Research reportMemory in aged mice is rescued by enhanced expression of the GluN2B subunit of the NMDA receptor
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)
Aging and the physiology of spatial memory
Neurobiology of Aging
(1988)- et al.
In vitro autoradiography of ionotropic glutamate receptors in hippocampus and striatum of aged Long-Evans rats: relationship to spatial learning
Neuroscience
(1996) - et al.
The effects of aging on N-methyl-d-aspartate receptor subunits in the synaptic membrane and relationships to long-term spatial memory
Neuroscience
(2009) - et al.
The neuroscience of remote spatial memory: a tale of two cities
Neuroscience
(2007) - et al.
A behavioural analysis of rats with damage to the medial prefrontal cortex using the Morris water maze: evidence for behavioural flexibility, but not for impaired spatial navigation
Brain Research
(1994) - et al.
The involvement of the orbitofrontal cortex in learning under changing task contingencies
Neurobiology of Learning and Memory
(2005) - et al.
Hippocampal connections and spatial discrimination
Brain Research
(1978) - et al.
The effects of hippocampal lesions upon spatial and non-spatial tests of working memory
Behavioural Brain Research
(1986) - et al.
Absence of sparing of spatial navigation, skilled forelimb and tongue use and limb posture in the rat after neonatal dopamine depletion
Physiology and Behavior
(1987) - et al.
Involvement of the posteroventral caudate-putamen in memory consolidation in the Morris water maze
Neurobiology of Learning and Memory
(1999)
Dissociation of hippocampal and striatal contributions to spatial navigation in the water maze
Neurobiology of Learning and Memory
The messenger RNAs for the N-methyl-d-aspartate receptor subunits show region-specific expression of different subunit composition in the human brain
Neuroscience
A novel NMDA antagonist, MK-801, impairs performance in a hippocampal-dependent spatial learning task
Pharmacology, Biochemistry and Behavior
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
Long-term potentiation in the hippocampus involves activation in N-methyl-d-aspartate receptors
Brain Research
The essential role of hippocampal CA1 NMDA receptor-dependent synaptic plasticity in spatial memory
Cell
MK-801 and AP5 impair acquisition, but not retention, of the Morris milk maze
Pharmacology, Biochemistry and Behavior
Effects of ketamine on tunnel maze and water maze performance in the rat
Behavioral and Neural Biology
Hippocampal long-term depression mediates spatial reversal learning in the Morris water maze
Neuropharmacology
Aging of glutamate receptors: correlations between binding and spatial memory performance in mice
Mechanisms of Ageing and Development
Influence of diet restriction on NMDA receptor subunits and learning during aging
Neurobiology of Aging
Differential effects of aging on NMDA receptors in the intermediate versus the dorsal hippocampus
Neurobiology of Aging
Development and aging of N-methyl-d-aspartate receptor expression in the prefrontal/frontal cortex of mice
Neuroscience
Loss of NMDA, but not GABA-A, binding in the brains of aged rats and monkeys
Neurobiology of Aging
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
Differential effects of aging on binding sites of the activated NMDA receptor complex in mice
Mechanisms of Ageing and Development
Influence of dietary restriction on ionotropic glutamate receptors during aging in C57B1 mice
Mechanisms of Ageing and Development
Effects of aging on NMDA and MK801 binding sites in mice
Brain Research
Age-related changes in excitatory amino acid receptors in two mouse strains
Neurobiology of Aging
[3H]MK-801 binding to the NMDA receptor complex, and its modulation in human frontal cortex during development and aging
Brain Research
Animal models of normal aging: relationship between cognitive decline and markers in hippocampal circuitry
Behavioural Brain Research
Spatial learning and motor deficits in aged rats
Neurobiology of Aging
Acetyl-l-carnitine: behavioral, electrophysiological, and neurochemical effects
Neurobiology of Aging
Changes in expression of splice cassettes of NMDA receptor GluN1 subunits within the frontal lobe and memory in mice during aging
Behavioural Brain Research
A nomenclature for ligand-gated ion channels
Neuropharmacology
Deficits in the expression of the NR2B subunit in the hippocampus of aged Fisher 344 rats
Neurobiology of Aging
Changes in the expression of the NR2B subunit during aging in macaque monkeys
Neurobiology of Aging
Age-related changes in the protein expression of subunits of the NMDA receptor
Brain Research. Molecular Brain Research
Electrophysiological analysis of NMDA receptor subunit changes in the aging mouse cortex
Mechanisms of Ageing and Development
Acute dissociation for analyses of NMDA receptor function in cortical neurons during aging
Journal of Neuroscience Methods
IRES-dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector
Molecular Therapy
Adenovirus gene transfer causes inflammation in the brain
Neuroscience
Reducing expression of GluN1(0XX) subunit splice variants of the NMDA receptor interferes with spatial reference memory
Behavioural Brain Research
Muscleblind-like 2-mediated alternative splicing in the developing brain and dysregulation in myotonic dystrophy
Neuron
Viral vector-mediated delivery of estrogen receptor-alpha to the hippocampus improves spatial learning in estrogen receptor-alpha knockout mice
Molecular Therapy
Susceptibility to induction of long-term depression is associated with impaired memory in aged Fischer 344 rats
Neurobiology of Learning and Memory
The nervous system: functional changes
Age-related changes in cognitive function
The effects of age: normal variation and its relation to disease
Timing of onset of cognitive decline: results from Whitehall II prospective cohort study
British Medical Journal
Cited by (63)
Investigating the effects of age and prior military service on fluid and crystallized cognitive functions using virtual morris water maze (vMWM) and NIH Toolbox tasks
2024, Archives of Gerontology and GeriatricsLong-lasting spatial memory deficits and impaired hippocampal plasticity following unilateral vestibular loss
2023, Progress in NeurobiologyAge-related memory decline, dysfunction of the hippocampus and therapeutic opportunities
2020, Progress in Neuro-Psychopharmacology and Biological PsychiatryAlpha-synuclein differentially reduces surface expression of N-methyl-D-aspartate receptors in the aging human brain
2020, Neurobiology of AgingCitation Excerpt :Thus, any alterations to GluN1 may affect the function of a complete NMDAR. In aging animal models and humans with age-related neurodegenerative diseases, both the function and expression of GluN1 were found to be disturbed in vulnerable brain regions, such as the hippocampus and striatum (Brim et al., 2013; Tong et al., 2013; Tsamis et al., 2013; Wang et al., 2017). Therefore, determining the mechanism underlying these alterations of GluN1 in aging and age-related neurodegenerative conditions may help elucidate how these conditions lead to the cognitive and motor abnormalities mediated by NMDARs.