Research ReportRelationship between mRNA expression of splice forms of the ζ1 subunit of the N-methyl-d-aspartate receptor and spatial memory in aged mice
Introduction
Age associated memory decline is a phenomenon that begins early in adulthood and overlaps with other aspects of cognitive aging (Salthouse, 2003). This decline in memory is so extensive that approximately 40% of people aged 65years of age or more can be diagnosed with some sort of age-related memory impairment (Larrabee and Crook, 1994). Age associated memory decline is usually modest compared to disorders associated with dementia and lacks the pathology observed in such disorders (Crook et al., 1990), but still represents a problem for quality of life and independent living in elderly individuals. Spatial memory is one type of memory that is affected by increasing age and can be studied in animals such as rodents and primates who experience age associated memory impairments (Barnes, 1988, Gage et al., 1984, Rapp et al., 1987).
There is evidence that brain regions such as the prefrontal cortex and hippocampal formation are associated with spatial memory functions (Gallagher et al., 2003, Greenwood, 2000, Tisserand and Jolles, 2003). All of these structures show greater decline in their volume in individuals with declining memory capabilities than the individuals with stable memory over time (Persson et al., 2006). This decline in prefrontal cortex volume has been speculated to alter the functional connectivity of cortical circuits contributing to cognitive aging (O'Donnell et al., 1999). A type of glutamate receptor known as the N-methyl-d-aspartate (NMDA) receptor is abundant in these areas of the brain (Bockers et al., 1994, Scherzer et al., 1998). These receptors are important for long term potentiation, one proposed mechanism for the formation of memory by strengthening of synapses (Cotman et al., 1989, Lynch, 1998). Inhibition of this receptor by antagonists results in impaired learning and memory abilities in rodents (Alessandri et al., 1989, Mondadori et al., 1989, Morris et al., 1986). Among the glutamate receptors, NMDA receptors are more susceptible to effects of aging (Magnusson, 1997a, Magnusson, 1997b). It has been observed that NMDA receptors experience changes in gating behavior, magnesium block and response to transmitter with increasing age in rodents and primates (see review Magnusson, 1998b). In autoradiographic experiments [3H] glutamate binding to the receptor decreases with increased age, suggesting a reduction in receptor activity with increased age (Kito et al., 1990).
The NMDA receptor is composed of four or five protein subunits from three different families of proteins, the ζ1 (NR1), ɛ (NR2) and NR3 families in the rodents. Four members in the ɛ family, one member in the ζ1, with eight splice variants, and two members in the NR3 family have been identified and cloned (Eriksson et al., 2002, Ikeda et al., 1992, Ishii et al., 1993, Kutsuwada et al., 1992, Matsuda et al., 2002, Meguro et al., 1992, Yamazaki et al., 1992). Expression studies in Xenopus oocytes indicate that the ζ1 subunit is sufficient for a functional channel (Ishii et al., 1993, Kutsuwada et al., 1992, Meguro et al., 1992, Yamazaki et al., 1992).
The ζ1 subunit contains three splicing sites, one in the N-terminal (N1 cassette) and the other two in the C-terminal (C1 and C2 cassettes) region (Anantharam et al., 1992, Durand et al., 1992, Nakanishi et al., 1992, Sugihara et al., 1992, Yamazaki et al., 1992). Since the C2 cassette contains the stop codon, in its absence, an additional sequence becomes a part of the protein and is known as the C2′ cassette (Zukin and Bennett, 1995). Depending on whether the N1, C1 or C2 cassettes are present or absent, they make eight different splice variants. The splice variants of NR1 (ζ1) present in the receptor complex determine a number of properties of the ion channel, such as affinity to agonist and antagonists, zinc modulation and spatio-temporal expression in the brain (Dingledine et al., 1999, Laurie and Seeburg, 1994, Prybylowski and Wolfe, 2000, Zhong et al., 1995). Due to these properties, differential effects of aging on the different splice variants could lead to important changes in the physiology and pharmacology of the NMDA receptor in the aged brain.
There is evidence that the ζ1 subunit of the NMDA receptor shows declines in mRNA and protein expression during the aging process in C57BL/6 mice (Magnusson, 2000, Magnusson et al., 2002), but some studies in the same strain of mice show no change in ζ1 during aging (Magnusson, 2001). One difference between these studies was the addition of a behavioral experience in the later study (Magnusson, 2001). The influence that behavioral testing may have on the ζ1 subunit needs to be further explored. Calorie restriction also leads to an upregulation of ζ1 subunits in middle-aged and aged mice (Magnusson, 2001). This suggests that the expression of the ζ1 subunit during aging is variable and potentially susceptible to intervention.
It has been suggested that learning experience stabilizes synaptic modification and improves NMDA receptor expression (Quinlan et al., 2004, Sun et al., 2005). Because the ζ1 subunit mRNA shows variability during aging and could possibly be influenced by learning experience, we postulated that the overall expression of ζ1 subunits during aging is a function of differential changes in individual splice forms and its stabilization is due to learning experience influencing expression of the individual splice forms. In the present study, one aim was to determine if expression of individual splice forms were differentially affected by aging in prefrontal and/or frontal regions. The other aim was to determine roles of individual splice forms in learning ability and whether they were affected by experience in learning tasks. The focus was on the splice forms that were found previously to be most affected by aging, ζ1-1 and ζ1-3 (Magnusson et al., 2005), and on the ζ1-a (− N1 cassette) and ζ1-b (+ N1 cassette), which had not been assessed yet in C57BL/6 mice across ages.
Section snippets
Spatial reference memory
There was a significant main effect of age on performance in the reference memory place (F(2, 30) = 7.1, p = 0.003; Fig. 1A) and probe trials (F(2, 30) = 4.5, p = 0.02; Fig. 1B). Twenty-six month old mice had significantly higher cumulative proximity scores than both 11 and 4-month olds in place trials (Fig. 1A) and higher average proximity scores than the 4-month old mice in probe trials (Fig. 1B) when averaged over all the respective trials. There was a significantly lower cumulative proximity value
Discussion
This study provides new evidence for a differential effect of behavioral experience on the expression of different N- and C-terminal splice forms of the ζ1 subunit during aging and evidence for a role of the ζ1-a splice form within orbitofrontal brain regions in working memory and associative memory performances in aged mice. The oldest mice showed significant deficits in both spatial reference and spatial working memory ability as compared to the young and middle-aged mice. There was little
Animals
A total of 72 C57BL/6 mice (National Institute on Aging, NIH) from three different age groups (four, ten and twenty-six months of age) were used for the study. They were fed ad libitum and housed in cages under 12 h light and 12 h dark cycle. The animals were divided into two behavioral groups; naïve and behaviorally-characterized, containing twelve animals from each age group. Animals in the behaviorally-characterized group were subjected to learning experience with the use of the Morris water
Acknowledgments
We sincerely acknowledge Dr. Karen Conneely for helping us with the corrections for the multiple correlation tests. This work was supported by NIH grant AG16322 to K.R.M. and P20RR16454-02 to BRIN.
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