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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 55:B530-B532 (2000)
© 2000 The Gerontological Society of America

Preservation of N-Methyl-D-Aspartate Receptor Binding Sites With Age in Rat Neocortex

Alan M. Palmera

a Departments of Psychiatry and Pharmacology, University of Pittsburgh School of Medicine, Pennsylvania

Alan M. Palmer, Vernalis Research Limited, 613 Reading Road, Winnersh, Wokingham, RG41 5UA, UK E-mail: a.palmer{at}vernalis.com.

Decision Editor: Jay Roberts, PhD


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
This study used [3H]dizocilpine ([3H]MK-801) binding to examine glycine, polyamine, and zinc subsites of the N-methyl-D-aspartate (NMDA) receptor in well-washed membranes derived from the neocortex of Fischer 344/Norwegian brown rats aged 3, 12, 24 and 37 months. [3H]dizocilpine binding in the presence of 100 µM glutamate was enhanced by the addition of 30 µM glycine. Binding in the presence of both glutamate and glutamate plus glycine were unaffected by age. The competitive polyamine site antagonist arcaine inhibited [3H]dizocilpine binding in a dose-dependent fashion and 50 µM spermidine caused a rightward shift in this dose response curve. IC50 values derived from these plots were not significantly affected by age. Similarly, zinc inhibited binding in a dose-dependent fashion and was also unaffected by age. These data indicate that the NMDA receptor is spared in aging.

HUMAN aging is associated with a mild slowing of both reaction time and motor speed, as well as a decline of complex cognitive skills (1). There is also an increased risk of ischemic stroke and neurodegenerative disease with advancing age (2). Excitatory amino acid (EAA) neurons and receptors, particularly the N-methyl-D-aspartate (NMDA) receptor, have been implicated in cognitive function, ischemic stroke, and neurodegenerative disease (3). Assessment of EAA neurons with aging has, because of difficulty assessing EAA neurons in postmortem human brain, largely relied on examination of experimental animals (2) whereas the NMDA receptor has been successfully assessed in both humans and experimental animals (2).

Investigations of the NMDA receptor have been greatly facilitated by the availability of compounds such as dizocilpine [(+)-5-methyl-10,11-dihydro-5H-dibenzola[a,d]cyclohepten-5,20-imine hydrogen maleate, MK-801], a use-dependent, open channel blocker of the NMDA receptor (4)(5)(6). Thus, in well-washed brain membrane preparations, [3H]-dizocilpine binding can be used to provide an index of the open state of the NMDA-operated channel and thus serve as a tool to examine the capacity of the receptor to respond to modulatory influences. The activity of the NMDA-receptor channel is regulated by a number of subsites, including a transmitter binding site that binds L-glutamate or L-aspartate (7), a co-agonist site that binds glycine (5)(6)(8), a voltage-dependent magnesium binding site (9)(10), an inhibitory divalent cation modulatory site that binds zinc (11)(12), and a positive modulatory site that binds polyamines such as spermine and spermidine (6)(13). [3H]-dizocilpine binding has been shown to be increased by glutamate, glycine, and polyamines and decreased by Zn2+ (5)(6)(13)(14). This study uses [3H]-dizocilpine binding to investigate the integrity of the co-agonist site of the NMDA receptor, in conjunction with polyamine and zinc modulatory sites, in the long-lived Fischer 344/Norwegian brown strain of rat aged 3, 12, 24, and 37 months. An assessment of excitatory amino acid nerve terminals in these animals has previously been published (15).


   Methods
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 Abstract
 Methods
Results
 Discussion
 References
 
Male hybrid Fischer 344/Norwegian Brown rats aged 3, 12, 24, and 37 months were obtained from the National Institute on Aging colonies (Bethesda, MD) and were used within 2 weeks of arrival. Animals were maintained on a 12-hour light-dark cycle with free access to food and water. Following decapitation, the neocortex was dissected free of white matter on an ice-chilled inverted petri dish and then frozen (-80°C). Membranes were prepared and assays performed, as described previously (16). Briefly, using well-washed membranes, binding assays were performed in a 0.4 ml final volume of 5 mM Tris-acetate (pH 7.4) for 1 hour at 25°C. Assays used 5 nM [3H]dizocilpine and drugs, dissolved in Tris-acetate buffer, as appropriate. Nonspecific binding was defined by 100 µM dizocilpine and represented 10% of total binding. Curves monitoring the effects of arcaine were performed in the presence of both 100 µM glutamate and 30 µM glycine to preclude actions at glutamate and glycine sites and to reduce the influence of variable amounts of amino acids in the tissue samples. It is unlikely that these conditions measure [3H]dizocilpine binding at equilibrium. However, by monitoring nonequilibrium conditions it is possible to detect the agonist actions of glycine and polyamines as well as the antagonist actions of the competitive polyamine site antagonist arcaine and the divalent cation zinc (5)(6)(17). Glycine enhancement of binding was determined as fold increase according to the following equation: fold increase = (maximal binding + basal binding)/basal binding, where maximal binding is binding in the presence of 100 µM glutamate and 30 µM glycine and basal binding is binding in the presence of 100 µM glutamate. Inhibition of binding was quantified by determining values from Hill plots using Accufit-competition software (Lundon Software, Chagrin Falls, OH). Protein concentrations were determined as described previously (16). The effect of age in animals aged 3, 12, 24, and 37 months was assessed by analysis of variance (ANOVA) (css:statistica, StatSoft, Tulsa, OK). Data are presented as mean ± SEM. Probabilities of p < .05 were considered significant.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
[3H]dizocilpine binding was assessed in rats aged 3, 12, 24, and 37 months and the results shown in Table 1 . [3H]dizocilpine binding in the presence of 100 µM glutamate was enhanced by the addition of 30 µM glycine as described previously (5)(6)(16). Binding in the presence of both glutamate and glutamate plus glycine was unaffected by age. The competitive polyamine site antagonist arcaine inhibited [3H]dizocilpine binding in a dose-dependent fashion, and 50 µM spermidine caused a rightward shift in this dose response curve, as shown previously (16)(17) (data not shown). IC50 values derived from these plots were not significantly affected by age. Zinc inhibited binding in a dose-dependent fashion, as shown previously (14), but this inhibition was unaffected by age. The absence of a statistically significant effect of age is supported by the absence of any clear trends when mean values are compared across the four age groups.


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  		Table 1. Preservation of Glycine, Polyamine, and Zinc Subsites of the NMDA Receptor in the Neocortex of Aging Rats

 

   Discussion
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 Abstract
 Methods
 Results
 Discussion
References
 
Using [3H]dizocilpine binding, this study has shown that there was no effect of aging on either the glycine co-agonist site or the polyamine and zinc modulatory sites of the NMDA receptor in neocortex of Fischer 344/Norwegian Brown rats aged 3, 12, 24 and 37 months. Binding in the presence of 100 µM glutamate alone was also unaffected by age. Together, these data suggest a preservation of the NMDA receptor with aging. This is in agreement with the study of Shimada and colleagues (18), who showed no change in [3H]dizocilpine binding in the frontal cortex of Sprague-Dawley rats aged 2, 8, and 25 months. However, Tamaru and colleagues (19) found diminished [3H]dizocilpine binding (in the presence of both glutamate plus glycine and glutamate plus glycine plus spermidine) in neocortex of Fischer 344 rats aged 7 and 29 months when compared with animals aged 3 months. These changes were more pronounced in the presence of spermidine than in the absence of this polyamine. The actions of polyamines at the NMDA receptor are complex and consist of both stimulatory and inhibitory components (6)(13)(17). It is therefore possible that this confounded the study of Tamaru and colleagues (19). Because arcaine appears to inhibit the stimulatory actions of polyamines (which predominate at lower concentrations) without affecting the inhibitory actions (17), its use in the present study probably provides a more reliable measure of the polyamine site of the NMDA receptor and is consistent with competitive antagonism of the increase in binding produced by spermidine. Age-associated reductions in [3H]CPP and [3H]glycine binding to the NMDA receptor were reported by Kito and colleagues (20), but because these changes reflected differences from animals aged 2 months of age and the process of brain maturation may not be complete until 12 months (21), it seems likely that their results reflect changes occurring as a result of brain maturation rather than senescence. Similarly, the autoradiographic study of Mitchell and Anderson (22) showed reduced [3H]dizocipline binding in the inner frontal cortex and the entorhinal cortex of animals aged 12 and 24 months when compared with animals aged 6 months. The possibility of such false positive data has been minimized in the present study by examining 3 groups of animals aged 12 months or older. It is also possible that the discrepancy between the autoradiographic study of Mitchell and Anderson and the present study of well-washed cortical membranes is attributable to methodological differences, or it may be a consequence of age-related changes occurring only in discrete regions of cortex and therefore not apparent when the whole neocortex is assessed.

In conclusion, like EAA nerve terminals ((15); which were assessed in the same animals), the glycine, polyamine and zinc subsites of the NMDA receptor in rat neocortex are spared in aging.


   Acknowledgments
 
This study was supported by National Institutes of Health Grant AG 08974-01. The author thanks M. Burns and P. Robichaud for their excellent technical support.

Received November 2, 1999

Accepted March 27, 2000


   References
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 Abstract
 Methods
 Results
 Discussion
 References
 

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