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.

Fig. 1

Analysis of total NMDA receptor subunits NR1 (n = 10), NR2A (n = 9), and NR2B (n = 9) in saccharin control (Sacc) and fetal alcohol-exposed (FAE) adult hippocampal tissue homogenates. (A) Representative Western blots for NR1 and β-actin in Sacc and FAE hippocampal samples. (B) Normalized optical density of anti-NR1 immunoreactivity in Sacc and FAE hippocampal samples. (C) Representative Western blots for NR2A and β-actin in Sacc and FAE hippocampal samples. (D) Normalized optical density of anti-NR2A immunoreactivity; significant decrease in FAE [t(16) = 2.305, *p < 0.05] NR2A subunit levels. (E) Representative Western blots for NR2B & β-actin in Sacc and FAE hippocampal samples. (F) Normalized optical density of anti-NR2B immunoreactivity. There was no significant (ns) difference between Sacc and FAE in NR2B levels. All data were expressed as the optical density of the anti-NMDA receptor subunit immunoreactive band normalized to the optical density of the anti-β-actin immunoreactive band.

Fig. 2

Synaptosomal membrane expression of NMDA receptor subunits in saccharin control (Sacc) and fetal alcohol-exposed (FAE) LP1 fractions. (A) Representative Western blots of NR1 and corresponding postsynaptic density protein-95 (PSD-95) in Sacc (n = 6) and FAE (n = 6) LP1 samples. (B) NR1 levels in FAE LP1 samples were significantly increased compared to controls [t(10) = 3.40, **p < 0.01]. (C) Representative Western blot of NR2A and corresponding PSD-95 in Sacc and FAE LP1 samples. (D) No significant difference between Sacc (n = 6) and FAE (n = 6) levels of surface NR2A. (E) Representative Western blot of NR2B and corresponding PSD-95 in Sacc and FAE LP1 samples. (F) No difference in NR2B expression between groups (n = 6). All data expressed relative to level of anti-PSD-95 immunoreactivity in each LP1 sample.

Fig. 3

Analysis of postsynaptic density-95 (PSD-95)-associated NMDA subunits in saccharin (Sacc) and fetal alcohol-exposed (FAE) hippocampal homogenates. (A) Representative Western blots of NR1 subunits that co-immunoprecipitated with PSD-95 in Sacc and FAE samples; PSD-95 levels in the corresponding samples are also shown. (B) PSD-95-associated NR1 levels in Sacc and FAE hippocampus (Sacc n = 12, FAEn = 11). No significant difference in NR1 was found between the groups. (C) Representative Western blots of anti-NR2A immunoreactivity that co-immunoprecipitated with PSD-95 in Sacc and FAE hippocampal samples; PSD-95 levels in the corresponding samples are also shown. (D) PSD-95-associated NR2A subunits (Sacc n = 13, FAE n = 14). (E) Representative Western blots of anti-NR2B immunoreactivity associated with anti-PSD-95 immunoprecipitates isolated from Sacc and FAE hippocampal samples; PSD-95 levels in the corresponding samples are also shown. (F) PSD-95-associated NR2B subunits (Sacc n = 14, FAE n = 13). ANOVA revealed a significant effect of prenatal diet [F(1,24) = 6.510, *p < 0.02]. In panels A, C, E the lanes labeled IgG were a pooled Sacc or FAE sample incubated with mouse IgG2a and served as the negative control, which was subtracted from the corresponding experimental optical densities. All NMDA receptor subunit data are expressed relative to the level of PSD-95 present within each immunoprecipitated sample (i.e., the same lane was probed for both anti-PSD-95 and anti-NMDA receptor subunit immunoreactivities).