doi:10.1016/j.brainres.2004.09.008 
Copyright © 2004 Elsevier B.V. All rights reserved.
Research Report
Effect of vitamin E treatment on N-methyl-D-aspartate receptor at different ages in the rat brain
Beatriz Martínez Villayandrea, Miguel Angel Paniaguab, Arsenio Fernández-Lópezb, Miguel Angel Chinchetrua and Pedro Calvoa, 
, 
aDpto. Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad de León, 24007 León, Spain
bDpto. Biología Celular y Anatomía, Universidad de León, 24007 León, Spain
Accepted 8 September 2004.
Available online 13 October 2004.
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Abstract
A comparative study using membrane homogenate binding, autoradiography, and Western blot assays was carried out to determine the age-related changes in N-methyl-D-aspartate (NMDA) receptors in 4-, 12- and 24-month-old male Wistar rats, treated or not with vitamin E. Vitamin E treatment was 20 mg/kg i.p. daily for 15 days. [3H] 5-methyl-10,11-dihydro-5H-dibenzo (a,d) cycloheptan-5,10-imine maleate (MK-801) binding was significantly increased in all areas studied (cortex and hippocampus) at all ages when rats received this treatment. A Western blot study in vitamin-E-treated rats and their controls did not reveal significant differences in the amounts of NR2A, an NMDA receptor subunit widely distributed in the brain mainly in cortex and hippocampus. We conclude that the effect of vitamin E on NMDA receptors is largely age independent. Previous reports and our data have described the presence of age-dependent NMDA receptor changes. The effect of vitamin E in aging is considered to be mediated by free radical scavenging, but from our data, we conclude that this mechanism is not relevant for age-dependent NMDA receptor changes. Our results also support that age or vitamin E treatment have no relevant effects on NR2A subunit, at least until 24 months in rats.
Keywords: NMDA receptor; Aging; [3H]MK-801; Brain; Vitamin E
Neuroscience classification codes: Neurotransmitters, modulators, transporters, and receptors, Excitatory amino acid receptors: structure, function and expression
Fig. 1. (A) [3H]MK-801 binding in the cortex of 4M, 12M and 24M control and vitamin-E-treated rats. For further details, see Table 1. 4M: 4-month-old rats; 12M: 12-month-old rats; 24M: 24-month-old rats. *p<0.05 vs. control rats; +p<0.05 vs. 4-month-old control rats; 
: p<0.05 vs. 4-month-old vitamin-E-treated rats (by two-way ANOVA). (B) [3H]MK-801 binding in the hippocampus of 4M, 12M and 24M control and vitamin-E-treated rats. For further details, see Table 1. *p<0.05 vs. control rats; +p<0.05 vs. 4-month-old control rats; #p<0.05 vs. 24-month-old control rats; : p<0.05 vs. 4-month-old vitamin-E-treated rats; ○: p<0.05 vs. 24-month-old vitamin-E-treated rats (by two-way ANOVA).
Fig. 2. Autoradiograms of [3H]MK-801 binding in rat brain in 4-month-old control rats (A), 12-month-old control rats (C) and 24-month-old control rats (E), and in 4-month-old vitamin-E-treated rats (B), 12-month-old vitamin-E-treated rats (D) and 24-month-old vitamin-E-treated rats (F). For further details, see Table 2. Bar: 2 mm.
Fig. 3. Expression of NR2A subunit in cortex (A) and hippocampus (B) of 4-, 12- and 24-month-old control and vitamin-E-treated rats (lanes 1, 3, 5 and 2, 4, 6, respectively).
Table 1.
[3H]MK-801 binding values in membrane homogenates from rat brain at different ages
Values are the mean±S.E.M.
The table is a supplement for Fig. 1A and B. Values are expressed as fmol/mg protein. Membranes of different groups of rats were incubated with 5 nM [3H]MK-801 in the presence of 2 mM L-glutamate and 0.3 mM glycine as described in Materials and methods. For further details, see the Experimental Procedure. C: control rats; E: vitamin-E-treated rats (20 mg/kg i.p., once daily for 15 days).
* p<0.05 vs. control rats (by two-way ANOVA).
Table 2.
Autoradiographical densities of [3H]MK-801 binding in rat brain at different ages
Values are the mean±S.E.M.
The table is a supplement for Fig. 2. Autoradiographical studies were performed using 10 nM [3H]MK-801 in the presence of 10 μM L-glutamate and 10 μM glycine. Values are indicated in fmol/mg tissue. See Results.
Cx: cortex; Cing: cingulate; Str: striatum; Ent: entorhinal; FP: fronto parietal; Olf: olfactory; L1–L3: from the most superficial to the deepest layers where densities were measured in the indicated structures; Hp: hippocampus; DG: dentate gyrus; Lac-Mol: stratum lacunosum moleculare.
* p<0.05 vs. control rats (by two-way ANOVA).
+ p<0.05 vs. 4-month-old control rats (by two-way ANOVA).
† p<0.05 vs. 4-month-old vitamin-E-treated rats (by two-way ANOVA).
‡ p<0.05 vs. 12-month-old vitamin-E-treated rats (by two-way ANOVA).
# p<0.05 vs. 12-month-old control rats (by two-way ANOVA).
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