Research report
The effects of aging on different C-terminal splice forms of the ζ1(NR1) subunit of the N-methyl-d-aspartate receptor in mice

https://doi.org/10.1016/j.molbrainres.2004.12.012Get rights and content

Abstract

Changes in NMDA receptors in the prefrontal/frontal cortex and hippocampus of C57BL/6 mice during aging show a relationship to declines in spatial learning. The present study was designed to determine whether aging influences the mRNA expression of different splice forms of the ζ1 subunit of the NMDA receptor. We examined the mRNA of 4 C-terminal splice forms with the use of in situ hybridization. The ζ1-1 splice form (+C1 and +C2 cassettes) overall showed a maintenance of mRNA density from 3 to 10 months of age, followed by a significant decline by 26 months of age. In contrast, the mRNA for the ζ1-3 splice form [+C1 and +C2′(−C2)] showed significant declines between 3- and 10-month-old mice. These declines were maintained in the old mice. The ζ1-2 splice form (−C1 and +C2) showed a near-significant decrease in expression during aging across all brain regions. The ζ1-4 subunit mRNA [−C1 and +C2′ (−C2)] showed no significant changes with increased age. These results indicate that there is a differential effect of aging on different splice variants of the ζ1 subunit of the NMDA receptor and those that are affected show a different temporal pattern of aging. This heterogeneity has implications for producing imbalances in the modulation of the remaining receptors.

Introduction

Memory is one of the earliest of the cognitive functions to show declines during the aging process [2]. Memory deficits associated with aging are seen in humans and non-human primates (see reviews [18], [19]), dogs [23], and rodents [6], [17], [34], [37], [51], [61]. The N-methyl-d-aspartate (NMDA) receptor, a subtype of glutamate receptors, is particularly important in learning and memory functions [10], [35]. NMDA antagonists inhibit memory performance [3], [24], [41], [44] and block the initiation of long-term potentiation in the hippocampus [8], [22], [44] and neocortex [4]. These studies suggest that detrimental changes to the NMDA receptor during the aging process should impact negatively on memory functions.

Aging animals do exhibit declines in NMDA receptor binding densities and functions, including memory-related functions. NMDA-stimulated release of transmitters is decreased with increasing age [21], [49]. Long-term potentiation is also altered in aged rodents [7], [11]. Age-related declines in binding of glutamate and [(±)-2-carboxypiperazin-4-yl] propyl-1-phosphonic acid (CPP) to NMDA binding sites have been reported in mice, rats, and monkeys [28], [36], [47], [54], [57]. Humans also exhibit declines with increased age in binding of [3H]MK801 to the NMDA receptor complex in the frontal cortex [48]. Changes in NMDA binding sites during aging have been correlated with poor performance in reference memory tasks, such as the Morris water maze, in both frontal cortical regions [34], [37] and in the hippocampus [34], [37], [47].

Functional subunits of the NMDA receptor complex have been cloned for rodents [26], [27], [29], [40], [42], [43], [52], [59]. There are two families of subunits identified for the NMDA complex in mice, the ζ1 (NR1 for other species) and the ε (NR2 for other species) families. The ζ1 subunit appears to be necessary and sufficient for the formation of functional channels [27], [29], [40], [42]. A decline in the expression of mRNA and protein for the ζ1 subunit have been seen in C57BL/6 mice [36], [38], but other studies on this same strain of mice show no effect of aging on the expression of the ζ1 subunit [37], [46].

There are eight different splice variants of the mRNA for the ζ1 subunit that exist in the brain [31], [60]. There is an insertion cassette (N1) near the 5′ end of the mRNA for the ζ1 subunit and two deletion cassettes (C1 and C2). A C2′ cassette is formed in the absence of the C2 cassette, because a stop codon is lost with the deletion of the C2 cassette [25], [53]. The eight splice variants exist as combinations of 2 splice forms involving the presence or absence of the N1 cassette and 4 splice forms involving the combinations of C-terminal cassettes [25], [53]. These splice variants exhibit heterogeneity with respect to agonist and antagonist affinity, zinc modulation, and regional and developmental expression patterns [25], [30], [60].

One study has shown that there is a significant decline in the protein expression of the C2 cassette in the hippocampus of aged rats [9]. However, this examination of protein cassettes was not able to differentiate between the two different C-terminal splice forms that express the C2 cassette in order to determine whether one or both of them contributed to the decline in the expression of the C2 cassette during aging. An examination of the mRNA, using oligonucleotides specifically designed by Seeburg and Laurie [30], allowed us to examine the combinations of the C-terminal cassettes, which brings us one-step closer to understanding the effects of aging on individual splice variants. This study was designed to examine the effects of aging on the 4 C-terminal splice forms with the use of oligonucleotides designed to identify ζ1-1 (+C1 and +C2 cassettes), ζ1-2 (−C1 and +C2), ζ1-3 [+C1 and +C2′(−C2)], ζ1-4 [−C1 and +C2′ (−C2)], and all ζ1 subunits (ζ1-pan). See Fig. 1 in [30] for sites of oligonucleotide targets for the splice forms. The ζ1-pan mRNA was examined in the same animals as the splice form mRNA in order to compare the age-related changes in the ζ1 C-terminal splice forms to changes in all ζ1 subunits.

Section snippets

Materials

Probe sequences for the ζ1 splice forms (40 bases) were obtained from Laurie and Seeburg [31] and were adjusted for the mouse sequence. Probe sequence for ζ1-pan (45 bases) was obtained from Watanabe and coworkers [55], [56]. Oligonucleotides were commercially prepared (Macromolecular Resources, Colorado State University, Fort Collins, CO). Sequences for the mouse were:

  • ζ1-1, TCCACCCCCGGTGCTCGTGTCTTTGGAGGACCTACGTCTC;

  • ζ1-2, TCCACCCCCGGTGCTCTGCAGGTTCTTCCTCCACACGTTC;

  • ζ1-3,

Overall brain region results

There was a significant main effect of age on ζ1-pan mRNA density over all the brain regions analyzed, F(2,20) = 4.995, P = 0.0174 (Fig. 1, Fig. 2). The 26-month-old mice had significantly less density of ζ1-pan mRNA than the 3-month-olds when all brain regions were averaged within age groups (Fig. 1, Fig. 2). There was a significant main effect of age on the density of ζ1-1 mRNA across all brain regions analyzed, F(2,21) = 3.992, P = 0.0339 (Fig. 1, Fig. 2). The 26-month-old mice had

Discussion

The major findings of this study are that there are different temporal patterns of aging for the different C-terminal splice forms of the ζ1 subunit of the NMDA receptor. The ζ1-1 splice form overall showed a maintenance of mRNA density from 3 to 10 months of age, followed by a significant decline by 26 months of age. In contrast, the mRNA for the ζ1-3 splice form showed significant declines between 3- and 10-month-old mice. These declines were maintained in the old mice. The ζ1-2 splice form

Acknowledgments

This work was supported by NIH grant AG16322 to K.R.M. and NIH Grant Number P20 RR16454 from the BRIN Program of the National Center for Research Resources.

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