Article Figures & Data
Figures
- Figure 1.
Ten days of IH increases hippocampal HIF1a and disrupts Barnes maze performance in wild-type mice but not in HIF1a+/−. A, left, Representative digitized Western blotting images for HIF1a (103 kDa) and PCNA (40 kDa) in hippocampal nuclear protein fractions from control (n = 4) and IH10 (n = 4). Right, Quantification of HIF1a protein normalized to PCNA revealed that nuclear HIF1a was increased in IH10 when compared with control (p = 0.019). B, Total latency to exit the Barnes maze during three training sessions in control (n = 10) and in IH10 (n = 11). Each blue (control) and red (IH10) line represents an individual performance during training. Training to the exit was conducted over three sessions. Each session was separated by 24 hours. C, Left, During the probe trial, the distance traveled to initially enter the exit zone was shorter in control when compared with IH10 (p = 0.048). Right, Latency to initial entry was smaller in control as well (p = 0.034). D, Heat maps of the mean entry probability across all false exits (1–19) and the exit zone during probe trial for the control and IH10. Comparison of entry probability into the exit zone during the probe trial reveals that control has a greater probability for entering the exit zone when compared with IH10 (p = 0.004). E, Left, Representative digitized Western blotting images HIF1a and PCNA in hippocampal nuclear protein fractions from 0-HIF1a+/− (n = 4) and 10-HIF1a+/− (n = 4). Right, Quantification of HIF1a protein normalized to PCNA revealed that nuclear HIF1a is similar between 0-HIF1a+/− and 10-HIF1a+/− (p = 0.84). F, Total latency to exit the Barnes maze during three training sessions in 0-HIF1a+/− (n = 7) and in 10-HIF1a+/− (n = 8). Each gray (0-HIF1a+/−) and yellow (10-HIF1a+/−) line represents an individual performance during training. All experimental groups exhibit decreased total latency over the course of training. G, Left, In HIF1a+/−, the distance initial to initial entry into the exit zone was similar between 0-HIF1a+/− and 10-HIF1a+/− (p = 0.55). Right, Latency to initial entry into the exit zone during the probe trial were similar between 0-HIF1a+/− and 10-HIF1a+/− (p = 0.39). H, Heat maps of the mean entry probability into all zones during the probe trial for 0-HIF1a+/− and 10-HIF1a+/−. Entry probability was similar between 0-HIF1a+/− and 10-HIF1a+/− (p = 0.21); *p < 0.05; **p < 0.01; N.S., p > 0.05.
- Figure 2.
IH suppresses NMDAr-dependent synaptic potentiation in wild-type hippocampal slices, but NMDAr-dependent LTP is unaffected by IH in the hippocampal slices from HIF1a+/−. A, LTP was evoked using HFS in control (blue, n = 6) is attenuated by AP5 (green, n = 5). LTPHFS is attenuated in following IH (IH10, red, n = 6) and is no longer sensitive to AP5 (IH+AP5, gold, n = 5). A comparison of LTPHFS magnitude (60 min following HFS) was performed to compare experimental conditions to control; **p < 0.01. B, LTPTBS is readily evoked in control (light blue, n = 5) and is completely blocked by AP5 (light green, n = 5). Following IH, LTPTBS is present (IH10, pink, n = 5). Following a one-way ANOVA, a post hoc comparison of LTPTBS magnitude (60 min following TBS) was performed to compare experimental conditions to control; ***p < 0.01. C, LTPHFS was evoked in both 0-HIF1a+/− (n = 8, gray) and 10-HIF1a+/− (n = 8, dark yellow). No difference was found when comparing LTPHFS magnitude between 0-HIF1a+/− and 10-HIF1a+/− (p = 0.94). D, LTPTBS was evoked in both 0-HIF1a+/− (n = 6, light gray), 10-HIF1a+/− (n = 5, light yellow), and 10-HIF1a+/− + AP5 (n = 5, light green). No difference was found when comparing LTPTBS magnitude of 0-HIF1a+/− and 10-HIF1a+/−. Representative traces illustrate baseline (black) and 60 min following HFS (colored trace). Scale bars: 0.2 mV/10 ms. In experiments using AP5, electrophysiological recordings began at 20 min before eliciting LTP (i.e., t = −20) while AP5 was applied 10 before eliciting LTP (i.e., t = −10). For all the experiment, the arrow represents the electric protocols: HFS or TBS; ***p < 0.001, **p < 0.01; N.S., p > 0.05.
- Figure 3.
The IH reduces the contribution of the NMDAr to fEPSP and GluN1 protein from wild-type mice but does not induce these changes in HIF1a+/−. A, Representative traces of the fEPSP from control, IH10, 0-HIF1a+/−, and 10-HIF1a+/− in: aCSF (black), Mg2+-free media (blue), and Mg2+-free media with AP5 (red). Scale bars: 0.4 mV/10 ms. B, top, Change in amplitude of the fEPSP from aCSF to Mg2+-free media. Bottom, Change in amplitude of the fEPSP from Mg2+-free media to Mg2+-free media with AP5; *p < 0.05; N.S., p > 0.05. C, left, Representative Western blottings of GluN1 and the housekeeping protein, GAPDH from control (n = 5), IH10 (n = 5), 0-HIF1a+/− (n = 5), and 10-HIF1a+/− (n = 5). Right, Comparisons of normalized GluN1 protein expression were performed to compare experimental conditions to control. This revealed that GluN1 was reduced in IH10 and unchanged in both 0-HIF1a+/− and 10-HIF1a+/−; *p < 0.05; N.S., p > 0.05. D, left, Representative Western blottings of PSD-95 and the housekeeping protein, GAPDH from control (n = 3), IH10 (n = 3), 0-HIF1a+/− (n = 3), and 10-HIF1a+/− (n = 3). Right, Comparisons of normalized PSD-95 protein expression were performed to compare experimental conditions to control; *p < 0.05; N.S., p > 0.05.
- Figure 4.
IH enhances protein carbonyl content and increase NOX4 expression in wild type but not in HIF1a+/−. A, Hippocampal homogenates from control (n = 4), IH10 (n = 4), 0-HIF1a+/− (n = 4), and 10-HIF1a+/− (n = 4). While IH10 displayed elevated protein, carbonyl content was not elevated in either 0-HIF1a+/− or 10-HIF1a+/−. B, Comparison of the pro-oxidant enzyme, NOX4, from control (n = 5), IH10 (n = 5), 0-HIF1a+/− (n = 5), and 10-HIF1a+/− (n = 5) reveals that NOX4 is increased in IH10; p < 0.01), but not elevated in either 0-HIF1a+/− or 10-HIF1a+/−; **p < 0.01; N.S., p > 0.05.
- Figure 5.
Antioxidant treatment mitigates the IH-dependent effects on GluN1 expression, LTPTBS, and performance in the Barnes maze. A, left, Representative Western blottings of GluN1 and GAPDH from Control, IH10, wild-type mice treated with saline during 10 d of IH (i.e., vehicle control exposed to IH, IHSaline, n = 4), wild-type mice treated with MnTMPyP during 10 d of IH (IHMnTMPyP). Right, Normalized GluN1 protein expression was examined in control (n = 4), IH10 (n = 4), IHSaline (n = 4), IHMnTMPyP (n = 4). No difference in GluN1 was evident between IH10 (open black circles in IH10 label) and IHSaline (open blue circles in IH10 label); therefore, the two groups were merged into the IH10 label for comparisons to control. Comparisons revealed that GluN1 was reduced only in IH10 and unchanged in IHMnTMPyP. B, In hippocampal slices from IHMnTMPyP, LTPTBS (n = 5) could be reliably evoked contrasting the effect of IH10 on LTPTBS (Fig. 2B). Scale bars: 0.2 mV/10 ms. The arrow represents the TBS protocol. C, The total latency to exit the Barnes maze progressively decreased in both IHSaline (n = 11, pink lines represent individual performance) and IHMnTMPyP (n = 10, purple lines represent individual performance), suggesting that both groups could learn the exit zone location. D, Heat maps of the mean entry probability across all false exits (1–19) and the exit zone during the probe trial for IHSaline and IHMnTMPyP. Comparison of entry probability into the exit zone during the probe trial reveals that IHMnTMPyP has a greater probability for entering the exit zone when compared with IHSaline (p = 0.006); ***p < 0.001, *p < 0.05; N.S., p > 0.05.
- Figure 6.
A mechanistic framework by which sleep apnea lowers the threshold for cognitive impairment. Schematic synthesizing our findings into a pathway by which IH promotes a pro-oxidant state in the hippocampus that impairs NMDAr-dependent plasticity and spatial memory.
Tables
Figure Statistical test Statistical values 1A Unpaired t test with Welch's correction p = 0.03; t = 2.789, df = 3 1B, left One-way ANOVA, Dunnett's multiple comparison test One-way ANOVA p = 0.0044, F = 7.191, 1 vs 2 (training session): p < 0.05, CI of diff = 23.74–217.9; 1 vs 3 (training session): p < 0.01, CI of diff = 47.11–241.3 1B, right One-way ANOVA, Dunnett's multiple comparison test. One-way ANOVA p = 0.0006, F = 11.68, 1 vs 2 (training session): p < 0.01, CI of diff = 63.81–239.3; 1 vs 3 (training session): p < 0.001, CI of diff = 66.88–242.3 1C, left Unpaired t test with Welch's correction p = 0.04, t = 2.85, df = 9 1C, right Unpaired t test with Welch's correction p = 0.03, t = 2.501, df = 9 1D Unpaired t test with Welch's correction p = 0.0037; t = 3.436, df = 15 1E Unpaired t test with Welch's correction p = 0.84; t = 0.2118, df = 5 1F, left One-way ANOVA, Dunnett's multiple comparison test One-way ANOVA p = 0.0136, F = 6.288, 1 vs 2 (training session): p > 0.05, CI of diff = –12.74 to 207.3; 1 vs 3 (training session): p < 0.01, CI of diff = 44.13–264.2 1F, right One-way ANOVA, Dunnett's multiple comparison test One-way ANOVA p = 0.0156, F = 5.688, 1 vs 2 (training session): p > 0.05, CI of diff = –3.202 to 221.3; 1 vs 3 (training session): p < 0.05, CI of diff = 36.56–261.1 1G, left Unpaired t test with Welch's correction p = 0.547, t = 0.6258, df = 9 1G, right Unpaired t test with Welch's correction p = 0.48, t = 0.7431, df = 9 1H, left Unpaired t test with Welch's correction p = 0.2120, t = 1.356, df = 8 2A One-way ANOVA, Dunnett's multiple comparison test One-way ANOVA p = 0.0004, F = 10.20; control vs AP5: p < 0.01, CI of diff = 21.28–62.13; control vs 10-IH: p < 0.01, CI of diff = 5.21–44.71; control vs 10-IH+AP5: p < 0.01, CI of diff = 11.04–51.89 2B One-way ANOVA, Dunnett's multiple comparison test One-way ANOVA p < 0.0001, F = 116.9; control vs AP5: p < 0.01, CI of diff = 54.80–80.72; control vs 10-IH: p < 0.01, CI of diff = 56.41–82.32 2C Unpaired t test with Welch’s correction p = 0.94; t = 0.065, df = 13.14 2D One-way ANOVA, Dunnett's multiple comparison test One-way ANOVA p < 0.0001, F = 54.50; 0-HIF1a+/− vs 10-HIF1a+/−: p > 0.05, CI of diff = –20.49 to 10.15; 10-HIF1a+/− vs 10-HIF1a+/−+ AP5: p < 0.01, CI of diff = 42.75–75.42 3B, top One-way ANOVA, Dunnett's multiple comparison test One-way ANOVA p = 0.56, F = 0.70; control vs IH10: p > 0.05, CI of diff = –24.53 to 54.60; control vs 0-HIF1a+/−: p > 0.05, CI of diff = –20.36 to 54.60; control vs 10-HIF1a+/−: p > 0.05, CI of diff = –33.12 to 39.06 3B, bottom One-way ANOVA, Dunnett's multiple comparison test One-way ANOVA p = 0.56, F = 0.70; control vs IH10: p < 0.05, CI of diff = –70.53 to –6.241; control vs 0-HIF1a+/−: p > 0.05, CI of diff = –56.75 to 3.840; control vs 10-HIF1a+/−: p > 0.05, CI of diff = –40.38 to 17.97 3C One-way ANOVA, Dunnett's multiple comparison test One-way ANOVA p = 0.014, F = 4.74; control vs IH10: p < 0.05, CI of diff = 0.96–0.78 ; control vs 0-HIF1a+/−: p > 0.05, CI of diff = –0.38 to 0.05; control vs 10-HIF1a+/−: p > 0.05, CI of diff = –0.27 to 0.157 3D One-way ANOVA, Dunnett's multiple comparison test One-way ANOVA p = 0.14, F = 2.39; control vs IH10: p > 0.05, CI of diff = –0.42 to 0.27 ; control vs 0-HIF1a+/−: p > 0.05, CI of diff = –0.63 to 0.0636; control vs 10-HIF1a+/−: p > 0.05, CI of diff = –0.35 to 0.33 4A One-way ANOVA, Dunnett's multiple comparison test One-way ANOVA p = 0.006 F = 6.871, control vs IH10: p < 0.01 CI of diff = –105.1 to –17.65; control vs 0-HIF1a+/−: p > 0.05 CI of diff = –52.12 to 35.32; control vs 10-HIF1a+/−: p > 0.05 CI of diff = –40.58 to 46.85 4B One-way ANOVA, Dunnett's multiple comparison test One-way ANOVA p = 0.003, F = 11.70; control vs IH10: p > 0.05, CI of diff = –1.85 to –0.28; control vs 0-HIF1a+/−: p > 0.05, CI of diff = –0.35 to 0.45; control vs 10-HIF1a+/−: p > 0.05, CI of diff = –0.28 to 0.52 5A One-way ANOVA, Dunnett's multiple comparison test One-way ANOVA p = 0.0023, F = 10.00; control vs IH10: p < 0.01, CI of diff = 0.09–0.75; control vs 10-MnTMPyP: p > 0.05, CI of diff = –0.49 to 0.26 5B One-way ANOVA, Dunnett's multiple comparison test One-way ANOVA p < 0.0001, F = 57.60, control vs IH: p < 0.001, CI of diff = 50.58–88.15; control vs IH+MnTMPyP: p > 0.05, CI of diff = –19.59 to 17.98 5C, top One-way ANOVA, Dunnett’s multiple comparison test One-way ANOVA p = 0.0008, F = 6.32, 1 vs 2 (training session): p > 0.05, CI of diff = –40.99 to 139.5; 1 vs 3 (training session): p < 0.001, CI of diff = 71.59–252.1 5C, bottom One-way ANOVA, Dunnett’s multiple comparison test One-way ANOVA p = 0.0056, F = 9.10, 1 vs 2 (training session): p > 0.05, CI of diff = –25.40 to 187.3; 1 vs 3 (training session): p < 0.01, CI of diff = 55.75–268.4 5D Unpaired t test with Welch’s correction p = 0.0005; t = 4.292, df = 16.7112
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