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. 2006 Aug;26(15):5908-20.
doi: 10.1128/MCB.00269-06.

Synaptic plasticity deficits and mild memory impairments in mouse models of chronic granulomatous disease

Kenneth T Kishida  1 , Charles A Hoeffer, Daoying Hu, Maryland Pao, Steven M Holland, Eric Klann
Affiliations

Synaptic plasticity deficits and mild memory impairments in mouse models of chronic granulomatous disease

Kenneth T Kishida et al. Mol Cell Biol. 2006 Aug.

Abstract

Reactive oxygen species (ROS) are required in a number of critical cellular signaling events, including those underlying hippocampal synaptic plasticity and hippocampus-dependent memory; however, the source of ROS is unknown. We previously have shown that NADPH oxidase is required for N-methyl-D-aspartate (NMDA) receptor-dependent signal transduction in the hippocampus, suggesting that NADPH oxidase may be required for NMDA receptor-dependent long-term potentiation (LTP) and hippocampus-dependent memory. Herein we present the first evidence that NADPH oxidase is involved in hippocampal synaptic plasticity and memory. We have found that pharmacological inhibitors of NADPH oxidase block LTP. Moreover, mice that lack the NADPH oxidase proteins gp91(phox) and p47(phox), both of which are mouse models of human chronic granulomatous disease (CGD), also lack LTP. We also found that the gp91(phox) and p47(phox) mutant mice have mild impairments in hippocampus-dependent memory. The gp91(phox) mutant mice exhibited a spatial memory deficit in the Morris water maze, and the p47(phox) mutant mice exhibited impaired context-dependent fear memory. Taken together, our results are consistent with NADPH oxidase being required for hippocampal synaptic plasticity and memory and are consistent with reports of cognitive dysfunction in patients with CGD.

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Figures

FIG. 1.
FIG. 1.
NADPH oxidase inhibitors block HFS-induced E-LTP. (A) A single 1-second 100-Hz train of HFS (indicated by the arrow) induced E-LTP in vehicle-treated wild-type hippocampal slices (control) but was blocked when HFS was delivered in the presence of either 10 μM DPI or 100 μM apocynin (n = 8 slices; four mice per treatment) (P < 0.0001 by one-way ANOVA) (apocynin versus control, P < 0.001; DPI versus control, P < 0.001; DPI versus apocynin, P > 0.05 by Newman-Keuls multiple comparison test). (B) Two representative fEPSP recordings from time points 1 and 2 in panel A were overlaid to demonstrate potentiation in control slices, which was absent in drug-treated slices. (C) Control slices with or without either 10 μM DPI or 100 μM apocynin applied to slices for 20 min while recording fEPSPs; 10 μM DPI or 100 μM apocynin did not affect baseline fEPSPs (P > 0.05 by one-way ANOVA). (D) Three representative fEPSPs from time points 1, 2, and 3 in panel C were overlaid, demonstrating the lack of change in baseline responses during 1 hour of recording with or without application of either 10 μM DPI or 100 μM apocynin for 20 min.
FIG. 2.
FIG. 2.
NADPH oxidase mutant mice do not express HFS-induced E-LTP. (A) A single 1-second 100-Hz train of HFS (indicated by the arrow) induced E-LTP in wild-type mice but failed to induce LTP in gp91phox KO mice (n = 11 slices; five mice per genotype) (P < 0.0001 by two-way ANOVA). (B) Two representative fEPSPs from time points 1 and 2 in panel A were overlaid to demonstrate potentiation in slices prepared from wild-type mice, which was absent in slices prepared from gp91phox KO mice. (C) A single 1-second 100-Hz train of HFS (indicated by the arrow) induced E-LTP in wild-type mice but failed to induce E-LTP in p47phox KO mice (n = 8 slices; four mice per genotype) (P < 0.0001 by two-way ANOVA). (D) Two representative fEPSPs from time points 1 and 2 in panel C were overlaid to demonstrate potentiation in slices prepared from wild-type mice, which was absent in slices prepared from p47phox KO mice.
FIG. 3.
FIG. 3.
NADPH oxidase mutant mice express L-LTP. (A) Four trains of HFS (1 second at 100 Hz; indicated by the arrows) spaced 5 min apart induced L-LTP in wild-type mice and in gp91phox KO mice (n = 8 slices; eight mice per genotype) (P > 0.05 by two-way ANOVA). (B) Two representative fEPSPs from time points 1 and 2 in panel A were overlaid to demonstrate potentiation in slices prepared from wild-type and gp91phox KO mice. (C) L-LTP in wild-type mice and slightly enhanced L-LTP in p47phox KO mice (n = 7 slices; seven mice per genotype) (P < 0.0001 by two-way ANOVA). (D) Two representative fEPSPs from time points 1 and 2 in panel C were overlaid to demonstrate potentiation in slices prepared from wild-type and gp91phox KO mice.
FIG. 4.
FIG. 4.
Normal hippocampal morphology and synaptic transmission in NADPH oxidase mutant mice. (A to C) Nissl-stained sagittal sections from the brains of adult wild-type (A), gp91phox KO (B), and p47phox KO (C) mice. Magnification, ×25. (D to G) The input-output relationship of hippocampal slices is similar between mutant animals and wild-type littermates (for all comparisons, P > 0.05 by two-way ANOVA for genotype and stimulus strength). (D) Fiber volley amplitude plotted against stimulus strength for wild-type (filled squares) and gp91phox KO (open squares) mice. (E) fEPSP slope plotted against stimulus strength for wild-type (filled squares) and gp91phox KO (open squares) mice. (F) Fiber volley amplitude plotted against stimulus strength for wild-type (filled squares) and p47phox KO (open squares) mice. (G) fEPSP slope plotted against stimulus strength for wild-type (filled squares) and p47phox KO (open squares) mice.
FIG. 5.
FIG. 5.
Normal NMDA receptor-mediated fEPSPs in NADPH oxidase mutant mice: isolation of NMDA receptor-mediated fEPSPs recorded in hippocampal area CA1. The change in fEPSP slope was monitored over time and plotted as a percentage of the baseline fEPSP. No significant difference was observed between NMDA receptor-mediated fEPSPs (P > 0.05, one-way ANOVA between all treatments). a, baseline fEPSP; b, fEPSP slope in the presence of 4 mM CaCl2 and 0 mM MgCl2; c, fEPSP after the addition of 20 μM CNQX; d, NMDA receptor-mediated fEPSPs verified by their sensitivity to 100 μM APV; e, fEPSP after the addition of 100 μM APV. n = 8 for each experimental condition.
FIG. 6.
FIG. 6.
Normal paired-pulse facilitation in NADPH oxidase mutant mice. PPF was unaltered in NADPH oxidase mutant mice before (A to C) and after (B and C) receiving a single 1-second 100-Hz train of HFS. The percentage of facilitation, calculated from the ratio of the second fEPSP slope to the first fEPSP slope, is shown at interpulse intervals ranging from 10 to 300 ms (n = 25 slices from seven mice for gp91phox wild-type littermates; n = 24 slices for seven mice for gp91phox KO and n = 18 slices for six mice for p47phox wild-type littermates; n = 19 slices for six mice for p47phox KO) (P > 0.05, one-way ANOVA). (B) PPF in p47phox KO mice compared to wild-type littermates before and after receiving a single 1-second 100-Hz train of HFS (n = 9 slices for six mice for p47phox wild-type littermates; n = 9 slices for six mice for p47phox KO) (P > 0.05, one-way ANOVA). (C) PPF in gp91phox KO mice compared to wild-type littermates before and after receiving a single 1-second 100-Hz train of HFS (n = 8 slices for six mice for gp91phox wild-type littermates; n = 8 slices for six mice for gp91phox KO) (P > 0.05, one-way ANOVA).
FIG. 7.
FIG. 7.
PTP is normal in p47phox KO mice but decreased in gp91phox KO mice. (A) PTP after a single 1-second 100-Hz train of HFS delivered in the presence of APV (100 μM) was deficient in gp91phox KO mice but was unaltered in p47phox KO mice (n = 13 slices for six wild-type mice; n = 10 slices for three gp91phox KO mice; n = 11 slices for three p47phox KO) (P < 0.0001 by one-way ANOVA) (wild-type versus gp91phox KO, P < 0.001; wild-type versus p47phox KO, P > 0.05; p47phox KO versus gp91phox KO, P < 0.001 by Newman-Keuls multiple comparison test). (B) PTP after a single 1-second 100-Hz train of HFS delivered in the presence of APV (100 μM) was deficient in wild-type slices treated with 10 μM DPI. (C) Synaptic fatigue is normal in gp91phox KO mice compared to wild-type littermates. A 3-second 10-Hz train of stimulation was delivered to hippocampal slices prepared from either gp91phox KO or wild-type littermates treated with 100 μM APV. fEPSPs were recorded, and the synaptic fatigue was measured by the steady decrease in fEPSP slope that occurred during the stimulus train. No significant difference was observed between gp91phox KO mice and wild-type littermates (P > 0.05, two-way ANOVA).
FIG. 8.
FIG. 8.
Conditioned fear memory in NADPH oxidase mutant mice. Mean freezing behavior is shown for contextual and cued fear conditioning tests performed 24 h after training. (A) Wild-type mice versus gp91phox KO mice showed no difference in mean freezing behavior during either the contextual or cued fear response test (n = 7 mice per genotype, three cohorts; P > 0.05 by Student's two-tailed t test). (B) p47phox KO mice showed a significant decrease in mean freezing behavior during the contextual fear response test (n = 7 mice per genotype; formula image indicates P < 0.05 by Student's two-tailed t test) but showed no difference in the cued fear response test (n = 7 mice per genotype; P > 0.05 by Student's two-tailed t test).
FIG. 9.
FIG. 9.
Morris water maze performance in NADPH oxidase mutant mice. (A to D) MWM performance of gp91phox KO mice compared to wild-type littermates. (A) Escape latencies of gp91phox KO mice versus wild-type littermates were significantly different in the MWM, plotted as a function of training day (n = 12 mice per genotype; P < 0.0001 by two-way ANOVA). (B and C) Mean proportion of time spent in each of the quadrants during probe test 1 (B) and probe test 2 (C) for gp91phox KO mice versus wild-type littermates (n = 12 mice per genotype; P > 0.05 by two-way ANOVA). (D) Mean number of platform location crossings during each of the probe trials comparing gp91phox KO mice to wild-type littermates (n = 12 mice per genotype) (formula image, P < 0.005 [first probe trial]; P was ≫0.05 for the second probe trial; two-tailed t test). (E to H) MWM performance of p47phox KO mice compared to wild-type littermates. (E) Escape latencies of p47phox KO mice versus wild-type littermates were significantly different in the MWM, plotted as a function of training day (n = 10 mice per genotype; P < 0.0001 by two-way ANOVA). (F and G) Mean proportion of time spent in each of the quadrants during probe test 1 (F) and probe test 2 (G) for p47phox KO mice versus wild-type littermates (n = 10 mice per genotype; P > 0.05 by two-way ANOVA). (H) Mean number of platform location crossings during each of the probe trials comparing p47phox KO mice to wild-type littermates (n = 10 mice per genotype) (P > 0.05 for the first probe trial; P > 0.05 for the second probe trial; two-tailed t test).
FIG. 10.
FIG. 10.
Open field analysis of NADPH oxidase mutant mice. (A to C) Open field analysis of gp91phox KO mice and wild-type littermates. (A) gp91phox KO mice showed no difference in total distance traveled compared to wild-type littermates (n = 8 for gp91phox KO mice and n = 11 for wild-type littermates; P > 0.05 by two-way ANOVA). (B) gp91phox KO mice showed no difference in their exploration of the center of the open field, expressed as the ratio of center distance to total distance traveled, compared to wild-type littermates (n = 8 for gp91phox KO mice and n = 11 for wild-type littermates; P > 0.05 by two-way ANOVA). (C) gp91phox KO mice reared significantly less than their wild-type littermates, as assessed by the number of vertical beam breaks (vertical activity; n = 8 for gp91phox KO mice and n = 11 for wild-type littermates; P < 0.0001 by two-way ANOVA). (D to F) Open field analysis of p47phox KO mice and wild-type littermates. (D) p47phox KO mice showed no difference in total distance traveled compared to wild-type littermates (n = 9 for p47phox KO mice and n = 8 for wild-type littermates; P > 0.05 by two-way ANOVA). (E) p47phox KO mice showed a slight increase in their exploration of the center of the open field, expressed as the ratio of center distance to total distance traveled, compared to wild-type littermates (n = 9 for p47phox KO mice and n = 8 for wild-type littermates; P < 0.0001 by two-way ANOVA). (F) p47phox KO mice showed no difference in rearing compared to their wild-type littermates as assessed by the number of vertical beam breaks (vertical activity; n = 9 for p47phoxKO mice and n = 8 for wild-type littermates; P > 0.05 by two-way ANOVA).
FIG. 11.
FIG. 11.
Rotating rod analysis of NADPH oxidase mutant mice. gp91phox and p47phox KO mice showed largely normal performance on the accelerating rotating rod compared to wild-type littermates. (A) Comparison of gp91phox KO mice and wild-type littermates, measuring time spent balanced on the accelerating rotating rod across eight trials over 2 days. n = 8 for wild-type mice, and n = 11 for gp91phox KO mice (P = 0.0268 by two-way ANOVA). (C) Comparison of p47phox KO mice and wild-type littermates, measuring time spent balanced on the accelerating rotating rod across eight trials over 2 days. n = 8 for wild-type mice, and n = 12 for p47phox KO mice (P > 0.05 by two-way ANOVA). (B and D) Weight comparisons of mutant and wild-type littermates showed no significant difference between knockout mice and wild-type littermates (P > 0.05, two-tailed t test).
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