-Ruiz et al, 2010). In contrast, Kervern and colleagues (2015) reported an upregulation in synaptic GluN2B protein expression following combined pre-and postnatal EtOH exposure, when measured in young adult rats.…”

“…Across postnatal development, GluN2A expression is up-regulated and GluN2B expression is slightly down-regulated (Monyer et al, 1994), producing a more "stable" phenotype that requires greater excitation to induce synaptic plasticity (Yashiro and Philpot, 2008). Aberrant NMDAR subunit expression has been documented in preweanling rats exposed to EtOH during the pre-and/or postnatal period (Nixon et al, 2002(Nixon et al, , 2004, as well as in adult mice following prenatal exposure (Samudio-Ruiz et al, 2010).…”

Impaired trace fear conditioning and diminished ERK1/2 phosphorylation in the dorsal hippocampus of adult rats administered alcohol as neonates.

“…In adult mice exposed to prenatal alcohol, PSD-95-associated NR2B subunit levels are downregulated (Samudio-Ruiz et al, 2010), likely reflecting fewer synaptic NRl/2A/2B-NMDARs. Prenatal exposure is further reported to reduce NMDAR-dependent LTP in the dentate gyms of adult rats, with males more impaired than females (Sickmann et al, 2014), as well as decrease ERKl/2 activation in the dentate gyrus of adult mice (Samudio-Ruiz et al, 2009).…”

Impact of combined prenatal ethanol and prenatal stress exposures on markers of activity-dependent synaptic plasticity in rat dentate gyrus

“…For example, there is substantial evidence to show that both prenatal ethanol and prenatal stress can alter the expression, subunit composition, and synaptic membrane localization of NMDA receptors (Barros et al, 2004; Brady et al, 2013; Kinnunen, Koenig, & Bilbe, 2003; Savage, Montano, Otero, & Paxton, 1991). Further, prenatal ethanol exposure has been shown to alter the levels of several phospholipases (Allan, Weeber, Savage, & Caldwell, 1997; Weeber et al, 2001), protein kinase C activity, (Galindo et al, 2004; Perrone-Bizzozero et al, 1998; Tanner et al, 2004) and ERK kinase (Samudio-Ruiz et al, 2010). More comprehensive knowledge of how prenatal insults alter these systems, particularly under activity-dependent conditions, is critical for a clearer understanding of the neurobiological bases of synaptic plasticity deficits, which could subsequently lead to the establishment of more rational therapeutic approaches for ameliorating the synaptic plasticity and learning deficits associated with these neurodevelopmental insults.…”

“…Tissue preparation was similar to the procedures described by Samudio-Ruiz, Allan, Sheema, & Caldwell (2010), as summarized in Fig. 2.…”