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. 2006 Apr 12;26(15):3933-41.
doi: 10.1523/JNEUROSCI.5566-05.2006.

Aging-dependent alterations in synaptic plasticity and memory in mice that overexpress extracellular superoxide dismutase

Daoying Hu  1 , Faridis Serrano, Tim D Oury, Eric Klann
Affiliations

Aging-dependent alterations in synaptic plasticity and memory in mice that overexpress extracellular superoxide dismutase

Daoying Hu et al. J Neurosci. .

Abstract

Oxidative damage caused by reactive oxygen species (ROS) has been proposed to be critically involved in several pathological manifestations of aging, including cognitive dysfunction. ROS, including superoxide, are generally considered as neurotoxic molecules whose effects can be alleviated by antioxidant enzymes. However, ROS also are known to be necessary components of the signal transduction cascades underlying normal synaptic plasticity. Therefore, we reasoned that the role that ROS and antioxidant enzymes play in modulating neuronal processes varies over the lifespan of an animal. We examined hippocampal long-term potentiation (LTP) and memory-related behavioral performance in transgenic mice overexpressing extracellular superoxide dismutase (EC-SOD) and their wild-type littermates at different ages. We found that aged EC-SOD transgenic mice exhibited enhanced hippocampal LTP, better cerebellum-dependent motor learning, and better hippocampus-dependent spatial learning compared with their wild-type littermates. We also found that EC-SOD overexpression impaired contextual learning, but the impairment was decreased in the aged transgenic mice. At the molecular level, aged EC-SOD transgenic mice had lower superoxide levels, a decrease in protein carbonyl levels, and a decrease in p38 and extracellular signal-regulated kinase 2 phosphorylation compared with aged wild-type mice. Our findings suggest that elevated levels of superoxide contribute to aging-related impairments in hippocampal LTP and memory, and that these impairments can be alleviated by overexpression of EC-SOD. We conclude that there is an age-dependent alteration in the role of superoxide in modulating synaptic plasticity and learning and memory.

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Figures

Figure 1.
Figure 1.
Baseline synaptic transmission and presynaptic plasticity are normal in aged EC-SOD transgenic mice. The 20-month-old mice were used in the experiments. A, Plots of fEPSP slope versus fiber volley amplitudes. There was no significant difference between aged wild-type (WT) and aged EC-SOD transgenic (EC-SOD TG) mice in baseline synaptic transmission. Error bars indicate SEM for seven determinations. B, Paired-pulse facilitation in aged WT and aged EC-SOD TG mice. Shown are responses to paired pulses in which the fEPSP slope of the response to the second stimulus was expressed as a percentage of the fEPSP slope of the response to the first stimulus plotted against the interpulse interval of the paired pulses. There was no significant difference between aged WT and aged EC-SOD TG mice. Error bars indicate SEM for six determinations. C, Posttetanic potentiation in aged WT and aged EC-SOD TG mice. Shown are points representing 10 responses measured before HFS and 15 responses measured immediately after HFS in the presence of the NMDA receptor antagonist APV (100 μm). There was no significant difference between aged WT and aged EC-SOD TG mice by two-way ANOVA, followed by Bonferroni's post hoc tests. Error bars indicate SEM for five determinations for WT mice and four determinations for EC-SOD TG mice.
Figure 2.
Figure 2.
Young EC-SOD transgenic mice have impaired LTP, whereas aged EC-SOD transgenic mice exhibit enhanced LTP when compared with wild-type littermates. A, Stable baseline responses were recorded in hippocampal area CA1 of slices from either young wild-type (WT) or young EC-SOD transgenic (EC-SOD TG) mice. LTP-inducing HFS consisted of one train of 100 Hz HFS for 1 s (3- to 4-month-old WT, n = 10; EC-SOD TG, n = 8). The traces shown on the right are representative fEPSPs recorded in stratum radiatum in area CA1 of hippocampal slices from young WT and young EC-SOD TG mice before and 40 min after HFS. Calibration: A, B, 3 mV, 5 ms. B, Similar to A, except that aged mice were used for this study (>20-month-old WT, n = 9; EC-SOD TG, n = 9). The traces shown on the right are representative fEPSPs recorded in stratum radiatum in area CA1 of hippocampal slices from aged WT and aged EC-SOD TG before and 40 min after HFS. C, Four 100 Hz trains (1 s) separated by 5 min was used to induce L-LTP in old WT and old EC-SOD TG slices (>20-month-old WT, n = 10; EC-SOD TG, n = 10). The traces shown on the right are representative fEPSPs recorded in stratum radiatum in area CA1 of hippocampal slices from aged WT and aged EC-SOD TG before and 150 min after the final train of HFS. D, Responses were recorded from aged WT and EC-SOD TG slices given LTP-inducing HFS (a single 100 Hz train for 1 s) in the presence of catalase (260 U/ml) as indicated by the bar (>20-month-old WT, n = 6; EC-SOD TG, n = 6). The traces shown on the right are representative fEPSPs recorded in stratum radiatum in area CA1 of hippocampal slices from aged WT and aged EC-SOD TG before and 40 min after HFS. In all panels, the cumulative data are plotted as mean ± SEM.
Figure 3.
Figure 3.
Aged EC-SOD transgenic mice do not exhibit a significant age-related decline in spatial memory in the Morris water maze. A, Time spent searching for the platform on each training day. Data are plotted as mean ± SEM. Wild-type (WT) and EC-SOD transgenic (EC-SOD TG) littermates were used. For each genotype: 3–4 month group, n = 20; 13–14 month group, n = 10; 19–20 month group, n = 18. B, Visible platform test. No significant difference was observed between WT and EC-SOD TG. Data are plotted as mean ± SEM. The 12-month-old mice were used for this test (WT, n = 9; EC-SOD TG, n = 6). C, Time spent in each of the quadrants in the probe trial on the last training day. *p < 0.05 compared with aged wild-type mice by Student's t test. The time spent in the target quadrant was not significantly different between wild-type and EC-SOD transgenic in the 3–4 month and 13–14 month groups. D, The number of platform crossings in the probe trial is shown for the target quadrant and the corresponding location in the other quadrants. The 19- to 20-month-old EC-SOD TG mice tend to cross the original hidden platform place more often than the wild-type littermates. p = 0.1 by Student's t test.
Figure 4.
Figure 4.
Impaired associative memory in EC-SOD transgenic mice. A, Freezing behavior was expressed as the percentage of time during training (train) and testing (test) for 3-month-old wild-type (WT) and EC-SOD transgenic (EC-SOD TG) mice. Bar graphs on the left show the percentage of time that the mice froze to context 24 h after training. Bar graphs on the right show the percentage of time that the mice froze to the auditory cue 25 h after training. (WT, n = 7; EC-SOD TG, n = 10). Data are plotted as mean ± SEM. ∗∗∗p < 0.001 compared with aged wild-type mice by Student's t test. B, Similar to A, except that aged (>20 months of age) mice were used for this study (WT, n = 20; EC-SOD TG, n = 22).∗p < 0.05 compared with aged wild-type mice by Student's t test.
Figure 5.
Figure 5.
Aged EC-SOD transgenic mice exhibit better motor performance than wild-type littermates. A, Body weight of wild-type mice (WT) and EC-SOD transgenic mice (EC-SOD TG). B, Motor coordination accessed by the rotating rod test. The time for the mice to remain on the rod on the second training day was plotted. Aged EC-SOD TG mice performed significantly better than wild-type mice. Data are plotted as mean ± SEM (3–4 month group, n = 20; 13–14 month group, n = 15; >19 month group, n = 20). ∗ p < 0.05, ∗∗p < 0.01 compared with age-matched wild-type mice by Student's t test. C–E, Motor learning ability was assessed by measuring the rate of improvement after repeated trials in the rotating rod test. The time for the mice to remain on the rod in each trial was plotted as mean ± SEM. Data were transformed using log 10, and then a test for homogeneity of slopes (ANCOVA) was performed to determine whether the slopes were parallel. At 13 months of age and at >19 months of age, EC-SOD transgenic mice learned at a higher rate than their wild-type littermates (†p < 0.0001, ANCOVA; §p < 0.0001, two-way ANOVA, effect of genotype). In the >19-month-old group, Bonferroni's post hoc comparisons indicated a significant difference between wild-type and EC-SOD transgenic mice from trial 5 to trial 8 (∗p < 0.05, ∗∗p < 0.001, two-way ANOVA, followed by Bonferroni's post hoc tests).
Figure 6.
Figure 6.
Endogenous levels of superoxide in young and old wild-type and EC-SOD transgenic mice. Brain sections were prepared from four 3-month-old (young) and four 20-month-old (aged) wild-type (WT) and EC-SOD transgenic (TG) mice that received two intraperitoneal injections of DHE (for details, see Materials and Methods). Mice that were not injected with DHE were used as controls for autofluorescence. A, Coronal sections of the hippocampus showing differences in ethidium fluorescence between young and aged WT and young and aged TG mice. Scale bar, 300 μm. B, Coronal sections through CA1 and CA3 of the hippocampus and cerebellum showing differences in ethidium fluorescence. The images were taken with the same settings, and a typical image for each region is shown. Scale bar, 75 μm. C, Quantification of fluorescence in hippocampal area CA1. DHE fluorescence was measured with Scion Image software, using 1 inch square throughout the soma of CA1 pyramidal cells from at least eight sections. Data were graphed as pixel intensity, mean ± SEM; n > 40. Tissue autofluorescence (control) from mice that received no DHE is shown by the left bar. Values were compared by one-way ANOVA, followed by Tukey's post hoc tests, indicating significantly enhanced levels in aged mice compared with young mice (**p < 0.01). Student's t test showed significant differences between young wild-type (young WT) and young transgenic (young TG) (#p < 0.05), as well as between aged wild-type (aged WT) and aged transgenic (aged TG) (###p < 0.0001) mice.
Figure 7.
Figure 7.
Decreased brain protein oxidation in aged EC-SOD transgenic mice. A, Spectrophotometric assays for protein carbonyls in brain homogenates. Levels of protein oxidation (protein carbonyls) were determined as described in Materials and Methods. Results were expressed as percentage of young wild-type (young WT) values. Error bars indicate SEM for five determinations. In this assay, young EC-SOD transgenic mice (young TG) exhibited similar protein carbonyl levels compared with young WT mice, whereas old EC-SOD transgenic mice (aged TG) exhibited significantly lower protein carbonyl levels than old wild-type mice (aged WT); **p < 0.05 by Student's t test. B, Western blot analysis for protein carbonyls in hippocampal CA1 homogenates. The hippocampal CA1 region of young (3 months) or old (>20 months) wild-type or EC-SOD transgenic mice was dissected and homogenized, and the lysates were centrifuged to remove insoluble material. Total protein (50 μg) was loaded in each lane. The experiment was performed four times; a representative blot is shown.
Figure 8.
Figure 8.
Decreased activation of p38 and ERK2 in the hippocampus of aged EC-SOD transgenic mice. Activation of p38 (A) and ERK2 (B) was assessed via Western blot analysis of phospho-p38 and phospho-ERK2 in hippocampal homogenates prepared from wild-type (WT) and EC-SOD transgenic (EC-SOD TG) mice at 3 months (young) and 20 months (aged) of age. Representative Western blots for phosphorylated p38 and total p38 and phosphorylated ERK2 and total ERK2 are shown at the top of each panel. For both p38 and ERK2, phospho-immunoreactivity was normalized to total immunoreactivity, and results were plotted as arbitrary units compared with young wild-type controls. Values are mean ± SEM for six determinations. **p < 0.01 compared with the same genotype by Student's t test. Ab, Antibody.
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