Article Figures & Data
Figures
- Figure 1.
Kv4.2 protein levels in the hippocampus are reduced 90 min following kainic acid-induced seizure, but no changes in miRNA-mediated silencing of Kv4.2 are detected. A, Timeline of kainic acid injection and tissue harvest. B, C, Hippocampal Kv4.2 protein levels are significantly reduced in female mice 90 min following injection of 15 mg/kg, i.p., kainic acid (B; unpaired t test, *p = 0.020; n(SAL) = 34, n(KAI) = 32), whereas mRNA levels are not significantly different (C; Mann–Whitney test, p = 0.641; n(SAL) = 27, n(KAI) = 26; 2 statistical outliers were removed from the saline group, and 3 statistical outliers were removed from the kainic acid group), similar to those observed in male mice (Gross et al., 2016). Kv4.2 protein levels were normalized to β3-tubulin levels on the same blot, and Kv4.2 mRNA was normalized to Gapdh mRNA in the same samples. D, In contrast to male mice, RISC association of Kv4.2 mRNA is not significantly increased after kainic acid in the hippocampus of female mice (Mann–Whitney test, p = 0.741; n = 27/group). E, F, Hippocampal RISC association of the Kv4.2-targeting miRNA miR-324-5p is reduced in female mice following kainic acid administration (E; Mann–Whitney test, *p = 0.038; n(SAL) = 25, n(KAI) = 18; 2 statistical outliers removed from the kainic acid group), while total miR-324-5p levels are unchanged (F; Mann–Whitney test, p = 0.735; n(SAL) = 26, n(KAI) = 22; 2 statistical outliers removed from the kainic acid group). In F, miR-324-5p total levels were normalized to RU19 or miR-191 in the same samples. SAL, saline; KAI, kainic acid; a.u., arbitrary units. Bars and error bars represent the mean ± SEM. Analyses in cortical tissue are shown in Extended Data Figure 1-1.
- Figure 2.
MiR-324-5p inhibition does not consistently increase latency to kainic acid-mediated seizure onset or reduce seizure severity in female mice. A, Illustration depicting the experimental timeline. B, Latency to and occurrence of status epilepticus are not altered in female mice by miR-324-5p antagomir treatment (log-rank (Cox–Mantel) test, p = 0.53; Fisher’s exact test, p = 0.31; n(SCR) = 7, n(a324) = 9). C–E, Latency to seizure onset as determined by EEG (C) and by behavior (D, Racine class 3 seizures; E, Racine class 5 seizures) is not significantly changed by antagomir treatment (C, unpaired t test, p = 0.191; n(SCR) = 8, n(a324) = 9; D, unpaired t test, p = 0.563; n(SCR) = 8, n(a324) = 10; E, Mann–Whitney test, p = 0.965; n(SCR) = 8, n(a324) = 10). F, Time spent seizing in the first 30 min after kainic acid is on average reduced after miR-324-5p inhibition but not significantly different compared with scrambled antagomir-treated mice (unpaired t test, p = 0.26; n = 7). G, H, Hippocampal (G) and cortical (H) Kv4.2 protein levels after kainic acid-induced seizures are not significantly changed by miR-324-5p antagomirs compared with scrambled antagomirs (G, unpaired t test, p = 0.868; n(SCR) = 9, n(a324) = 10; H, p = 0.13, unpaired t test; n(SCR) = 8, n(a324) = 10). One statistical outlier from the scrambled group in H was removed. Kv4.2 protein levels were normalized to β3-tubulin levels on the same blot. scr, scrambled antagomir; a324, anti-miR-324-5p antagomir. Bars and error bars represent the mean ± SEM. Additional EEG analyses, levels, and RISC association of Kv4.2 mRNA as well as correlation of biochemical measures with seizure severity are shown in Extended Data Figure 2-1.
- Figure 3.
Stereotaxically delivered antagomirs combined with kainic acid-induced seizures do not change Kv4.2 protein levels, Kv4.2 mRNA levels, or RISC association of Kv4.2 mRNA in the hippocampus of female mice. A, Illustration depicting the experimental timeline. MiR-324-5p-specific or scrambled antagomirs were stereotaxically intracerebroventricularly injected. Twenty-four hours later, 15 mg/kg kainic acid was intraperitoneally injected, and brains were dissected 90 min later. B, Kv4.2 protein levels were not significantly changed in the hippocampus of female mice by antagomir or kainic acid treatment (two-way ANOVA; interaction: F(1,20) = 0.04, p = 0.846; antagomir: F(1,20) = 0.34, p = 0.564; kainic acid: F(1,20) = 0.28, p = 0.61; n = 6/group). Kv4.2 protein levels were normalized to β3-tubulin levels on the same blot. C, D, Likewise, RISC association with (C) and levels of (D) Kv4.2 mRNA were not significantly changed in the hippocampus of female mice by antagomir or kainic acid treatment (two-way ANOVA; C: interaction: F(1,24) = 0.82, p = 0.37; effect of antagomir: F(1,24) = 3.51, p = 0.073; effect of kainic acid: F(1,24) = 0.33, p = 0.569; n = 7; D: interaction: F(1,24) = 0.64, p = 0.431; effect of antagomir: F(1,24) = 0.4, p = 0.533; effect of kainic acid: F(1,24) = 1.08, p = 0.308; n = 7). In B, Kv4.2 protein was normalized to β-tubulin on the same blot, and in D, Kv4.2 mRNA levels were normalized to Gapdh mRNA. scr, scrambled antagomir; a324, anti-miR-324-5p antagomir; a.u., arbitrary units. Bars and error bars represent the mean ± SEM. Analyses in cortical tissue are shown in Extended Data Figure 3-1. Analyses of control sham surgeries are shown in Extended Data Figure 3-2.
- Figure 4.
MicroRNA-induced silencing of Kv4.2 in the hippocampus is correlated with plasma levels of progesterone and 17β-estradiol. A–C, Kv4.2 mRNA association with the RISC in hippocampal lysates from female mice is negatively correlated with plasma levels of progesterone (A; Pearson’s correlation: r = −0.50, R2 = 0.25, *p = 0.012; n = 24), positively correlated with plasma levels of 17β-estradiol (B; Pearson’s correlation: r = 0.66, R2 = 0.43, *p = 0.006; n = 16), and negatively correlated with progesterone/17β-estradiol ratios (C; Pearson’s correlation: r = −0.71, R2 = 0.51, *p = 0.002; n = 16). D–F, By contrast, RISC association of the Kv4.2-targeting miRNA miR-324-5p only shows a trend toward a negative correlation with progesterone (D; Pearson’s correlation: r = −0.36, R2 = 0.13, p = 0.098; n = 22) and no correlation with 17β-estradiol (E; Pearson’s correlation: r = −0.004, R2 = 0.00,001, p = 1.00; n = 14) or progesterone/17β-estradiol ratios (F; Pearson’s correlation: r = −0.19, R2 = 0.04, p = 0.52; n = 14). Dashed lines indicate 95% confidence intervals. Pearson’s correlation statistics are also shown in the figure. Absence of significant correlations among progesterone, 17β-estradiol, or progesterone/17β-estradiol ratios and two other potassium channels, Kv7.2 and Kv7.3, is shown in Extended Data Figure 4-1.
- Figure 5.
MicroRNA-induced silencing of Kv4.2 in the cortex is correlated with plasma levels of progesterone and 17β-estradiol. A–C, There is no significant correlation of Kv4.2 mRNA association with the RISC in cortical lysates from female mice with plasma levels of progesterone (A; Pearson’s correlation: r = −0.10, R2 = 0.01, p = 0.65; n = 24) or progesterone/17β-estradiol ratios (C; Pearson’s correlation: r = −0.30, R2 = 0.09, p = 0.248; n = 17); however, Kv4.2 mRNA association with Ago2 is positively correlated with plasma levels of 17β-estradiol (B; Pearson’s correlation: r = 0.52, R2 = 0.27, *p = 0.034; n = 17) in cortical lysates, similarly to what was observed in hippocampus. D–F, Similarly as Kv4.2 mRNA, RISC association of the Kv4.2-targeting miRNA miR-324-5p is not correlated with progesterone (D; Pearson’s correlation: r = −0.26, R2 = 0.07, p = 0.176; n = 28) and progesterone/17β-estradiol ratios in the cortex (F; Pearson’s correlation: r = −0.28, R2 = 0.08, p = 0.240; n = 20). Like Kv4.2 mRNA, miR-324-5p mRNA association with Ago2 is positively correlated with 17β-estradiol (E; Pearson’s correlation: r = 0.48, R2 = 0.24, *p = 0.030; n = 20). Dashed lines indicate 95% confidence intervals. Pearson’s correlation statistics are also shown in the figure.
- Figure 6.
Treatment with kainic acid alters correlations of miRNA-induced silencing of Kv4.2 in the hippocampus with plasma levels of progesterone and 17β-estradiol. A–C, Ninety minutes following kainic acid treatment, Kv4.2 mRNA association with the RISC in hippocampal lysates from female mice is not correlated with plasma levels of progesterone (A; Pearson’s correlation: r = −0.26, R2 = 0.067, p = 0.223; n = 24), is negatively correlated with plasma levels of 17β-estradiol (B; Pearson’s correlation: r = −0.58, R2 = 0.34, *p = 0.023; n = 15) and is not significantly correlated with progesterone/17β-estradiol ratios (C; Pearson’s correlation: r = −0.05, R2 = 0.003, p = 0.85; n = 15). D–F, RISC association of the Kv4.2-targeting miRNA miR-324-5p is not significantly correlated with progesterone (D; Pearson’s correlation: r = 0.31, R2 = 0.10, p = 0.210; n = 18) but shows a trend toward positive correlation with progesterone/17β-estradiol ratios (F; Pearson’s correlation: r = 0.61, R2 = 0.37, p = 0.06; n = 10). No significant correlation with 17β-estradiol was detected (E; Pearson’s correlation: r = −0.08, R2 = 0.006, p = 0.83; n = 10). Dashed lines indicate 95% confidence intervals. Pearson’s correlation statistics are also shown in the figure. Analyses in the cortex are shown in Extended Data Figure 6-1.
- Figure 7.
No significant differences in Kv4.2 protein expression or RISC association of Kv4.2 mRNA across estrous cycle stages and depending on kainic acid treatment in the hippocampus. A, Kv4.2 protein levels in the hippocampus are not significantly different across estrous stages in female saline-treated or kainic acid-treated mice, and no significant interaction between the treatment stage and estrous stage was detected. Consistent with results shown in Figure 1, a trend toward reduced protein levels after kainic acid was observed (two-way ANOVA; interaction: F(2,30) = 0.36, p = 0.70; effect of estrous stage: F(2,30) = 0.77, p = 0.474; effect of kainic acid: F(1,30) = 3.6, p = 0.07; n(proestrus+estrus saline) = 5, n(proestrus+estrus kainic acid) = 6, n(diestrus saline) = 8, n(diestrus kainic acid) = 5, n(metestrus saline) = 6, n(metestrus kainic) = 6). Kv4.2 was normalized to β3-tubulin signal on the same blot. B, Kv4.2 mRNA association with the RISC in the hippocampus is not significantly different across estrous stages in female saline-treated or kainic acid-treated mice (two-way ANOVA; interaction: F(2,22) = 0.04, p = 0.96; effect of estrous stage: F(2,22) = 0.20, p = 0.824; effect of kainic acid: F(1,22) = 2.7, p = 0.11, n(proestrus+estrus saline) = 4, n(proestrus+estrus kainic acid) = 6, n(diestrus saline) = 4, n(diestrus kainic acid) = 4, n(metestrus saline) = 5, n(metestrus kainic) = 5). P + E, Proestrus and estrous; D, diestrus; M, metestrus. No mice in proestrus were identified in the saline group. Mice in proestrus in the kainic group are indicated as gray triangles in the bar diagram. Mice used for this analysis are a subset of the mice analyzed in Figures 1, 4, and 6. Analyses of cortical tissue as well as of miR-324-5p association with the RISC are shown in Extended Data Figure 7-1.
- Figure 8.
No significant differences in Kv4.2 expression or miRNA-induced silencing after kainic acid treatment in ovariectomized female mice. A–J, Mice were ovariectomized, injected intraperitoneally with kainic acid after 14 d of recovery, and hippocampal (A–E) and cortical (F–J) tissue was collected 90 min later (Fig. 1A, timeline). A, F, Kv4.2 protein levels were not significantly different following kainic acid treatment in hippocampus (A; unpaired t test, p = 0.135; n(SAL) = 9, n(KAI) = 10) or cortex (F; unpaired t test, p = 0.821; n(SAL) = 9, n(KAI) = 10) in ovariectomized mice. B, G, Likewise, no changes in Kv4.2 mRNA levels were observed in hippocampus (B; unpaired t test, p = 0.101; n(SAL) = 9, n(KAI) = 10) or cortex (G; unpaired t test, p = 0.935; n(SAL) = 9, n(KAI) = 9; one statistical outlier removed). C, H, No changes in Kv4.2 mRNA association with the RISC in hippocampus (C; Mann–Whitney test, p = 0.604, n(SAL) = 9, n(KAI) = 10) or cortex (H; Mann–Whitney test, p = 0.447; n(SAL) = 9, n(KAI) = 10) were detected. D, I, Likewise, miR-324-5p levels in hippocampus (D; unpaired t test, p = 0.545; n = 9) or cortex (I; unpaired t test, p = 0.623; n = 8) were unchanged after kainic acid in ovariectomized mice. E, J, RISC association of miR-324-5p was also not different after kainic acid in hippocampus (E; Mann–Whitney test, p = 0.757; n(SAL) = 7, n(KAI) = 9) or cortex (J; unpaired t test, p = 0.556; n = 8) of ovariectomized mice. Kv4.2 protein was normalized to β-tubulin on the same blot, Kv4.2 mRNA levels were normalized to Gapdh mRNA, and miR-324-5p total levels were normalized to RU19 or miR-91 in the same samples. SAL, saline; KAI, kainic acid; a.u., arbitrary units. Bars and error bars represent the mean ± SEM.
Tables
- Table 1
No significant correlations between Kv4.2 protein and mRNA levels and 17β-estradiol or progesterone in hippocampus or cortex of 6- to 8-week-old saline-treated female mice
17β-
EstradiolProgesterone Proestrus/
estrousHippocampus Protein r = −0.20 r = −0.09 r = 0.27 R2 = 0.04 R2 = 0.00 R2 = 0.08 p = 0.38 p = 0.6 p = 0.2 n = 21 n = 29 n = 21 mRNA r = −0.12 r = 0.13 r = −0.06 R2 = 0.01 R2 = 0.02 R2 = 0.004 p = 0.64 p = 0.55 p = 0.81 n = 17 n = 25 n = 17 Cortex Protein r = 0.03 r = 0.16 r = −0.02 R2 = 0.00 R2 = 0.03 R2 = 0.00 p = 0.89 p = 0.42 p = 0.92 n = 20 n = 28 n = 20 mRNA r = −0.18 r = −0.08 r = −0.05 R2 = 0.03 R2 = 0.006 R2 = 0.003 p = 0.48 p = 0.71 p = 0.84 n = 17 n = 24 n = 17
Figure 1-1
Kv4.2 protein levels and microRNA-induced silencing of Kv4.2 mRNA are unchanged in the cortex of female mice 90 min following kainic acid-induced seizure. A, Timeline of kainic acid injection and tissue harvest. B, C, Cortical Kv4.2 protein levels are unchanged in female mice 90 min following injection of 15 mg/kg, i.p., kainic acid (B; unpaired t test, p = 0.937, n = 32/group). Similarly, Kv4.2 mRNA levels are unchanged (C; unpaired t test, p = 0.372, n = 26; 3 statistical outliers removed from the saline group). Kv4.2 protein levels were normalized to β3-tubulin levels on the same blot, and Kv4.2 mRNA was normalized to Gapdh mRNA in the same samples. D, RISC association of Kv4.2 mRNA is not changed after kainic acid in the cortex of female mice (unpaired t test, p = 0.226; n(SAL) = 29, n(KAI) = 30; 1 statistical outlier removed from kainic acid group). E, F, Cortical RISC association of the Kv4.2-targeting microRNA miR-324-5p shows a trend towards being reduced in female mice following kainic acid (E; unpaired t test, p = 0.062; n(SAL) = 32, n(KAI) = 29), and miR-324-5p levels are unchanged (F; unpaired t test, p = 0.838, n(SAL) = 31, n(KAI) = 27; 2 statistical outliers removed from the kainic acid group). In F, miR-324-5p total levels were normalized to RU19 or miR-91 in the same samples. a.u., Arbitrary units. Bars and error bars represent the mean ± SEM. Analyses in hippocampal tissue are shown in Figure 1. Download Figure 1-1, TIF file.
Figure 2-1
MiR-324-5p inhibition does not change numbers or duration of seizures after kainic acid treatment in female mice, and miRNA-induced silencing of Kv4.2 does not correlate with seizure severity. A, B, Number (A) and duration (B) of seizures within the first 30 min after kainic acid injection are not significantly changed after treatment with miR-324-5p antagomirs compared with scrambled controls (unpaired t tests, n = 7; A, p = 0.35; B, p = 0.79). C, D, Association of Kv4.2 mRNA with the RISC is significantly increased after kainic acid-induced seizure in miR-324-5p antagomir-treated mice compared with scrambled antagomir-treated mice in the hippocampus (C), but no the cortex (D; unpaired t tests, n = 7; C, p = 0.965). Bars and error bars represent the mean ± SEM. E–H, No significant correlations of Kv4.2 mRNA RISC association (E, G) and Kv4.2 protein (F, H) in hippocampus or cortex with latency to seizure (E, F) or time seizing (G, H; Pearson’s correlations; E: hippocampus: r = 0.46, R2 = 0.21, p = 0.11; n = 13; cortex: r = 0.41, R2 = 0.17, p = 0.14; n = 14; F: hippocampus: r = –0.14, R2 = 0.02, p = 0.63; n = 14; cortex: r = 0.02, R2 < 0.001, p = 0.94; n = 15; G: hippocampus: r = –0.34, R2 = 0.11, p = 0.31; n = 11; cortex: r = 0.16, R2 = 0.02, p = 0.65; n = 11; H: hippocampus, r = 0.02, R2 < 0.01, p = 0.94; n = 11; cortex: r = 0.11, R2 = 0.01, p = 0.73; n = 12). I, J, Likewise, no significant correlations between hippocampal or cortical Kv4.2 mRNA and latency to seizure (I; Pearson’s correlations; hippocampus: r = –0.43, R2 = 0.18, p = 0.13; n = 14; cortex: r = 0.42, R2 = 0.42, p = 0.14; n = 13) or time seizing (J; Pearson’s correlations; hippocampus: r = 0.08, R2 < 0.01, p = 0.80; n = 12; cortex: r = –0.30, R2 = 0.41, p = 0.37; n = 11). Dashed lines indicate 95% confidence intervals. MiR-324-5p antagomir-treated mice are represented as red dots, and scrambled antagomir-treated mice as black dots. Pearson’s correlation statistics are also shown in the graphs in E–J. Sample sizes in E–J differ because in some groups, certain samples were lost or excluded due to experimental failure, or lack of EEG (see Materials and Methods). Additional EEG analyses, as well as Kv4.2 protein levels in hippocampus and cortex after antagomir and kainic acid treatment are shown in Figure 2. Download Figure 2-1, TIF file.
Figure 3-1
Stereotaxically delivered antagomirs combined with kainic acid-induced seizures do not change Kv4.2 protein levels or RISC association of Kv4.2 mRNA in the cortex of female mice but change Kv4.2 mRNA levels. A, Illustration depicting the experimental timeline. MiR-324-5p-specific or scrambled antagomirs were stereotaxically intracerebroventricularly injected. Twenty-four hours later, 15 mg/kg kainic acid was intraperitoneally injected and brains were dissected 90 min later. B, Kv4.2 protein levels were not significantly changed in the cortex of female mice by antagomir or kainic acid treatment (2-way ANOVA; interaction: F(1,20) = 0.04, p = 0.846; effect of antagomir: F(1,20) = 0.34, p = 0.564; effect of kainic acid: F(1,20) = 0.28, p = 0.61; n = 6/group). Kv4.2 protein levels were normalized to β3-tubulin levels on the same blot. C, Likewise, RISC association of Kv4.2 mRNA was not significantly changed in the cortex of female mice by antagomir or kainic acid treatment (2-way ANOVA; interaction: F(1,28) = 0.03, p = 0.873; effect of antagomir: F(1,28) = 0.42, p = 0.521; effect of kainic acid: F(1,28) = 2, p = 0.168; n = 8). D, In contrast, Kv4.2 mRNA levels were significantly increased by seizure in the cortex of scrambled antagomir-injected female mice, but not in those of miR-324-5p-specific antagomir-injected female mice (2-way ANOVA with Dunnett’s multiple-comparison tests; interaction: F(1,27) = 16.4, *p = 0.004; effect pf antagomir: F(1,27) = 4.02, p = 0.0.055; effect of kainic acid: F(1,27) = 2, p = 0.168, *p = 0.0015; n = 8, except for a324/kA, n = 7). Kv4.2 mRNA levels were normalized to Gapdh mRNA. a.u., Arbitrary units. Bars and error bars represent the mean ± SEM. Analyses in hippocampal tissue are shown in Figure 3. Download Figure 3-1, TIF file.
Figure 3-2
Sham surgeries blunt the effect of kainic acid-induced seizures on Kv4.2 protein levels in female mice. A, Illustration depicting the experimental timeline. ACSF (vehicle) was stereotaxically intracerebroventricularly injected. Twenty-four hours later, 15 mg/kg kainic acid was intraperitoneally injected, and brains were dissected 90 min later. B, Kv4.2 protein levels were not significantly changed in the hippocampus or cortex of female mice by kainic acid treatment 24 h after a sham surgery (hippocampus: unpaired t test, p = 0.413; n = 8; cortex: Mann–Whitney test, p = 0.593; n = 8). Kv4.2 protein levels were normalized to β3-tubulin levels on the same blot. Example blots shown on top. C, Likewise, RISC association of Kv4.2 mRNA was not significantly changed in the hippocampus or cortex of sham female mice by kainic acid treatment (unpaired t tests: p(hippocampus) = 0.598, p(cortex) = 0.460; n = 8). D, Kv4.2 mRNA levels were unchanged in hippocampus or cortex of sham intracerebroventricularly injected female mice after kainic acid-induced seizures (unpaired t tests: p(hippocampus) = 0.932, p(cortex) = 0.633; n = 8). Kv4.2 mRNA levels were normalized to Gapdh mRNA. E, RISC association of miR-324-5p was not significantly changed in the hippocampus or cortex of sham female mice by kainic acid treatment (unpaired t tests: p(hippocampus) = 0.714, n(SAL) = 7, n(KAI) = 8; p(cortex) = 0.076; n = 8). F, MiR-324-5p levels were unchanged in hippocampus of sham intracerebroventricularly injected female mice after kainic acid-induced seizures, but significantly reduced in the cortex (unpaired t tests; hippocampus: p = 0.176; n(SAL) = 7, n(KAI) = 8; one statistical outlier removed from the saline group; cortex: unpaired t test: p = 0.050, n = 8). MiR-324-5p levels were normalized to miR-191. a.u., Arbitrary units. Bars and error bars represent the mean ± SEM. Analyses of antagomir-injected mice in hippocampal and cortical tissue are shown in Figure 3 and Extended Data Figure 3-1, respectively. Download Figure 3-2, TIF file.
Figure 4-1
RISC association of the voltage-gated potassium channels Kv7.2 and Kv7.3 in the hippocampus are not correlated with plasma levels of progesterone or 17β-estradiol. A–C, Kv7.2 mRNA association with the RISC in hippocampal lysates from female mice is not significantly correlated with plasma levels of progesterone (A; Pearson’s correlation: r = 0.22, R2 = 0.05, p = 0.54; n = 10), 17β-estradiol (B; Pearson’s correlation: r = 0.41, R2 = 0.17, p = 0.24; n = 10), and progesterone/17β-estradiol ratios (C; Pearson’s correlation: r = –0.03, R2 = 0.001, p = 0.93; n = 10). D–F, Likewise, RISC association of Kv7.3 mRNA is not significantly correlated with progesterone (D; Pearson’s correlation: r = 0.24, R2 = 0.06, p = 0.54; n = 9), 17β-estradiol (E; Pearson’s correlation: r = 0.48, R2 = 0.23, p = 0.19; n = 9), or progesterone/17β-estradiol ratios (F; Pearson’s correlation: r = –0.07, R2 = 0.006, p = 0.85; n = 9). Dashed lines indicate 95% confidence intervals. Pearson’s correlation statistics are also shown in the figure. Corresponding analyses of Kv4.2 are shown in Figure 4. Download Figure 4-1, TIF file.
Figure 6-1
In the cortex, treatment with kainic acid abolishes all significant correlations of microRNA-induced silencing of Kv4.2 with plasma levels of progesterone and 17β-estradiol. A–C, Ninety minutes following kainic acid treatment, Kv4.2 mRNA association with the RISC in cortical lysates from female mice is not significantly correlated with plasma levels of progesterone (A; Pearson’s correlation: r = –0.05, R2 = 0.003, p = 0.80; n = 27), 17β-estradiol (B; Pearson’s correlation: r = 0.02, R2 = 0.0004, p = 0.94; n = 16), and progesterone/17β-estradiol ratios (C; Pearson’s correlation: r = –0.20, R2 = 0.04, p = 0.47; n = 16). D–F, Likewise, no significant correlations between RISC association of miR-324-5p with plasma progesterone (D; Pearson’s correlation: r = –0.20, R2 = 0.04, p = 0.33; n = 25), 17β-estradiol was detected (E, Pearson’s correlation: r = 0.07, R2 = 0.005, p = 0.79; n = 16), and progesterone/17β-estradiol ratios (F; Pearson’s correlation: r = –0.11, R2 = 0.01, p = 0.70; n = 16) was observed. Dashed lines indicate 95% confidence intervals. Pearson’s correlation statistics are also shown in the figure. Analyses in hippocampal tissue are shown in Figure 6. Download Figure 6-1, TIF file.
Figure 7-1
No significant differences in miR-324-5p association with the RISC in hippocampus or cortex, and no changes in Kv4.2 protein expression and Kv4.2 mRNA association with the RISC in the cortex across estrous cycle stages and depending on kainic acid treatment. A, Kv4.2 protein levels in the cortex not significantly different across estrous stages in female saline-treated or kainic acid-treated mice (two-way ANOVA; interaction: F(2,29) = 1.4, p = 0.26; effect of estrous stage: F(2,29) = 1.2, p = 0.307; effect of kainic acid: F(1,29) = 0.06, p = 0.807; n(proestrus+estrus saline) = 5, n(proestrus+estrus kainic acid) = 7, n(diestrus saline) = 8, n(diestrus kainic acid) = 5, n(metestrus saline) = 5, and n(metestrus kainic) = 5). Kv4.2 was normalized to β3-tubulin signal on the same blot. B, Kv4.2 mRNA association with the RISC in the cortex is not significantly different across estrous stages in female saline-treated or kainic acid-treated mice (two-way ANOVA; interaction: F(2,24) = 1.8, p = 0.19; effect of estrous stage: F(2,24) = 1.5, p = 0.23; effect of kainic acid: F(1,24) = 0.8, p = 0.37; n(proestrus+estrus saline) = 2, n(proestrus+estrus kainic acid) = 7, n(diestrus saline) = 7, n(diestrus kainic acid) = 5, n(metestrus saline) = 5, and n(metestrus kainic) = 4). C, MiR-324-5p association with the RISC in the hippocampus is not significantly different across estrous stages in female saline-treated or kainic acid-treated mice (two-way ANOVA; interaction: F(2,14) = 1, p = 0.39; effect of estrous stage: F(2,14) = 0.58, p = 0.57; effect of kainic acid: F(1,14) = 1.5, p = 0.24; n(proestrus+estrus saline) = 3, n(proestrus+estrus kainic acid) = 4, n(diestrus saline) = 3, n(diestrus kainic acid) = 3, n(metestrus saline) = 5, and n(metestrus kainic) = 2). D, MiR-324-5p association with the RISC in the cortex is not significantly different across estrous stages in female saline-treated or kainic acid-treated mice (two-way ANOVA; interaction: F(2,27) = 1.4, p = 0.272; effect of estrous stage: F(2,27) = 0.29, p = 0.75; effect of kainic acid: F(1,27) = 1.5, p = 0.22; n(proestrus+estrus saline) = 5, n(proestrus+estrus kainic acid) = 6, n(diestrus saline) = 7, n(diestrus kainic acid) = 4, n(metestrus saline) = 5, and n(metestrus kainic) = 6). +E, Proestrus and estrous; D, diestrus; M, metestrus. No mice in proestrus were identified in the saline group. Mice in proestrus in the kainic group are indicated as gray triangles in the bar diagram. The mice used for this analysis are a subset of the mice analyzed in Figures 1, 4, and 6. Analyses of Kv4.2 protein and Kv4.2 mRNA association with the RISC are shown in Figure 7. Download Figure 7-1, TIF file.
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