Increasing adult-born neurons protects mice from epilepsy

Abstract

Neurogenesis occurs in the adult brain in the hippocampal dentate gyrus, an area that contains neurons which are vulnerable to insults and injury, such as severe seizures. Previous studies showed that increasing adult neurogenesis reduced neuronal damage after these seizures. Because the damage typically is followed by chronic life-long seizures (epilepsy), we asked if increasing adult-born neurons would prevent epilepsy. Adult-born neurons were selectively increased by deleting the pro-apoptotic gene Bax from Nestin-expressing progenitors. Tamoxifen was administered at 6 weeks of age to conditionally delete Bax in Nestin-CreER^T2 Bax ^fl/fl mice. Six weeks after tamoxifen administration, severe seizures (status epilepticus; SE) were induced by injection of the convulsant pilocarpine. After mice developed epilepsy, seizure frequency was quantified for 3 weeks. Mice with increased adult-born neurons exhibited fewer chronic seizures. Postictal depression was reduced also. These results were primarily in female mice, possibly because they were the more affected by Bax deletion than males, consistent with sex differences in Bax. The female mice with enhanced adult-born neurons also showed less neuronal loss of hilar mossy cells and hilar somatostatin-expressing neurons than wild type females or males, which is notable because these two hilar cell types are implicated in epileptogenesis. The results suggest that selective Bax deletion to increase adult-born neurons can reduce experimental epilepsy, and the effect shows a striking sex difference. The results are surprising in light of past studies showing that suppressing adult-born neurons can also reduce chronic seizures.

Keywords: Bax; dentate gyrus; ectopic granule cell; mossy cell; pilocarpine; sex differences; somatostatin; temporal lobe epilepsy.

Figure 1.. Pilocarpine-induced SE in Cre+ and Cre− mice.

A. The experimental timeline is shown.

1. Tamoxifen was injected 1/day for 5 days in 6 week-old Nestin-CreER^T2 Bax^fl/fl mice. Six weeks after the last tamoxifen injection, mice were injected with pilocarpine (Pilo) at a dose that induces status epilepticus (SE).

2. On the day of pilocarpine injection, one group of mice without EEG electrodes were monitored for behavioral seizures for 2 hr after pilocarpine injection. Another group of mice were implanted with EEG electrodes 3 weeks prior to pilocarpine injection. In these mice, video-electroencephalogram (video-EEG) was used to monitor SE for 10 hr after pilocarpine injection.

B. Locations to implant EEG electrodes are shown. Four circles represent recording sites: left frontal cortex (Lt FC), left hippocampus (Lt HC), right hippocampus (Rt HC) and right occipital cortex (Rt OC). Two diamonds represent ground (GRD) and reference (REF) electrodes. D. Pooled data for mice that were implanted with EEG electrodes and unimplanted mice. These data showed no significant genotypic differences but there was a sex difference.

1. The latency to the onset of first seizure was similar in both genotypes (t-test, p=0.761). The seizure was a behavioral seizure ≥stage 3 of the Racine scale (unilateral forelimb jerking). For this figure and all others, detailed statistics are in the Results.

2. The number of seizures in the first 2 hr after pilocarpine injection was similar in both genotypes (t-test, p=0.377).

3. After separating males and females, females showed a shorter latency to the onset of the first seizure compared to males (two-way ANOVA, p=0.043); Cre+ females had a shorter latency to the first seizure relative to Cre+ males (Bonferroni’s test, p=0.010).

4. The number of seizures in the first 2 hr after pilocarpine injection were similar in males and females (two-way ANOVA, p=0.436).

E. Implanted mice. These data showed a significant protection of Cre+ mice on SE duration.

1. The severity of the first seizure (non-convulsive or convulsive) was similar between genotypes (Chi-square test, p=0.093).

2. Cre+ mice had a shorter duration of SE than Cre− mice (t-test, p=0.007).

3. After separating males and females, the first seizure was mostly non-convulsive in Cre+ females compared to Cre− females (60% vs. 14%) but no groups were statistically different (Fisher’s exact tests, p>0.05).

4. Once sexes were separated, there was no effect of sex by two-way ANOVA but a trend in Cre+ males to have a shorter SE duration than Cre− males (Bonferroni’s test, p=0.078).

Figure 2.. Reduced chronic seizures in Cre+ mice.

A. The experimental timeline is shown. Six weeks after pilocarpine injection, continuous video-EEG was recorded for 3 weeks to capture chronic seizures. Mice that were unimplanted prior to SE were implanted at 2–3 weeks after pilocarpine injection. B. Representative examples of 2 min-long EEG segments show a seizure in a Cre− (1, 3) and Cre+ (2, 4) mouse. C. Numbers of chronic seizures.

1. Pooled data of females and males showed no significant effect of genotype on chronic seizure number. The total number of seizures during 3 weeks of recording were similar between genotypes (t-test, p=0. 882).

2. After separating data based on sex, females showed fewer seizures. Cre+ females had fewer seizures than Cre− females (Bonferroni’s test, p=0.004). There was a sex difference in control mice, with fewer seizures in Cre− males compared to Cre− females (Bonferroni’s test, p<0.001).

D. Chronic seizure frequency.

1. Pooled data of females and males showed no significant effect of genotype on or chronic seizure frequency. The frequency of chronic seizures (number of seizures per day) were similar (Welch’s t-test, p=0.717).

2. Seizure frequency was reduced in Cre+ females compared to Cre− females (Bonferroni’s test, p=0.004). There was a sex difference in control mice, with lower seizure frequency in Cre− males compared to Cre− females (Bonferroni’s test, p<0.001).

E. Seizure duration per mouse.

1. Each data point is the mean seizure duration for a mouse. Pooled data of females and males showed no significant effect of genotype on seizure duration (t-test, p=0.379).

2. There was a sex difference in seizure duration, with Cre− males having longer seizures than Cre− females (Bonferroni’s test, p=0.005). Because females exhibited more postictal depression (see Fig. 3), corresponding to spreading depolarization (Ssentongo et al. 2017), the shorter female seizures may have been due to truncation of seizures by spreading depolarization.

F. Seizure durations for all seizures.

1. Every seizure is shown as a data point. The durations were similar for each genotype (Mann-Whitney U test, p=0.079).

2. Cre+ females showed longer seizures than Cre− females (Dunn’s test, p<0.001). Cre+ females may have had longer seizures because they were protected from spreading depolarization.

Figure 3.. Reduced postictal depression in Cre+ female mice.

A.

1. A seizure of a male mouse and female mouse are shown to illustrate postictal depression starting at the end of the seizure (red arrow).

2. All 4 channels are shown for the male (left) and female mouse (right). Rt OC, right occipital cortex; Lt FC, left frontal cortex; Lt HC, left hippocampus; Rt HC, right hippocampus. The red arrows point to the end of the seizure.

3. The areas in A2 marked by the red bar are expanded. The blue double -sided arrows reflect the mean EEG amplitude before (a, c) and after the seizure (b,d).

B. For all spontaneous recurrent seizures (SRS) in the 3 week-long recording period, there was a significant difference between groups, with number of SRS with PID reduced in Cre+ females compared to Cre− females (Fisher’s exact test, all p <0.05). Males had very little postictal depression and there was no significant effect of genotype. C. The same data are plotted but the percentages are shown instead of the numbers of seizures.

Figure 4.. Temporal dynamics of chronic seizures.

A. Each day of the 3 weeks-long EEG recording periods are shown. Each row is a different mouse. Days with seizures are coded as black boxes and days without seizures are white. B.

1. The number of days with seizures were similar between genotypes (t-test, p=0.822).

2. The maximum seizure-free interval was similar between genotypes (t-test, p=0.107).

3. After separating females and males, two-way ANOVA showed no effect of genotype or sex on days with seizures.

4. Two-way ANOVA showed no effect of genotype or sex on the maximum seizure-free interval.

C. Then same data are shown but days with ≥3 seizures are black, days with < 3 seizures as grey, and are white. Clusters of seizures are reflected by the consecutive black boxes. D.

1. The cluster durations were similar between genotypes (Mann-Whitney’s U test, p=0.723).

2. The maximum inter-cluster interval was similar between genotypes (t-test, p=0.104.

3. Cre+ females had significantly fewer clusters than Cre− females (two-way ANOVA followed by Bonferroni’s test, p=0.009). There was a sex difference, with females having more clusters than males. Cre− females had more days with >3 seizures than control males (Cre− females: 6.3 ± 1.4 days; Cre− males: 2.3 ± 0.5 days; Bonferroni’s test, p < 0.001).

4. There was no significant effect of genotype or sex on the maximum inter-cluster interval. However, there was a trend for the inter-cluster interval to be longer in Cre+ females relative to than Cre− females.

Figure 5.. Increased DCX in Cre+ mice.

A-B. The experimental timelines are shown. A. Mice were perfusion-fixed 6 weeks after tamoxifen injection, just before SE. Sections were then stained for DCX. B. Mice were tested 2 months after SE, after EEG recording. Then mice were perfused and staining was conducted for DCX. C. DCX quantification. DCX-ir within a region of interest (ROI; yellow lines) including the SGZ and GCL was thresholded. DCX-ir above the threshold is shown in red. Calibration, 100 μm (a); 50 μm (b). The inset is expanded to the right. D. The area of DCX-ir relative to the area of the ROI (referred to as area fraction) was greater in Cre+ mice compared to Cre− mice. Two-way ANOVA followed by Tukey pot-hoc tests, all p<0.05). E. Cre+ mice had increased DCX-ir relative to Cre− mice 2 months after SE.

1. Sexes were pooled. The area fraction of DCX-ir was greater in Cre+ than Cre− mice (t-test, p=0.041).

2. When sexes were separated, Cre+ females showed greater DCX-ir than Cre− females (two-way ANOVA followed by Bonferroni’s test, p=0.015). There was a sex difference, with Cre− males showing more DCX-ir than Cre− females (Bonferroni’s test, p=0.007). DCX-ir was similar in Cre− and Cre+ males (Bonferroni’s test, p=0.498).

F. Representative examples of DCX-ir 2 months after SE.

1. Cre− female mouse.

2. Cre+ female mouse. The red boxes in a are expanded in b. Arrows point to DCX-ir cells. Calibration, 100 μm (a); 50 μm (b).

Figure 6.. Hilar Prox1-ir cells increased in Cre+ mice.

A. Representative examples of hilar Prox1-ir in Cre− (1) and Cre+ (2) mice are shown. The boxes in a are expanded in b. Arrows point to hilar Prox1-ir cells, corresponding to hilar ectopic GCs. Calibration, 100 μm (a); 50 μm (b). B. Prox1-ir is shown, within a hilar ROI. The area of the ROI above the threshold, relative to the area of the ROI, is red. This area is called the area fraction, and was used to quantify hilar Prox1-ir. Calibration, 100 μm. C.

1. Cre+ mice had more hilar Prox1-ir cells than Cre− mice (t-test, p<0.001).

2. When sexes were divided, Cre+ mice had more hilar Prox1-ir cells than Cre− mice in both female (two-way ANOVA followed by Bonferroni’s test, p<0.001) and male mice (Bonferroni’s test, p=0.001).

D. Correlations between hilar Prox1-ir cells and measurements of chronic seizures.

1. All Cre− and Cre− mice were compared regardless of sex. For the Cre− mice there was a significant inverse correlation between the # of Prox1-ir cells and # of chronic seizures (R²=0.296). Thus, the more Prox1-ir cells there were, the fewer chronic seizures there were. However, that was not true for Cre+ mice (R²=0.072).

2. There was an inverse correlation between the number of hilar Prox1-ir cells and the seizure-free interval for Cre+ mice (R²=0.467) but not Cre− mice (R²=0.008). Thus, the more hilar Prox1-ir cells there were, the shorter the seizure-free periods were. However, this was not true for Cre− mice.

3. When data were divided by genotype and sex there was no significant correlation between hilar Prox1-ir cells and # of seizures (Cre− F, R²=0.0035; Cre+ F, R²=0.043; Cre− M, R²=0.104; Cre+ M, R²=0.083).

4. When data were divided by genotype and sex, there was a significant inverse correlation for the # of hilar Prox1-ir cells and seizure-free interval, but only for male Cre+ mice (R²=0.704). Cre+ females showed a trend (R²=0.395) and Cre− mice did not (Cre− F, R²=0.007, Cre− M, R²=0.046).

Figure 7.. Preserved mossy cells and hilar SOM cells in Cre+ female mice but not parvalbumin interneurons.

A.

1. 1–2. Representative examples of GluR2/3 labelling of Cre− (1) and Cre+ mice (2). Calibration, 50 μm.

2. 3. Cre+ mice had more hilar GluR2/3-immunofluorescent (positive; +) cells than Cre− mice (t-test, p=0.022). Sexes were pooled.

3. 4. After separating females and males, Cre+ females showed more hilar GluR2/3+ cells than Cre− females (Bonferroni’s test, p=0.011). Hilar GluR2/3+ cells were similar between genotypes in males (Bonferroni’s test, p=0.915).

B.

1. 1–2. Representative examples of SOM labelling in Cre− and Cre+ mice are shown. Calibration, 100 μm (a); 20 μm (b).

2. 3. In pooled data, Cre+ mice had more hilar SOM cells than Cre− mice (t-test, p=0.008).

3. 4. After separating females and males, Cre+ females showed more hilar SOM cells than Cre− females (Bonferroni’s test, p=0.019). Hilar SOM cells were similar between genotypes in males (Bonferroni’s test, p=0.897).

C.

1. 1–2. Representative examples of parvalbumin labelling in Cre− and Cre+ mice are shown. Calibration, 100 μm.

2. 3. The number of parvalbumin+ cells in the DG were similar in Cre− and Cre+ mice in pooled data (t-test, p=0.095).

3. There was no effect of genotype (p=0.096) or sex (p=0.616) on the number of DG parvalbumin+ cells.

Figure. 8.. Cre+ female mice had less neuronal loss in hippocampus after SE.

A. A timeline is shown to illustrate when mice were perfused to examine Fluorojade-C staining. All mice were perfused 10 days after SE, a time when delayed cell death occurs after SE, mainly in area CA1 and subiculum. Note that prior studies showed hilar and CA3 neurons, which exhibit more rapid cell death after SE, are protected from cell loss in Cre+ mice examined 3 days after SE (Jain et al., 2019). Also, there was protection of CA1 at 3 days (Jain et al., 2019). B. Quantification. Fluorojade-C was thresholded using ImageJ and the pyramidal cell layer outlined in yellow. The fraction above threshold relative to the entire ROI (area fraction) was calculated (see Methods) 2. The Fluorojade-C area fraction was greater in Cre− mice than Cre+ mice. Statistical comparisons showed a trend for CA1 of Cre− mice to exhibit more Fluorojade-C than Cre+ mice (Mann-Whitney U test, p=0.060). Cre− mice had a significantly greater area fraction in the subiculum than Cre+ mice (Mann-Whitney U test, p=0.032). C. Examples of Fluorojade-C staining in CA1 (top) and subiculum (bottom) of Cre+ female (1) and Cre− female (2) mice. SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum; SLM, stratum lacunosum-moleculare. Arrows point to numerous Fluorojade-C-stained neurons in Cre− mice but not Cre+ mice. Calibration, 200 μm. D.

1. Comparisons of female mice by two-way ANOVA showed an effect of genotype (F(1,15)=11.97, p=0.004) with less Fluorojade C in Cre+ mice for CA1 (p=0.016) and subiculum p=0.016).

2. Comparisons of male mice showed no significant effect of genotype on Fluorojade C in either CA1 or the subiculum (F(1,8)=0.002, p=0.965; CA1, p=0.828, subiculum, p=0.973, respectively).

3. When genotypes were pooled, female mice did not have significantly more damage than males (two-way ANOVA, sex (F(1,34)=3.16, p=0.085) and there was no effect of subfield (F(1,34)=0.0016, p=0.968).