Pilocarpine-induced SE in Cre+ and Cre-mice

  1. The experimental timeline is shown.

    1. Tamoxifen was injected 1/day for 5 days in 6 week-old Nestin-CreERT2 Baxfl/flmice. 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 groups 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.

  2. 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.

  3. Representative examples of 10 hr-long EEG for a Cre-(1) and Cre+ mouse (2).

  4. 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).

  5. 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).

Reduced chronic seizures in Cre+ mice

  1. 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.

  2. Representative examples of 2 min-long EEG segments show a seizure in a Cre-(1, 3) and Cre+ (2, 4) mouse.

  3. 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).

  4. 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).

  5. 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.

  6. 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.

Reduced postictal depression in Cre+ female mice

  1. Postictal depression in a male mouse.

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

    2. The baseline (a) and postictal period (b) are expanded. Postictal depression (PID) is defined by a decrease in the peak -to-trough amplitude (double-sided blue arrows) of the baseline relative to the postictal period. For the male mouse, there was no clear change.

  2. Postictal depression in a female mouse

    1. A seizure in a female mouse.

    2. Comparisons of the baseline and postictal period show a postictal depression.

  3. Comparisons of postictal depression in each experimental group.

    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.

  4. The same data are plotted as in C, but the percentages are shown instead of the numbers of seizures.

Temporal dynamics of chronic seizures

  1. 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.

  2. 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.

  3. 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.

  4. 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.

Increased DCX in Cre+ mice

  1. The experimental timeline is shown.

  2. Representative examples of doublecortin (DCX) immunoreactivity (ir).

    1. Cre-mouse.

    2. Cre+ mouse. The red boxes in a are expanded in b. Calibration, 100 μm (a); 50 μm (b).

  3. DCX quantification.

    DCX-ir within a region of interest (ROI) including the GCL and SGZ (yellow lines denote the ROI) was thresholded and DCX-ir is shown in red. Calibration, 100 μm (a); 50 μm (b).

  4. Cre+ mice had increased DCX-ir.

    1. The area of DCX-ir relative to the area of the ROI (referred to as area fraction) was significantly greater in Cre+ than Cre-mice (t-test, p=0.041).

    2. When sexes were separated, Cre+ females showed significantly greater DCX-ir than Cre-females (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 (p=0.498).

Hilar Prox1-ir cells increased in Cre+ mice

  1. 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).

  2. 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. Red arrows point to hilar ectopic granule cells. Calibration, 100 μm.

    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).

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

  1. Preserved GluR2/3+ hilar cells (mossy cells) in Cre+ female mice.

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

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

    • 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).

  2. Preserved hilar SOM cells in Cre+ female mice.

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

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

    • 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).

  3. Cre+ and Cre-mice have similar numbers of parvalbumin interneurons.

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

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

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

Cre+ mice had less neuronal loss in hippocampus after SE

  1. 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).

  2. Examples of Fluorojade-C staining in CA1 of Cre+ (1) and Cre-(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.

  3. Examples of Fluorojade-C staining in the subiculum of Cre+ (1) and Cre-(2) mice. Arrows point to numerous Fluorojade-C-stained neurons in Cre-mice but not Cre+ mice. Calibration, 200 μm.

  4. Quantification.

    1. Fluorojade-C was thresholded and the pyramidal cell layer outlined in yellow. The fraction above threshold relative to the entire ROI (area fraction) was calculated.

    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).

Associated with Fig. 1. Examples of EEG during SE

  1. Representative examples of a 10 hr-long EEG recording are shown for Cre-(1) and Cre+ (2) mice.

  2. 10 min-long EEG recording segments from the left hippocampus A are shown with higher temporal gain.

    1. Cre-mouse.

      1. Part of the baseline is shown. The area surrounded by the red box is expanded in C1a.

      2. The time when DZP was injected is shown. The area surrounded by the red box is expanded in C1b.

      3. The time following the seizure is shown. The area surrounded by the red box is expanded in C1c.

    2. Cre+ mouse.

      1. Part of the baseline is shown. The area surrounded by the red box is expanded in C2a. Note the baselines are similar in the two mice, suggesting no effect of genotype.

      2. The time when DZP was injected is shown. The area surrounded by the red box is expanded in C2b. Note there was a reduction in EEG amplitude in the Cre+ mouse.

      3. The time at the end of SE is shown. The area surrounded by the red box is expanded in C2c. Note that these two mice were similar after SE ended.

  3. The areas surrounded by the red boxes in B are expanded. The traces are 1 min-long.

Associated with Fig. 1. Incidence of SE in the unimplanted and implanted mice

  1. The experimental timelines of pilocarpine injection and surgery.

    1. Unimplanted mice.

    2. Implanted mice. Only the timing of surgery with respect to pilocarpine injection was different.

  2. The incidence of SE in unimplanted and implanted mice is shown based on numbers of mice. The incidence of SE was significantly higher in the unimplanted mice (Fisher’s exact test, p<0.0001). Genotype had no effect on the incidence of SE.

  3. The incidence of SE is shown as percentages.

Associated with Fig. 1. Power during SE in Cre+ female and Cre-female mice

  1. Power was calculated for consecutive 20 min-long bins before and during SE. Power in the 1-4 Hz band was decreased during SE in female Cre+ mice (blue triangles) relative to Cre-mice (red circles) but it was not statistically significant.

  2. Power in the 4-8 Hz band.

  3. Power in 8-30 Hz range.

  4. Power in 30-80 Hz range

  5. Power between 80 and 100 Hz.

Associated with Fig. 2. Results were independent of the time of implantation

  1. The number of chronic seizures were similar between mice that were implanted before and after SE (p=0.265).

  2. The frequency of seizures was similar between mice implanted before or after SE (p=0.276).

Associated with Fig. 2. Mortality was not significantly affected by genotype or sex

  1. The timeline of measurements of mortality is shown. Mice were categorized as dying during SE or within 3 days of SE (0-3 days), 3 days to 7 weeks, or both (Sum). Mice are included whether they were implanted with EEG electrodes before SE or implanted 3 weeks after SE.

    1. The numbers of mice that died were not significantly different between genotypes or sexes (Fisher’s Exact test, all p>0.05).

    2. The percent of mice that died is shown.

Associated with Figure 5. DCX before and after SE

  1. Normal conditions

    1. Timeline of experiments to confirm that Cre+ mice had increased DCX-ir compared to Cre-mice in normal conditions.

    2. Cre+ mice had significantly more DCX-ir in the GCL/SGZ compared to Cre-mice. The grey bar is for comparison: data were from a previous study with the same mice (Jain et la., 2019).

  2. After SE and chronic seizures

    1. Timeline of experiments for DCX-ir analysis after SE and chronic seizures. Mice were perfused after the 3 week-long recording session, 7 weeks after SE.

    2. Data are shown from Fig. 5 to compare mice after SE next to mice before SE (left, A2). Cre-female mice after SE had reduced DCX-ir compared to before SE (∼2 after vs. ∼4% before). Cre-males did not show this reduction (∼4% before and after SE). Therefore, there was an inherent difference in sexes in that Cre-females lost DCX-ir in chronic epilepsy but males did not. If loss of DCX-ir is reflects loss of immature GCs, Cre-females may have had more loss of young GCs after SE than males. If the death was due to a Bax-dependent process (apoptosis), the results could be explained by a greater effect of Bax deletion in the females than males.

  3. A model is used to illustrate the suggested explanation for data in A2 and B2. Before SE, all Cre-mice had similar DCX-ir. Area fraction was ∼4% so 4 cells are shown for simplicity. Only young-adult born neurons are shown (blue circles, each reflecting 1%). After SE, there is proliferation of new cells so 6 cells are shown instead of 4. It is assumed that SE-induced proliferation is similar in females and males because the data reported to date have not suggested any sex difference to our knowledge. After chronic epilepsy, the data in B2 show that Cre-females had reduced DCX-ir (∼2%) relative to Cre-males (∼4%). This could be due to programmed cell death of some of the neurons that proliferated after SE, which has been suggested (Parent et al., 1997). Based on these assumptions, more cells died of apoptosis after SE in female Cre-females than Cre-males, a sex difference. Black is used to indicate the young-adult-born neurons that died. This greater sensitivity to apoptosis, a Bax dependent process, may explain why Cre+ females with Bax deletion showed more DCX-ir after SE than Cre-females, but males did not.

Associated with Figure 6. Hilar ectopic GCs have different effects depending on survival of hilar mossy cells and SOM cells

  1. A hypothesis is diagrammed to explain how hilar ectopic GCs can promote seizures in some conditions but not do so in other conditions.

    1. In control conditions, there are mature (grey) and immature (blue) GCs in the GCL. Immature GCs are primarily at the border of the GCL and hilus. In the hilus, the primary cell types are glutamatergic mossy cells (green) and SOM-expressing cells (black).

    2. After SE, more immature cells arise, and some are in the hilus where they form abnormal circuitry in response to the loss of mossy cells and SOM cells.

    3. Hypothesis. Hilar ectopic GCs develop and their axons form excitatory projections to other excitatory cells, such as GCL GCs. Hilar cell loss occurs, and stimulates hilar ectopic GCs to form synapses on areas of GCL GCs that are vacated, such as the inner molecular layer. Indeed hilar ectopic GCs do form such connections(Scharfman et al. 2000; Pierce et al. 2005). GCL GCs form dense synapses on hilar ectopic GCs (Pierce et al. 2005). These new excitatory feedback circuits could contribute to more seizures (Scharfman & Hen 2007; Parent & Murphy 2008; Scharfman & McCloskey 2009).

    4. In mice with suppressed adult neurogenesis, few immature cells are in the GCL.

    5. After SE, there are fewer hilar GCs. There is loss of hilar ectopic GCs according to studies of Cho et al. (2015).

    6. Hypothesis: With few hilar ectopic GCs there are fewer recurrent excitatory circuits and therefore fewer seizures.

    7. In mice with deletion of Bax in Nestin-expressing progenitors, there are more immature GCs in the GCL, reflected by DCX. The data are from the present study.

    8. After SE, there are more hilar GCs, reflected by hilar Prox1. Hilar mossy and SOM cells are preserved based on hilar GluR2/3 and SOM immunofluorescence. There may be little seizure-promoting effect of hilar GCs in light of mossy and SOM cell preservation.

    9. Hypothesis: There are fewer seizures because less hilar damage, there is less available space for axons to find new targets. This hypothesis is based in part on long-standing notions that axonal innervation is a competitive process, and on prior studies considered hilar cell loss a stimulus for GCL GCs to sprout new axon collaterals (mossy fiber sprouting) in animal models of epilepsy and TLE (Isokawa et al. 1993; Buckmaster 2012; Schmeiser et al. 2017).

Associated with Figure 7. Additional analyses of GluR2/3, SOM and parvalbumin-expressing cells

  1. GluR2/3 hilar cells lacked Prox1 expression.

    1. Cre-female mouse. Left: several GluR2/3+ cells (green) are located in the hilus within the box (marked by dotted white lines). Calibration, 70 µm. Right: The area within the box in the left panel is expanded. The merged image of GluR2/3+ (green) and Prox1+ (red) cells shows no double labeling. Calibration, 40 µm.

    2. Cre + female mouse. Similar results are shown as for the Cre-mouse. White arrows mark ectopic GCs. Calibrations are the same as for the Cre-mouse.

  2. A comparison of dorsal and ventral measurements for Cre- and Cre+ male mice show no significant genotype effects.

    1. GluR2/3. A two-way ANOVA showed no effect of dorsal or ventral location (F(1,13) = 3.38; p = 0.089) or genotype (F(1,13) = 1.158; p = 0.302).

    2. SOM. A two-way ANOVA showed no effect of dorsal or ventral location (F(1,10) = 0.172; p = 0.687) or genotype (F(1,10) = 0.014; p = 0.908).

    3. Parvalbumin. A two-way ANOVA showed no effect of dorsal or ventral location (F(1,13) = 0.358; p = 0.560) or genotype (F(1,13) = 1.068 p = 0.320).

  3. A comparison of ventral measurements for both Cre- and Cre+ female and male mice.

    1. There were significantly more GluR2/3+ hilar cells in Cre+ female mice compared to Cre-female mice, like the dorsal hippocampus (Fig. 7). Thus, GluR2/3+ hilar cells were spared in Cre+ females in dorsal and ventral hippocampus. A two-way ANOVA showed o effect of sex (F(1,18) = 0.744; p = 0.400) or genotype F(1,18) = 0.386; p = 0.542) but there was a significant interaction F(1,18) = 5.433; p = 0.0316, and post-hoc tests showed that Cre+ females had significantly more GluR2/3+ cells than Cre-females (p = 0.045).

    2. There were no significant differences among groups for SOM+ cells. Thus, there was an effect in dorsal (Fig. 7) but not ventral hippocampus. Thus, SOM cells were spared in Cre+ females dorsally but not ventrally. A two-way ANOVA showed no effect of sex (F(1,17) = 0.718; p = 0.408) or genotype F (1, 17) = 0.769; P=0.393).

    3. There were no significant differences in numbers of parvalbumin+ cells, like dorsal hippocampus (Fig. 7). Thus, parvalbumin cells were similar regardless of genotype in dorsal and ventral hippocampus. A two-way ANOVA showed no significant effect of sex (F(1,16) = 0.401; p = 0.536) or genotype (F(1,16) = 0.221; p = 0.645).