There were three main findings from this study: (i) there were age-related changes in NMDA receptor binding density in motor and somatosensory cortices and in GluN1 subunit expression in many rostral cortical regions and intermediate dentate gyrus, when both GHRKO and control mice were averaged together; (ii) there was a slight sparing of NMDA receptor binding density within medial prefrontal and motor cortices of aged GHRKO mice; (iii) there was a greater age-related decline in the GluN1 mRNA expression in GHRKO mice in all of the same regions that showed the sparing in NMDA receptor binding density, as compared with controls.
Three out of ten rostral cortical regions showed a significant decline in NMDA-displaceable [3
H]glutamate binding between 6 and 24 months of age in these mice, regardless of knockout-related genotype. Primary motor and somatosensory cortices appeared to be more susceptible to aging than prefrontal regions across both genotypes. An age-related decrease in NMDA binding site densities has also been demonstrated within the cortex and/or hippocampal formation of C57Bl/6 and BALB/c mice (55
), Fischer 344 (52,53,97–101, but see 102
), Long–Evans (54
), Wistar (103
), and Sprague–Dawley (104
) rats, dogs (56
), and rhesus monkeys (51
). Declines in the binding within the channel of the NMDA receptor have also been described in the frontal cortex of humans during aging (57
). The lack of age-related changes in glutamate binding within the hippocampal formation regions is consistent with a lesser effect of aging on NMDA binding sites in these regions as compared with the cerebral cortex in C57BL/6 mice (81
). The mice in the current study, from a heterogeneous strain background, thus showed similar age-related changes in NMDA receptor binding density to many other strains and species. The heterogeneity in the effects of aging on NMDA receptor binding between different brain regions seen in the present study has also been documented in C57BL/6 mice (59
There was a slight sparing of NMDA-displaceable [3
H]glutamate binding in the old GHRKO dwarf mice (9–17% better maintenance of binding density across aging), as compared with control, normal-sized mice, within the deep layers of the medial prefrontal and secondary motor cortices and in all layers of the primary motor cortex. In these regions, normal-sized aged mice showed a significant decline in binding density (21–30%), as compared with the normal-sized young mice. The decreases in binding between young and old GHRKO mice (8–14% of young GHRKO binding) did not reach significance in the same regions. It is possible that the differences in percent of age-related decline in NMDA receptor binding would have been greater in magnitude if the GHRKO mice had been compared with wild-type mice, rather than heterozygotes. Caloric restriction also produced a similar pattern with respect to significant changes in horizontal sections of the rostral cerebral cortex; ad libitum-fed mice showed significant aging changes (13–22% reduction from young ad libitum-fed) but calorie-restricted mice did not (5–14% reduction from young ad libitum-fed; 58
). Caloric restriction in aged mice led to 3–15% better maintenance of NMDA receptor binding densities within rostral cortex across aging, as compared with the old ad libitum-fed mice (58
). Thus, alteration of the growth hormone signaling pathways led to a similar pattern to caloric restriction within rostral cortical regions with respect to improvement in glutamate binding to NMDA receptors in aged animals, both in the degree of reduction of the age-related change, as compared with old controls, and the percent decrease from binding densities found in the young of the same genotype.
Seven out of 10 of the cortical regions and both blades of the intermediate dentate gyrus showed a significant decline in mRNA expression of the GluN1 subunit of the NMDA receptor during aging when both GHRKO and control mice were considered together. The only regions that overlapped with the NMDA receptor binding changes across genotype were all layers of the primary motor cortex. Antagonism of NMDA receptors with MK801 can produce motor ataxias (105
), so it is possible that receptor declines in motor cortex may contribute to movement problems in aged individuals (106
). Age-related declines in GluN1 mRNA have also been seen within frontal cortex of C57BL/6 mice (55
), but other studies with this same strain showed no aging changes in the mRNA for this subunit (73
). Fischer 344 and Fischer 344 × Brown Norway F1 rats show declines in protein expression of the GluN1 subunit in the hippocampal formation during aging (67
). Wistar and Long–Evans rats, however, show no age-related change in the expression of the GluN1 subunit in hippocampal formation (76
). A decrease in the protein expression of the GluN1 subunit protein is also observed in the distal dendrites of the dentate granule cells in aged macaque monkeys, as compared with young adults (72
). The mice in the current study, with a heterogeneous strain background, showed a strong effect of the aging process on mRNA expression of the GluN1 subunit of the NMDA receptor.
More significant effects of aging were seen in the intermediate dentate gyrus, as compared to the dorsal, regardless of knockout genotype. This pattern is also seen in the hippocampal formation of C57BL/6 mice with NMDA receptor binding and GluN2B subunit mRNA (74
). Rat studies also show no significant effect of aging on NMDA receptor binding densities in the dorsal hippocampal formation (53
). It is not known why NMDA receptors in the intermediate hippocampal formation should be more susceptible to aging than the dorsal. It is also not clear exactly what the functional consequences of this change are. Declines in NMDA receptor binding in the intermediate hippocampus of C57BL/6 mice show a significant correlation to spatial memory deficits during aging (58
). NMDA receptors are important for the acquisition of spatial memories (8
), but the dorsal hippocampal formation is sufficient for acquiring spatial learning (110
). The intermediate hippocampal formation, along with the dorsal, is involved in retrieval of spatial memories (111
), but NMDA receptors are not involved in retrieval, at least not in young animals (8
). The role of NMDA receptors within the aged intermediate hippocampus in memory remains to be elucidated.
The deep layers of medial prefrontal, secondary motor, and somatosensory cortices and the whole thickness of the primary motor cortex exhibited a significant decrease in GluN1 mRNA in the GHRKO mice between young and old, but the changes were not significant in the control mice. Four of these five regions are the same regions that showed more significant age-related changes in NMDA receptor binding density in control mice than the GHRKO mice; the opposite pattern. The strain of Wistar-Kyoto rats show markedly higher binding of [3
H]MK801 to NMDA receptors than the Wistar strain of rats (112
) and C57BL/6 mice show higher surface expression of another NMDA receptor subunit, GluN2B, than the 129S6/SvEv strain (113
). Thus, it is possible that the heterogeneous strain background, combined with a small N, may have produced variability between individuals with respect to the NMDA receptor that interfered with detecting significant differences in both binding and mRNA within the same regions within the present study. The fact that there appear to be differences in the magnitude of the change between binding and mRNA densities within the knockout and control groups, however, suggests that variability was not the major issue. Although C57Bl/6 and FVB/N mice do not show differences in basal expression of glutamate receptor subunits, they do show strain differences in the change in receptor expression following manipulation with kainate injections (114
). It is possible that the manipulation of the GH receptor on a heterogeneous strain background may have differentially affected some of the knockouts with respect to the NMDA receptor.
There still remains the fact that within each genotype group, the age-related changes in NMDA receptor binding differed in magnitude from the changes in GluN1 mRNA (i.e., in the knockouts, when GluN1 mRNA decreased significantly with increased age, the NMDA-displaceable glutamate binding was not significantly different across ages and vice versa in the controls). Several possibilities might explain this opposing relationship: (i) the decline in GluN1 subunit expression could preferentially affect certain splice variants that could lead to an increase in agonist affinity for the receptor (115
) in GHRKO mice, (ii) mRNA expression could be inversely related to the protein expression of the GluN1 subunit, or (iii) there may be other associated changes in the GluN2 family of NMDA receptor subunits that could have influenced either the number or the affinity of glutamate binding sites.
Growth hormone treatments in normal Sprague–Dawley rats lead to declines in GluN1 subunit mRNA in young rats and increases in middle-aged rats (116
). In Brown Norway rats, growth hormone treatment enhances microvascular density and spatial working and reference memory (117
). Aged Fischer 344 × Brown Norway F1 rats, however, show no influence of insulin-like growth factor-1 on GluN1 subunit expression, despite improvements in GluN2A and GluN2B subunits (78
). These results suggest that growth hormone and its downstream factors can be beneficial during aging in rodents, but do not consistently act on the GluN1 subunit. The use of GH in aged humans is not currently recommended by the Growth Hormone Research Society, but they do encourage further study in model systems, such as the GHRKO mouse (118
The enhanced decline in the GluN1 subunit mRNA within some regions of the rostral cerebral cortex in GHRKO mice compared with normal-sized control mice is the opposite of the effect of caloric restriction on this subunit mRNA. Within both medial and lateral rostral cortex, caloric restriction in middle-aged and old C57BL/6 mice resulted in an increase in GluN1 subunit mRNA as compared with middle-aged, old, and/or young ad libitum-fed mice (81
). However, it is possible that behavioral testing experience is necessary to induce the increase (81
). The mRNA for the GluN1-a splice variants was upregulated following a behavioral testing experience in the water maze (73
Rodents undergoing caloric restriction resemble the GHRKO mice in many different ways. Both have increased life spans, decreased insulin-like growth factor-1 plasma expression, and show similar pathologies (119
). In addition, caloric restriction was unable to further extend life span in the GHRKO mice, as it did in the Ames dwarf mice (120
), suggesting that there is a shared mechanism. Although caloric restriction and GHRKO mice show many phenotypic similarities, there are many differences being discovered in the gene expression profiles that are altered by caloric restriction in control mice versus GHRKO mice (120
). There are also differences in stress tolerance in fibroblasts and Akt phosphorylation in cardiac muscle cells between the diet intervention and the knockout mice (126
). One major shared cellular mechanism between GHRKO and normal calorie-restricted mice appears to be increased insulin sensitivity (120
Insulin can enhance NMDA receptor activity (128
), at least in part via phosphorylation of the GluN2A and GluN2B subunits (128
). The similar degree of sparing of NMDA receptor binding density in GHRKO mice, as compared with calorie-restricted aged mice, suggest that altered insulin sensitivity may have had some effect on NMDA receptor expression. The opposite effects of the two interventions on the mRNA expression of the GluN1 subunit suggest that it is unlikely that the effects of increased insulin sensitivity alone account for the GluN1 subunit expression enhancements of caloric restriction. As mentioned above, it may require an interaction between increased insulin sensitivity and behavioral experience to enhance GluN1 subunit expression.
The life span of C57BL/6 mice is extended by caloric restriction to a greater extent than DBA/2 mice (131
). These two strains also show differences in metabolic and oxidative stress markers following caloric restriction (133
). Although the DBA/2 strain was not part of the background of the heterogeneous strain mice, the differences seen in GluN1 subunit expression between caloric restriction in C57BL/6 and ad libitum-fed GHRKO mice could also be due to strain differences.
Aged GHRKO mice show enhanced spatial and inhibitory avoidance memory and better locomotor function, as compared with normal-sized mice (24
). The small effect of the GHRKO genotype on NMDA-displaceable glutamate binding might suggest that the memory and motor improvements are unlikely to be related to NMDA receptors. However, the higher NMDA agonist binding that was seen in old, calorically restricted C57BL/6 mice showed a significant association with better reference memory with a similar degree of sparing of receptor binding density (58
). It remains to be seen if the changes seen here in the NMDA receptor are associated with the memory and/or locomotor improvements in aged GHRKO mice. It is also possible that the reduction in GluN1 mRNA in the GHRKO mice might enhance memory performance in old mice. There is increasing evidence of a negative relationship between this subunit and memory within groups of old mice (59
). Additional examples, involving antioxidant therapy, high blood pressure, and obesity, also suggest that not all changes that occur during aging are detrimental to health and longevity (135
). The best learners among old Fischer 344 rats appear to be able to upregulate some transmitter or signaling molecules in the face of age-related declines in others (136
). Therefore, it is possible that the improved function in the aged GHRKO mice might be due to an enhanced ability to compensate for other declines.
In conclusion, there was a slight sparing of NMDA receptor density in GHRKO mice, similar to that seen with caloric restriction in C57BL/6 mice. This suggests that the enhanced memory and locomotion of the old GHRKO mice may be due, at least in part, to an enhancement of NMDA receptors. However, in contrast to caloric restriction, there was a significant decline in mRNA expression of the major subunit, GluN1. These results suggest that alterations in growth hormone signaling pathways may enhance the expression of NMDA receptors in old age, but not through the upregulation of mRNA expression for the GluN1 subunit.