Elsevier

Behavioural Brain Research

Volume 238, 1 February 2013, Pages 211-226
Behavioural Brain Research

Research report
Memory in aged mice is rescued by enhanced expression of the GluN2B subunit of the NMDA receptor

https://doi.org/10.1016/j.bbr.2012.10.026Get rights and content

Abstract

The GluN2B subunit of the N-methyl-d-aspartate (NMDA) receptor shows age-related declines in expression across the frontal cortex and hippocampus. This decline is strongly correlated to age-related memory declines. This study was designed to determine if increasing GluN2B subunit expression in the frontal lobe or hippocampus would improve memory in aged mice. Mice were injected bilaterally with either the GluN2B vector, containing cDNA specific for the GluN2B subunit and enhanced green fluorescent protein (eGFP); a control vector or vehicle. Spatial memory, cognitive flexibility, and associative memory were assessed using the Morris water maze. Aged mice, with increased GluN2B subunit expression, exhibited improved long-term spatial memory, comparable to young mice. However, memory was rescued on different days in the Morris water maze; early for hippocampal GluN2B subunit enrichment and later for the frontal lobe. A higher concentration of the GluN2B antagonist, Ro 25-6981, was required to impair long-term spatial memory in aged mice with enhanced GluN2B expression, as compared to aged controls, suggesting there was an increase in the number of GluN2B-containing NMDA receptors. In addition, hippocampal slices from aged mice with increased GluN2B subunit expression exhibited enhanced NMDA receptor-mediated excitatory post-synaptic potentials (EPSP). Treatment with Ro 25-6981 showed that a greater proportion of the NMDA receptor-mediated EPSP was due to the GluN2B subunit in these animals, as compared to aged controls. These results suggest that increasing the production of the GluN2B subunit in aged animals enhances memory and synaptic transmission. Therapies that enhance GluN2B subunit expression within the aged brain may be useful for ameliorating age-related memory declines.

Highlights

► Enhanced GluN2B expression improves memory in aged animals, similar to young. ► Enhanced GluN2B expression in the frontal cortex improves later long-term memory. ► Enhanced GluN2B expression in the hippocampus improves early long-term memory. ► Enhanced GluN2B expression increases NMDA receptor-mediated synaptic transmission.

Introduction

Aging is associated with multiple functional declines, including declines in strength, balance, motor coordination, cognitive flexibility and memory [1], [2]. One of the earliest cognitive functions to show declines with increasing age is memory; deterioration is evident by the fifth decade in humans and is associated with significant impairment in memory recall [3], [4]. Spatial memory, which is responsible for the navigation of organisms within their environment, is particularly affected by the aging process [5], [6], [7]. In particular, spatial long-term and delayed short-term memory, as well as cognitive flexibility (i.e. the ability to switch a behavioral response according to the context of a situation), decline with age [8], [9], [10], [11], [12], [13].

Specific brain regions are important for the acquisition, consolidation and retrieval of spatial memory, including the hippocampus and the prefrontal cortices within the frontal lobe [14], [15], [16], [17], [18], [19], [20]. Specifically, the medial and orbital prefrontal cortices are important for both spatial long-term and short-term memory in the radial arm and Morris water mazes [20], [21]. The frontal lobe has also been shown to be necessary for the encoding of delayed short-term memories, as well as contributing to long-term memory by maintaining the learned information [22], [23]. The medial prefrontal cortex has also been shown to be important for the retrieval of spatial information stored in the hippocampus [24]. The frontal lobe, including the orbital and medial prefrontal cortices, has also been shown to be important for cognitive flexibility [25], [26], [27], [28]. In contrast, the hippocampus is responsible for the consolidation of less stable short-term memories into more stable long-term memories, which can be stored in the frontal lobe [19], [29], [30]. In particular, spatial long-term memory has been shown to require the hippocampus in a variety of studies using hippocampal lesions [14], [31], [32], [33]. In particular, selective electrolytic lesions of the entorhinal projection to the CA1 disrupt the consolidation of long-term memory [15]. In contrast, lesions of the dentate gyrus (DG) and CA3, suggest that these regions of the hippocampus may be important for the acquisition of spatial memory [16], [17]. The caudate nucleus is also believed to be involved in the acquisition and consolidation of spatial memory in the Morris water maze [34], [35], [36]. However, the striatum and medial temporal lobe are also believed to be involved in the formation of associative memory, which does not appear to be affected by aging [37], [38].

One subtype of glutamate receptor, the N-methyl-d-aspartate (NMDA) receptor, is highly expressed throughout the frontal lobe, caudate nucleus and hippocampus [39], [40] and has been shown to be important for learning and memory [41], [42]. Specifically, NMDA receptors are especially important for spatial memory [43] and are also thought to be involved in cognitive flexibility [44]. Antagonists of the receptor block initiation of long-term potentiation (LTP), a cellular mechanism believed to underlie learning and memory, in both the hippocampus and regions of the frontal lobe [45], [46], [47], [48], [49], [50], [51]. Memory and learning, including spatial memory, are also impaired by use of NMDA receptor specific antagonists such as AP5, MK801, ketamine and Ro 25-6981 [43], [52], [53], [54], [55], [56], [57], [58], [59], [60]. In addition, correlations have been seen between NMDA-displaceable [3H]glutamate binding and NMDA subunit expression and spatial memory performance in the Morris water maze [61], [62], [9], [63], [64], [65], [66], [67].

Aging animals exhibit declines in NMDA receptor binding densities. A number of binding studies, employing [3H]glutamate or glutamate analogs, have shown that the NMDA receptors are more susceptible to the effects of aging than any other type of glutamate receptor in the ventrolateral frontal cortex, including orbital and insular cortices, and in the hippocampus of the mouse brain [68], [69], [70], [71]. Similar results were also observed in other species, including rats [11], [44], [72], canids [65], non-human primates [66] and humans [73]. Several studies have used spatial memory tasks to characterize the relationship between age-related declines in memory and NMDA receptor expression [8], [9], [10], [11], [12], [61], [62], [67], [74], [75], [76], [77], [78]. Specifically, the age-related decline in densities of NMDA receptor binding and expression of its subunits in regions of the frontal lobe and hippocampus of the rodent brain have been shown to be associated with declines in spatial memory, including spatial long-term and delayed short-term memory, during aging [8], [9], [10], [11], [12], [44], [61], [62], [67], [78].

NMDA receptors are heteromeric tetramers composed of combinations of subunits from different families of proteins, now termed GluN1 (NR1), GluN2 (NR2) and GluN3 (NR3) subunit families [79], [80]. Of the NMDA receptor subunits, the GluN2B subunit is most affected by the aging process. The GluN2B subunit mRNA has been shown to be especially vulnerable to the effects of aging in the cerebral cortex and dentate gyrus of the hippocampus [63], [65]. Significant declines in GluN2B mRNA expression have also been observed in the prefrontal cortex and caudate nucleus of aged macaques and in the hippocampus of aged rats [81], [82]. In the frontal lobes of C57BL/6 mice, it appears that the decline in mRNA during adult aging may be a continuation of the developmental decline and, therefore, may be programmed [64]. Protein expression of the GluN2B subunit also declines with age across regions of the frontal lobe and hippocampus [9], [81], [83]. However, the protein levels of the GluN2B subunit show a greater decline with age within the synaptic membrane of the frontal lobe than in the tissue as a whole [12]. Within the hippocampus, there is a significant decline in GluN2B subunit expression in the synapse with age, similar to the tissue changes [12]. Functional studies of NMDA receptors also suggest that aging results in a decrease in or loss of functional GluN2B-containing receptors. Specifically, NMDA receptors from aged animals appear to be less sensitive to ifenprodil, a GluN2B specific antagonist, and have an increased rate of deactivation (faster channel closing) [84], [85].

The GluN2B subunit has also been shown to be important for spatial memory. Its decrease, via experimental manipulation, has been shown to be sufficient to account for the degree of spatial long-term memory impairment seen in aged rodents [86]. Moreover, aging studies have shown that there is a significant correlation between decreased GluN2B subunit expression and impaired spatial long-term memory in aged animals [9]. Specifically, age-related decreases in the protein expression of the GluN2B subunit within crude synaptosomes of the frontal cortex of C57BL/6 mice show a relationship to the declines in performance in the long-term spatial memory task across age groups. However, those expressing the highest levels of the GluN2B subunit within the synaptic membrane of the hippocampus, among aged mice, are the poorest performers in the same task [12]. Previous research has shown that increasing GluN2B subunit expression throughout multiple brain regions from birth is beneficial to memory, including spatial long-term and delayed short-term memory, and remains beneficial even into middle-age (18 months) [87], [88]. These data suggest that maintaining higher levels of the GluN2B subunit during aging, than are seen in normal aged mice, could be beneficial for memory.

The present paper explored the effects of increasing the expression of the GluN2B subunit within the frontal lobe or the hippocampus of aged mice using a replication deficient adenoviral vector to deliver cDNA specific to the GluN2B subunit. The orbital cortex was targeted because the ventrolateral frontal cortex, which includes the orbital and insular cortices, exhibits consistent declines in GluN2B subunit mRNA during aging in C57BL/6 mice [65] and shows significant relationships between age-related declines in NMDA receptor binding and spatial memory [61]. In addition, the orbital cortex is centrally located within the frontal lobe, which provided the best target for enhancement throughout the lobe.

Adenoviral vectors have previously been used to effectively deliver genes to the central nervous system (CNS) [89]. An adenoviral vector was chosen because of its size (packaging capacity) [90]. Specifically, the GluN2B vector expresses two transgenes, GluN2B and eGFP, from two independent promoters. This cannot be accomplished in an adeno-associated viral vector because the packaging capacity is too small [91]. A lentivector, such as feline immunodeficiency virus (FIV), can be engineered to express two transgenes using an internal ribosomal entry site (IRES) sequence; however, the expression levels of the gene under the control of the IRES is much lower [92]. Finally, higher infectious titers can be obtained with adenoviral vectors, as compared to lentivectors [93].

The purpose of the present study was to determine whether the decline in spatial memory and cognitive flexibility observed during aging could be improved by regionally increasing the expression of the GluN2B subunit within the aged brain. This would help determine whether a therapy aimed at enhancing the number of NMDA receptors that contain the GluN2B subunit during old age would be beneficial to cognitive functions.

Section snippets

Adenoviral vectors

Custom adenoviral vectors were designed by Viraquest, Inc. (North Liberty, IA). A plasmid containing the cDNA for the GluN2B subunit, a gift from Dr. M. Mishina, University of Tokyo, was subcloned into a human replication deficient type 5 adenoviral vector with cytomegalovirus (CMV) promoters and reporter gene for enhanced green fluorescent protein (eGFP). The GluN2B vector contains the cDNA of the GluN2B subunit gene and eGFP. The control vector contains only the cDNA of eGFP. The GluN2B

Effect of the GluN2B vector in the frontal lobe on spatial and associative memory

There was an overall effect of age on cumulative proximity scores in place trials within the long-term spatial memory task, with young animals spending more time closer to the platform than aged animals (F(1,76) = 26.3, p < 0.0001; young = 6546 ± 1092 cm, aged = 9435 ± 1172 cm). There was also an age by treatment interaction (F(2,76) = 4.7, p = 0.01). For the cognitive flexibility task, where the location of the platform was moved to the opposite quadrant from that used in the long-term spatial memory task,

Discussion

This study provides compelling evidence that increasing the expression of the GluN2B subunit of the NMDA receptor within the frontal lobe, caudate nucleus and hippocampus of aged mice can improve synaptic transmission and memory. Aged mice with enhanced GluN2B expression across these brain regions exhibited superior long-term spatial memory over their aged counterparts, and exhibited memory similar to young mice, but on different days of training for the different injection regions. In the

Conclusions

In conclusion, increasing the expression of the GluN2B subunit of the NMDA receptor in the frontal lobe or hippocampus appeared to restore long-term spatial memory of aged mice back to the level of young, rescuing it in different phases of learning in a region specific manner. Moreover, aged mice with enhanced GluN2B subunit expression in the hippocampus exhibited enhanced NMDA receptor-mediated EPSP responses. This study further substantiates the important role of the GluN2B subunit in

Disclosure statement

There is potential conflict of interest. R.H. has a financial interest in Viraquest, Inc., the company that provided the viral vector.

Acknowledgements

We thank M. Mishina, University of Tokyo, for the gift of the plasmid containing the cDNA of the GluN2B subunit and V. Elias, M. Shumaker, R. Bochart, C. Lehmann, O. La Faix and R. Jensen for their technical assistance. This work was funded by NIH grant AG016322 to K.R.M. and by NIH Grants AG014979, AG037984, AG036800 and the Evelyn F. McKnight Brain Research Grant to T.C.F.

References (130)

  • B.D. Devan et al.

    Dissociation of hippocampal and striatal contributions to spatial navigation in the water maze

    Neurobiology of Learning and Memory

    (1996)
  • M. Rigby et al.

    The messenger RNAs for the N-methyl-d-aspartate receptor subunits show region-specific expression of different subunit composition in the human brain

    Neuroscience

    (1996)
  • E.R. Butelman

    A novel NMDA antagonist, MK-801, impairs performance in a hippocampal-dependent spatial learning task

    Pharmacology, Biochemistry and Behavior

    (1989)
  • M.L. Escobar et al.

    In vivo effects of intracortical administration of NMDA and metabotropic glutamate receptors antagonists on neocortical long-term potentiation and conditioned taste aversion

    Behavioural Brain Research

    (2002)
  • E.W. Harris et al.

    Long-term potentiation in the hippocampus involves activation in N-methyl-d-aspartate receptors

    Brain Research

    (1984)
  • J.Z. Tsien et al.

    The essential role of hippocampal CA1 NMDA receptor-dependent synaptic plasticity in spatial memory

    Cell

    (1996)
  • V. Heale et al.

    MK-801 and AP5 impair acquisition, but not retention, of the Morris milk maze

    Pharmacology, Biochemistry and Behavior

    (1990)
  • B. Alessandri et al.

    Effects of ketamine on tunnel maze and water maze performance in the rat

    Behavioral and Neural Biology

    (1989)
  • Z. Dong et al.

    Hippocampal long-term depression mediates spatial reversal learning in the Morris water maze

    Neuropharmacology

    (2013)
  • K.R. Magnusson

    Aging of glutamate receptors: correlations between binding and spatial memory performance in mice

    Mechanisms of Ageing and Development

    (1998)
  • K.R. Magnusson

    Influence of diet restriction on NMDA receptor subunits and learning during aging

    Neurobiology of Aging

    (2001)
  • K.R. Magnusson et al.

    Differential effects of aging on NMDA receptors in the intermediate versus the dorsal hippocampus

    Neurobiology of Aging

    (2006)
  • T. Ontl et al.

    Development and aging of N-methyl-d-aspartate receptor expression in the prefrontal/frontal cortex of mice

    Neuroscience

    (2004)
  • G.L. Wenk et al.

    Loss of NMDA, but not GABA-A, binding in the brains of aged rats and monkeys

    Neurobiology of Aging

    (1991)
  • S.R. Das et al.

    Relationship between mRNA expression of splice forms of the zeta1 subunit of the N-methyl-d-aspartate receptor and spatial memory in aged mice

    Brain Research

    (2008)
  • K.R. Magnusson

    Differential effects of aging on binding sites of the activated NMDA receptor complex in mice

    Mechanisms of Ageing and Development

    (1995)
  • K.R. Magnusson

    Influence of dietary restriction on ionotropic glutamate receptors during aging in C57B1 mice

    Mechanisms of Ageing and Development

    (1997)
  • K.R. Magnusson et al.

    Effects of aging on NMDA and MK801 binding sites in mice

    Brain Research

    (1993)
  • K.R. Magnusson et al.

    Age-related changes in excitatory amino acid receptors in two mouse strains

    Neurobiology of Aging

    (1993)
  • M.A. Piggott et al.

    [3H]MK-801 binding to the NMDA receptor complex, and its modulation in human frontal cortex during development and aging

    Brain Research

    (1992)
  • M. Gallagher et al.

    Animal models of normal aging: relationship between cognitive decline and markers in hippocampal circuitry

    Behavioural Brain Research

    (1993)
  • F.H. Gage et al.

    Spatial learning and motor deficits in aged rats

    Neurobiology of Aging

    (1984)
  • S. Davis et al.

    Acetyl-l-carnitine: behavioral, electrophysiological, and neurochemical effects

    Neurobiology of Aging

    (1993)
  • S.R. Das et al.

    Changes in expression of splice cassettes of NMDA receptor GluN1 subunits within the frontal lobe and memory in mice during aging

    Behavioural Brain Research

    (2011)
  • G.L. Collingridge et al.

    A nomenclature for ligand-gated ion channels

    Neuropharmacology

    (2009)
  • D.A. Clayton et al.

    Deficits in the expression of the NR2B subunit in the hippocampus of aged Fisher 344 rats

    Neurobiology of Aging

    (2001)
  • L. Bai et al.

    Changes in the expression of the NR2B subunit during aging in macaque monkeys

    Neurobiology of Aging

    (2004)
  • K.R. Magnusson et al.

    Age-related changes in the protein expression of subunits of the NMDA receptor

    Brain Research. Molecular Brain Research

    (2002)
  • M.C. Kuehl-Kovarik et al.

    Electrophysiological analysis of NMDA receptor subunit changes in the aging mouse cortex

    Mechanisms of Ageing and Development

    (2000)
  • M.C. Kuehl-Kovarik et al.

    Acute dissociation for analyses of NMDA receptor function in cortical neurons during aging

    Journal of Neuroscience Methods

    (2003)
  • H. Mizuguchi et al.

    IRES-dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector

    Molecular Therapy

    (2000)
  • A.P. Byrnes et al.

    Adenovirus gene transfer causes inflammation in the brain

    Neuroscience

    (1995)
  • S.R. Das et al.

    Reducing expression of GluN1(0XX) subunit splice variants of the NMDA receptor interferes with spatial reference memory

    Behavioural Brain Research

    (2012)
  • K. Charizanis et al.

    Muscleblind-like 2-mediated alternative splicing in the developing brain and dysregulation in myotonic dystrophy

    Neuron

    (2012)
  • T.C. Foster et al.

    Viral vector-mediated delivery of estrogen receptor-alpha to the hippocampus improves spatial learning in estrogen receptor-alpha knockout mice

    Molecular Therapy

    (2008)
  • T.C. Foster et al.

    Susceptibility to induction of long-term depression is associated with impaired memory in aged Fischer 344 rats

    Neurobiology of Learning and Memory

    (2007)
  • P.S. Timiras

    The nervous system: functional changes

  • M.S. Albert

    Age-related changes in cognitive function

  • M.S. Albert et al.

    The effects of age: normal variation and its relation to disease

  • A. Singh-Manoux et al.

    Timing of onset of cognitive decline: results from Whitehall II prospective cohort study

    British Medical Journal

    (2012)
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