Temporal and regional alterations in NMDA receptor expression in Mecp2-null mice

Abstract

Our previous postmortem study of girls with Rett Syndrome (RTT), a development disorder caused by MECP2 mutations, found increases in the density of N-Methyl-D-aspartate (NMDA) receptors in the prefrontal cortex of 2-8-year-old girls, whereas girls older than 10 years had reductions in NMDA receptors compared with age-matched controls (Blue et al., Ann Neurol 1999b;45:541-545). Using [(3)H]-CGP to label NMDA-type glutamate receptors in 2- and 7-week old wild-type (WT), Mecp2-null, and Mecp2-heterozygous (HET) mice (Bird model), we found that frontal areas of the brain also exhibited a bimodal pattern in NMDA expression, with increased densities of NMDA receptors in Mecp2-null mice at 2 weeks of age but decreased densities at 7 weeks of age. Visual cortex showed a similar pattern, while other cortical regions only exhibited changes in NMDA receptor densities at 2 weeks (retrosplenial granular) or 7 weeks (somatosensory). In thalamus of null mice, NMDA receptors were increased at 2 and 7 weeks. No significant differences in density were found between HET and WT mice at both ages. Western blots for NMDAR1 expression in frontal brain showed higher levels of expression in Mecp2-null mice at 2 weeks of age but not at 1 or 7 weeks of age. Our mouse data support the notion that deficient MeCP2 function is the primary cause of the NMDA receptor changes we observed in RTT. Furthermore, the findings of regional and temporal differences in NMDA expression illustrate the importance of age and brain region in evaluating different genotypes of mice.

Figure 1

Pseudocolor images of [³H] CGP binding to NMDA receptors in sections from 2 week-old WT mice illustrate the different rostro-caudal levels where density measurements were made. At each level, the regions where densities were measured, are outlined in white and labeled. At prefrontal (A.) and frontal (B.) levels, the regions include frontal motor cortex, somatosensory cortex (Som), the insula (Ins) and the striatum (Str). At mid-frontal (C.) and dorsal hippocampus (D.) levels, NMDA densities were measured in the thalamus (Thal) and hippocampus (Hippo). NMDA receptor densities in the retrosplenial granular (RS) cortex, hippocampus and thalamus were measured at the level of the ventral hippocampus (E.).

Figure 2

Images of [³H] CGP binding to NMDA receptors (A. and C.) and the corresponding Nissl stained section (B.) that illustrates the different regions and layers of the hippocampal formation that were measured. This section is from a 2 week-old WT mouse and is at the level of the ventral hippocampus.

Figure 3

Pseudocolor images of [³H] CGP binding to NMDA receptors in sections through the frontal (A. and B.) and prefrontal levels (C. and D.) of the brain from WT (A. and C.) and Mecp2-null mice (B. and D.) at 2 and 7 weeks of age. The regions where densities were measured, are outlined in white in each section. These regions include frontal motor cortex, somatosensory cortex (Som), the insula (Ins) and the striatum (Str), and are labeled in A.

Figure 4

Pseudocolor images of [³H] CGP binding to NMDA receptors in sections through the dorsal hippocampus level of the brain from WT (A. and C.) and Mecp2-null mice (B. and D.) at 2 and 7 weeks of age. The regions where densities were measured are outlined in white in each section. These regions include visual cortex, retrosplenial granular (RS) cortex, hippocampus and thalamus, and are labeled in A.

Figure 5

Line graphs comparing the density of NMDA receptors (percent WT; mean ± s.e.m.) between WT and Mecp2-null mice at 2 and 7 weeks of age in different brain regions, which illustrate the different regional and developmental differences in NMDA receptor expression between WT and Mecp2-null mice. P values: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ^#p=0.055; ^p=0.075.

Figure 6

Line graphs comparing the density of NMDA receptors between WT and Mecp2-null mice at 2 (A.) and 7 (B.) weeks of age at different rostrocaudal levels of the insula (percent WT; mean ± s.e.m.). P values: *p<0.05; **p<0.01; ***p<0.001.

Figure 7

Western blots of the NMDAR1 subunit of the NMDA receptor and β-actin from WT and Mecp2-null (null) mice at 1, 2 and 7 weeks of age are shown in A. Mean densities of NMDA1 protein (percent WT; mean ± s.e.m.) show a significant increase in NMDAR1 expression in Mecp2-null mice at 2 weeks of age but no genotype differences at 1 or 7 weeks of age (B.). P value: *p<0.05.