Lower sensitivity to stress and altered monoaminergic neuronal function in mice lacking the NMDA receptor epsilon 4 subunit

Abstract

NMDA receptors, an ionotropic subtype of glutamate receptors (GluRs), play an important role in excitatory neurotransmission, synaptic plasticity, and brain development. They are composed of the GluRzeta subunit (NR1) combined with any one of four GluRepsilon subunits (GluRepsilon1-GluRepsilon4; NR2A-NR2D). Although the GluRzeta subunit exists in the majority of the CNS throughout all stages of development, the GluRepsilon subunits are expressed in distinct temporal and spatial patterns. In the present study, we investigated neuronal functions in mice lacking the embryonic GluRepsilon4 subunit. GluRepsilon4 mutant mice exhibited reductions of [(3)H]MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate] binding and (45)Ca(2+) uptake through the NMDA receptors. The expression of GluRzeta subunit protein, but not GluRepsilon1 and GluRepsilon2 subunit proteins, was reduced in the frontal cortex and striatum of the mutant mice. A postmortem examination in GluRepsilon4 mutant mice revealed that tissue contents of norepinephrine, dopamine, serotonin, and their metabolites were reduced in the hippocampus and that dopamine, as well as serotonin, metabolism was upregulated in the frontal cortex, striatum, hippocampus, and thalamus. To clarify the phenotypical influences of the alteration in neuronal functions, performances in various behavioral tests were examined. GluRepsilon4 mutant mice showed reduced spontaneous locomotor activity in a novel environment and less sensitivity to stress induced by the elevated plus-maze, light-dark box, and forced swimming tests. These findings suggest that GluRepsilon4 mutant mice have dysfunctional NMDA receptors and altered emotional behavior probably caused by changes in monoaminergic neuronal activities in adulthood.

Fig. 1.

[³H]MK-801 binding in forebrain synaptic membranes of adult GluRε4 mutant mice. Triton X-100-treated forebrain synaptic membranes were incubated with 5 nm [³H]MK-801 at 30°C for 16 hr, in the presence or absence of 10 μm glutamate (Glu), Glu plus 10 μm glycine (Gly), or Glu plus Gly plus 1 mm spermidine (SPD). Each column represents the mean ± SEM (n = 6). *p < 0.05 and **p < 0.01 versus wild-type (+/+).

Fig. 2.

NMDA-stimulated⁴⁵Ca²⁺ uptake into forebrain synaptosomes of adult GluRε4 mutant mice. The forebrain synaptosomes were preincubated at 37°C for 10 min, in the presence or absence of 100 μm MK-801. The assay was initiated by adding prewarmed buffer containing 1 μCi/ml ⁴⁵CaCl₂for 5 min, in the presence of 100 μm NMDA, NMDA plus 10 μm glycine (Gly), or NMDA plus Gly plus 1 mm spermidine (SPD). Eachcolumn represents the mean ± SEM (n = 8 for −MK-801 group; n = 6 for +MK-801 group). *p < 0.05, **p < 0.01, and ***p < 0.001 versus wild-type (−MK-801).

Fig. 3.

Expression of NMDA receptor subunit proteins in various regions of the adult GluRε4 mutant mouse brain. The amount of each NMDA receptor subunit was determined by Western blot analysis using antibodies against GluRζ, GluRε1, or GluRε2. The synaptosomal protein samples were prepared from various regions of the adult GluRε4 mutant mouse brain. FC, Frontal cortex;HIP, hippocampus; STR, striatum;THA, thalamus. Each column represents the mean ± SEM (n = 4). *p < 0.05 versus wild-type (+/+).

Fig. 4.

Monoamine metabolism in various regions of the adult GluRε4 mutant mouse brain. The tissue contents of monoamines and their metabolites in various regions were measured by HPLC with an electrochemical detector. a, MHPG/NE; b, DOPAC/DA; c, HVA/DA; d, 5-HIAA/5-HT. Each column represents the mean ± SEM (n = 8). *p < 0.05 and **p < 0.01 versus wild-type (+/+).

Fig. 5.

Locomotor activity in a novel environment in adult GluRε4 mutant mice. Locomotor activity and the number of rearing events in a novel environment were measured every 5 min for 30 min. Each column represents the mean ± SEM (n = 10–11). An ANOVA with repeated measures revealed no difference in the time course of locomotion (F_(1,19) = 0.192; p= 0.9651) and rearing (F_(1,19) = 1.180;p = 0.3253). **p < 0.01 versus wild-type (+/+).

Fig. 6.

Emotional behavior of adult GluRε4 mutant mice.A, Elevated plus-maze test; the frequencies of entries to open and closed arms were recorded for 5 min. B, Light–dark box test; the amounts of time spent in the light and dark boxes were measured for 10 min. C, Forced swimming test; the duration of immobility was measured for 10 min. The data represent the mean ± SEM (n = 8–10). **p < 0.01 and ***p < 0.001 versus corresponding wild-type (+/+).