The first experiment addressed the effect of increasing adult-born neurons on pilocarpine-induced SE in Nestin-CreER^T2Bax^fl/fl mice (called ‘Cre+,’ below). To produce Nestin-CreER^T2Bax^fl/fl mice, hemizygous Nestin-CreER^T2 mice were bred with homozygous Bax^fl/fl mice. Littermates of Cre+ mice that lacked Cre (called ‘Cre-,’ below) were also treated with tamoxifen and were controls.

Figure 1A1 shows the experimental timeline. Tamoxifen was injected s.c. once per day for 5 days to delete Bax from Nestin-expressing progenitors. After 6 weeks, a time sufficient for a substantial increase in adult-born neurons (Drew et al., 2016; Jain et al., 2019), pilocarpine was injected s.c. to induce SE.

Figure 1A2 shows the experimental timeline during the day of pilocarpine injection. The location of electrodes for EEG are shown in Figure 1B. Mice monitored with EEG were implanted with electrodes 3 weeks before SE (see Methods). Examples of the EEG are shown in Figure 1C for Cre- and Cre+ mice and details are shown in Figure 1—figure supplement 1.

The latency to the first seizure after pilocarpine injection was measured for all mice (with and without EEG electrodes; Figure 1D) or just those that had EEG electrodes (Figure 1E). When mice with and without electrodes were pooled, the latency to the onset of first seizure was similar in both genotypes (Cre-: 47.2±4.8 min, n=27; Cre+: 45.3±3.9 min, n=28; Student’s t-test, t(53)=0.3, p=0.761; Figure 1D1).

The total number of seizures was quantified until 2 hr after pilocarpine injection because at that time diazepam was administered to decrease the severity of SE. The total number of seizures were similar in both genotypes (Cre-: 3.0±0.2 seizures; Cre+: 2.8±0.1 seizures; Student’s t-test, t(54)=0.9, p=0.377; Figure 1D2).

Interestingly, when the sexes were separated, Cre+ females had a shorter latency to the first seizure than all other groups (Figure 1D3). Thus, a two-way ANOVA with genotype (Cre- and Cre+) and sex (female and male) as main factors showed a main effect of sex (F(1,51)=4.31; p=0.043) with Cre+ females exhibiting a shorter latency compared to Cre+ males (Cre+ females, 34.3±3.4 min, n=15; Cre+ males, 57.9±5.6 min, n=13; Tukey’s post-hoc test, p=0.026) but not other groups (Cre- female, 46.7±9.3 min, n=12; Cre- males, 47.4±4.4 min, n=15; all p>0.344; Figure 1D3). There was no effect of genotype (F(1,49)=0.75; p=0.305) or sex (F(1,49)=0.62; p=0.436) on the total number of seizures by two-way ANOVA (Figure 1D4).

When adult neurogenesis was suppressed by thymidine kinase activation in GFAP-expressing progenitors, the severity of the first seizure was worse, meaning it was often convulsive rather than non-convulsive (Iyengar et al., 2015). Therefore, we examined the severity of the first seizure. These analyses were conducted only with mice implanted with electrodes because only with the EEG can one determine if a seizure is non-convulsive. A non-convulsive seizure was defined as an EEG seizure without movement. When sexes were pooled, the proportion of mice with a non-convulsive first seizure was not different (Cre-: 18.2%, 2/11 mice; Cre+: 28.6%, 4/14 mice Fisher’s exact test, p>0.999; Figure 1E1). However, when sexes were separated, the first seizure was non-convulsive in 60% of Cre+ females (3/5 mice) whereas only 25% of Cre- females had a first seizure that was nonconvulsive (1/4 mice), 14% of Cre- males (1/7 mice), and 11% of Cre+ males (1/9 mice; Figure 1E3). Although the percentages suggest differences, i.e., Cre+ females were protected from an initial severe seizure, the differences were not significantly different (Fisher’s exact test, p=0.166; Figure 1E3).

The duration of SE was shorter in Cre+ mice compared to Cre- mice (Cre-: 280.5±14.6 min, n=11; Cre+: 211.4±17.2 min, n=14; Student’s t-test, t(23)=0.30, p=0.007; Figure 1E2). When the sexes were separated, the effects of genotype were modest. A two-way ANOVA showed that the duration of SE was significantly affected by genotype (F(1,21)=6.7; p=0.017) but not sex (F(1,21)=5.04; p=0.487). Cre+ males showed a trend for a shorter SE duration compared to Cre- males (Cre- males: 280.1±22.8 min, n=7; Cre+ males: 199.1±24.1 min, n=9; Tukey’s post-hoc test, p=0.078; Figure 1E4). Cre+ females had a mean SE duration that was shorter than Cre- females, but it was not a significant difference (Cre- females: 281.0±11.8 min, n=4; Cre+ females: 233.4±20.5 min, n=5; p=0.485; Figure 1E4). More females would have been useful, but the incidence of SE in females was only 42.8% if they were implanted with EEG electrodes (Figure 1—figure supplement 2). In contrast, the incidence of SE in the unimplanted females was 100%, a significant difference by Fisher’s exact test (p<0.0001; Figure 1—figure supplement 2). In males, the incidence of SE was also significantly different in implanted and unimplanted mice (implanted males, 70.4%; unimplanted males, 100%; Fisher’s exact test, p<0.0001, Figure 1—figure supplement 2).

We also investigated power during SE (Figure 1—figure supplement 3). The baseline was measured, and then power was assessed for 5 hr, after SE had ended. Power was assessed in 20-min consecutive bins. Females were used in this analysis (Cre- and Cre+). Two-way RMANOVA with genotype and time as main factors showed no effect of genotype for any frequency range: delta (1–4 Hz, F(1,7)=1.61; p=0.245); theta (4–8 Hz, F(1,7)=1.75; p=0.227); beta (8–30 Hz, F(1,7)=1.65; p=0.240); low gamma (80 Hz, F(1,7)=0.29; p=0.174); high gamma (80–100 Hz, F(1,7)=0.17; p=0.689). There was a significant effect of time for all bands (delta, p=0.003; theta, p=0.002, beta, low gamma, and high gamma, p<0.001), which is consistent with the declining power in SE with time.

Diazepam was administered earlier in females during SE than in males, and this could have influenced the results. However, the timing of SE was not significantly different in females than in males (Figure 1—figure supplement 4). Also, diazepam was administered the same way in all Cre+ and Cre- females similarly but only the Cre+ females were protected as discussed below.

In summary, Cre+ mice did not show extensive differences in SE except for SE duration, which was shorter.

Continuous video-EEG was recorded for 3 weeks to capture chronic seizures (Figure 2A). Representative examples of chronic seizures are presented in Figure 2B. All chronic seizures were convulsive. First, we analyzed data with sexes pooled (Figure 2C) and the total number of chronic seizures were similar in the two genotypes (Cre-: 22.6±3.0 seizures, n=18; Cre+: 21.3±1.6 seizures, n=17; Student’s t-test, t(33)=0.15, p=0.882; Figure 2C1). The frequency of chronic seizures were also similar among genotypes (Cre-: 1.1±0.14 seizures/day, n=18; Cre+: 1.0±0.08 seizures/day, n=17; Welch’s t-test, t(26)=0.37, p=0.717; Figure 2D1).

Data were then segregated based on sex and a two-way ANOVA was conducted with genotype and sex as main factors. There was a main effect of genotype (F(1,32)=4.26; p=0.047) and sex (F(1,32)=12.46; p=0.001) on the total number of chronic seizures and a significant interaction between sex and genotype (F(1,32)=8.54; p=0.006). Tukey’s post-hoc tests showed that Cre+ females had ~half the chronic seizures of Cre- females (Cre- female: 44.6±10.2 seizures, n=7; Cre+ female: 22.6±2.0 seizures, n=9; p=0.004; Figure 2C). However, Cre+ males and Cre- males had a similar number of chronic seizures (Cre- male: 16.1±1.6 seizures, n=12; Cre+ male: 19.9±2.7 seizures, n=8; p>0.999; Figure 2C2). Cre- females also had more seizures than Cre- males (p<0.001) and Cre+ males (p=0.005; Figure 2C2).

Results for seizure frequency were similar to results comparing total numbers of seizures. There was a main effect of genotype (F(1,32) 4.18; p=0.049) and sex (F(1, 32)=11.96; p=0.002) on chronic seizure frequency, and a significant interaction between sex and genotype (F(1,32)=8.29; p=0.007). Cre+ female mice had approximately half the seizures per day as Cre- females (Tukey’s post-hoc test, Cre- female: 2.1±0.5 seizures/day; Cre+ female: 1.1±0.1 seizures/day; p=0.004; Figure 2D2). Cre- females also had more seizures than Cre- males (p<0.001) and Cre+ males (p=0.005; Figure 2D2).

While reviewing the data for each mouse plotted in Figure 2C2 and D, one point appeared spurious in the Cre-females, potentially influencing the comparison. The seizures in this mouse were more than 2 x the standard deviation of the mean. Although not an outlier using the ROUT method (see Methods), we were curious if removing the data of this mouse would lead to a difference in the statistical results. There was still a main effect of sex (F(1,31)=16.04; p=0.0004) with a significant interaction between sex and genotype (F(1,31)=9.20; p=0.005) and Cre+ females had significantly fewer seizures than Cre- female mice (p=0.020; Figure 2—figure supplement 1A1). Tests for seizure frequency led to the same conclusions (Figure 2—figure supplement 1A2). These data suggest that spurious data point was not the reason for the results.

All mice were included in the analyses above, both those implanted and unimplanted during SE. Mice which were unimplanted prior to SE were implanted at approximately 2–3 weeks after pilocarpine to study chronic seizures. Because implantation affected the incidence of SE (discussed above), we asked if chronic seizures were different in implanted and unimplanted mice. The total number of chronic seizures (F(1,17)=1.33, p=0.265) and seizure frequency (F(1,17)=1.27, p=0.276) were similar, suggesting that implantation did not influence chronic seizures (Figure 2—figure supplement 1B).

There were no significant differences in mortality associated with SE or chronic seizures. For quantification, we examined mortality during SE and the subsequent 3 days, 3 days until the end of the 3-week-long-EEG recording period, or both (Figure 2—figure supplement 2A). Graphs of mouse numbers (Figure 2—figure supplement 2B1) or percentages of mice (Figure 2—figure supplement 2B2) were similar: groups (Cre- females, Cre+ females, Cre- males, Cre+ males) were not significantly different (Fisher’s exact tests, all p >0.05).

To evaluate the duration of individual seizures at the time mice were epileptic, two measurements were made. First, the durations of each seizure of a given mouse were averaged, and then the averages for Cre- mice were compared to the averages for Cre+ mice (Figure 2E1). There was no difference in the genotypes (Cre-: 46.8±2.9 s, n=17; Cre+: 43.4±2.5 s, n=16; Student’s t-test, t(31)=0.89, p=0.379; Figure 2E1).

When separated by sex, a two-way ANOVA showed that female seizure durations were shorter than males (F(1,29)=12.42; p=0.001). However, this was a sex difference, not an effect of genotype (F(1,29)=0.033; p=0.856; Figure 2E2), with Cre- female seizure duration shorter than Cre- male seizure duration (Tukey’s post-hoc test, p=0.005), and the same for Cre+ females compared to Cre+males (p=0.001; Figure 2E2). One reason for the sex difference could be related to the greater incidence of postictal depression in females (see below), because that suggests spreading depolarizations truncated the seizures in females but not males.

The second method to compare seizure durations compared the duration of every seizure of every Cre- and Cre+ mouse. In the previous comparison (Figure 2E1), every mouse was a data point, whereas here every seizure was a data point (Figure 2F1). The data were similar between genotypes (Cre-: 41.9±0.9 s; Cre+: 36.8±0.7 s; Mann-Whitney U test, U statistic, 18873, p=0.079; Figure 2F1). When the sexes were separated, a Kruskal-Wallis test was significant (Kruskal-Wallis statistic, 69.30, p<0.001). Post-hoc tests showed that Cre+ females had longer seizure durations than Cre- females (Cre- female: 33.2±0.7 s; Cre+ female: 39.3±0.6 s, p<0.001; Figure 2F2). Cre+ females may have had longer seizures because they were protected from spreading depolarizations that truncated seizures in Cre- females. Seizure durations were not significantly different in males (Cre- male: 51.4±1.7 s; Cre+ male: 44.0±1.3 s, p=0.298; Figure 2F2).

Postictal depression is a debilitating condition in humans where individuals suffer fatigue, confusion, and cognitive impairment after a seizure. In the EEG, it is exhibited by a decrease in the EEG amplitude immediately after a seizure ends relative to baseline. In recent years the advent of DC amplifiers made it possible to show that postictal depression is often associated with spreading depolarization (Ssentongo et al., 2017), a large depolarization shift that is accompanied by depolarization block. As action potentials are blocked there are large decreases in input resistance leading to cessation of synaptic responses. As ion pumps are activated to restore equilibrium, there is recovery and the EEG returns to normal (Somjen, 2001; Hartings et al., 2017; Herreras and Makarova, 2020; Lu and Scharfman, 2021).

We found that males had little evidence of postictal depression but it was common in females (Figure 3), a sex difference that is consistent with greater spreading depolarization in females (Eikermann-Haerter et al., 2009; Bolay et al., 2011; Kudo et al., 2023). As shown in Figure 3A1, a male and a female showed a robust spontaneous seizure (selected from the 3-week-long recording period when mice are epileptic). However, the end of the seizure in the male did not exhibit a decrease in the amplitude of the EEG (relative to baseline) but it did in the female (Figure 3A1-3). In Figure 3A2-3, the male seizure is on the left and the female seizure is on the right. For quantification (Figure 3B-C), the mean peak-to-trough amplitude of the EEG 25–30 s before the seizure was compared to the mean amplitude of the EEG during the maximal depression of the EEG after the seizure. If the depression was more than half, the animal was said to have had postictal depression.

When all chronic seizures were analyzed (n=274), the number and percentages of seizures with postictal depression were reduced in Cre+ females compared to Cre- females (Fisher‘s exact test, p=0.009; Figure 3B). There was a sex difference, with all females showing more postictal depression (108/154 seizures, 70.5%) than all males (17/120 seizures, 14.2%; Fisher’s exact tests, p<0.0001, Figure 3B-C). Postictal depression in males was relatively rare and genotypic differences were not robust (Figure 3B-C).

Next, we asked if the distribution of seizures during the 3 weeks of video-EEG was affected by increasing adult-born neurons. In Figure 4A, a plot of the day-to-day variation in seizures is shown with each day of recording either black (if there were seizures) or white (if there were no seizures).

The number of days with seizures were similar between genotypes (Cre-: 8.6±0.6 days, n=18; Cre+: 8.4±0.6 days, n=17; Student’s t-test, t(33)=0.23, p=0.822; Figure 4B1). The number of consecutive days without seizures, called the seizure-free interval, was also similar between genotypes (Cre-: 6.4±0.4 days, n=19; Cre+: 7.7±0.6 days, n=17; Student’s t-test, t(34)=1.65, p=0.107; Figure 4B2). When data were segregated based on sex, a two-way ANOVA showed no effect of genotype (F(1,32)=1.18, p=0.286) or sex on the number of days with seizures (F(1,32)=0.86, p=0.361; Figure 4B3). There also was no effect of genotype (F(1,32)=2.86, p=0.100) or sex (F(1,32)=0.53, p=0.471) on seizure-free interval (Figure 4B4).

Clustering is commonly manifested in consecutive days with frequent seizures. Clusters of seizures can have a substantial impact on the quality of life (Haut, 2015; Jafarpour et al., 2019) so they are important. In humans, clusters are defined as at least 3 seizures within 24 hr (Goffin et al., 2007; Jafarpour et al., 2019). Therefore, we defined clusters as >1 consecutive day with ≥3 seizures/day (Figure 4C). The duration of clusters were similar between genotypes (Cre-: 3.8±0.7 days, n=19; Cre+: 3.0±0.3 days, n=18; Mann-Whitney’s U test, U statistic 159, p=0.723; Figure 4D1). Next, we calculated the number of days between clusters, which we call the intercluster interval. Genotypes were similar (Cre-: 7.2±0.8 days, n=13; Cre+: 9.2±0.8 days, n=9; Student’s t-test, t(20)=1.70, p=0.104; Figure 4D2).

Two-way ANOVA was then performed on data segregated by sex. For cluster duration, there was no effect of genotype (F(1,33)=3.36, p=0.076) but there was a main effect of sex (F(1,33)=7.66, p=0.009) and a significant interaction of genotype and sex (F(1,33)=0.66, p=0.009). Cre+ females had fewer days with ≥3 seizures than Cre- females (Cre- females: 6.3±1.4 days; Cre+ females: 3.0±0.4 days; Tukey’s post-hoc test, p=0.009; Figure 4D3). These data suggest Cre+ females were protected from the peak of a cluster, when seizures increase above 3 /day.

There was no effect of genotype (F(1,17)=2.72, p=0.117) or sex (F(1,17)=2.72, p=0.117) on the intercluster interval (Figure 4D4). However, this result may have underestimated effects because Cre+ females often had such a long interval that it was not captured in the 3-week-long recording period. That led to fewer Cre+ females that were included in the measurement of intercluster interval. In 5 out of 9 (i.e. 55%) Cre+ females, there was only one cluster in 3 weeks, so intercluster interval was too long to capture. Of those mice where intercluster interval could be measured, Cre- females had an interval of 5.7±1.0 days (n=7) and Cre+ females had a 9.0±1.1 day interval (n=4). That difference was not significant.

In summary, Cre+ females had fewer seizures, fewer days with ≥3 seizures, reduced postictal depression, and appeared to have a long period between clusters of seizures.

We first confirmed that prior to pilocarpine treatment, Cre+ mice had more young adult-born neurons compared to Cre- mice (Figure 5A, Figure 5—figure supplement 1A–D). To that end, we quantified the adult-born GCs associated with the GCL/SGZ in both Cre+ and Cre- mice. DCX was used as a marker because it is highly expressed in immature neurons (Brown et al., 2003; Couillard-Despres et al., 2005). The area of the GCL/SGZ that exhibited DCX-ir was calculated and expressed as a percent of the total area of the GCL/SGZ (Figure 5C).

A two-way ANOVA with sex and genotype as factors showed a significant effect of genotype (F(1,9)=60.78, p<0.001) but not sex (F(1,9)=1.20, p=0.301; Figure 5D). Post-hoc comparisons showed that Cre+ females had more DCX than Cre- females (p=0.001) and the same was true for males (p=0.003). Cre+ males had more DCX than Cre- females (p<0.001), and Cre+ females had more DCX than Cre- males (p=0.006). Cre- females and males were not different (p=0.774). The results are consistent with studies using the same methods which showed that Cre+ males have more DCX compared to Cre- males (Jain et al., 2019). Together the data suggest that Cre+ mice had more young adult-born neurons than Cre- mice immediately before SE.

We also quantified DCX at the time when epilepsy had developed, after the 3-week-long EEG recording (Figure 5B). Representative examples of DCX expression in the GCL/SGZ are presented in Figure 5F and Figure 5E shows the area fraction of DCX in the GCL/SGZ was significantly greater in Cre+ mice than Cre- mice (Cre-: 3.1±0.4%, n=20; Cre+: 4.2±0.3%, n=17; Student’s t-test, t(35)=2.13, p=0.041; Figure 5D1). Therefore, Cre+ mice had increased DCX in the GCL/SGZ after chronic seizures had developed.

To investigate a sex difference, a two-way ANOVA was conducted with genotype and sex as main factors. There was a significant effect of genotype (F(1,33)=12.62, p=0.001) and sex (F(1,33)=11.68, p=0.002), with Cre+ females having more DCX than Cre- females (Cre- female: 1.8±0.3, n=7; Cre+ female: 3.8±0.4, n=9; Tukey’s post-hoc test, p=0.015; Figure 5E). In contrast, DCX levels were similar between Cre+ and Cre- male mice (p=0.498, Figure 5E). Therefore, elevated DCX occurred after chronic seizures had developed in Cre+ mice but the effect was limited to females. Because Cre+ epileptic females had increased immature neurons relative to Cre- females at the time of SE, and prior studies show that Cre+ females had less neuronal damage after SE (Jain et al., 2019), female Cre+ mice might have had reduced chronic seizures because of high numbers of immature neurons. However, the data do not prove a causal role.

It is notable that the Cre+ male mice did not show increased numbers of immature neurons at the time of chronic seizures but Cre+ females did. It is possible that there was a ‘ceiling’ effect in DCX expression that would explain why male Cre+ mice did not have a significant increase in immature neurons relative to male Cre- mice.

Mice were anesthetized by isoflurane inhalation (3% isoflurane for induction and 1.75–2% isoflurane for maintenance during surgery; NDC# 07-893-1389, Patterson Veterinary) and placed in a stereotaxic apparatus (David Kopf Instruments). Prior to surgery, the analgesic Buprenex (Buprenorphine hydrochloride; NDC# 1296-0757-5; Reckitt Benckheiser) was diluted in sterile saline (0.9% sodium chloride solution) to yield a 0.03 mg/ml stock solution and 0.2 mg/kg was injected s.c. During surgery, mice were placed on a heating blanket with a rectal probe for automatic maintenance of body temperature at 31 °C.

Before electrode implantation, hair over the skull was shaved and then the scalp was cleaned with 70% ethanol. A midline incision was made to expose the skull with a sterile scalpel. To implant subdural screw electrodes (0.10’ stainless steel screws, Cat# 8209, Pinnacle Technology), six holes were drilled over the exposed skull. The coordinates were: right occipital cortex (anterior-posterior or AP −3.5 mm from Bregma, mediolateral or ML, 2.0 mm from the midline); left frontal cortex (Lt FC, AP −0.5 mm; ML −1.5 mm); left hippocampus (AP −2.5 mm; ML −2.0 mm) and right hippocampus (AP −2.5 mm; ML 2.0 mm). An additional screw was placed over the right olfactory bulb as ground (AP 2.3 mm; ML 1.8 mm) and another screw over the cerebellum at the midline as reference (relative to Lambda: AP −1.5 mm; ML −0.5 mm). Here, ‘ground’ refers to the earth ground, and ‘reference’ refers to the reference for all 4 screw electrode recordings (Moyer et al., 2017). An eight-pin connector (Cat# ED85100-ND, Digi-Key Corporation) was placed over the skull and secured with dental cement (Cat# 51459, Dental Cement Kit; Stoelting Co.).

After surgery, mice were injected with 50 ml/kg warm (31 °C) lactated Ringer’s solution (s.c.; NDC# 09355000476, Aspen Veterinary Resources). Mice were housed a clean cage on a heating blanket for 24 hr. Moistened food pellets were placed at the base of the cage to encourage food intake. Afterwards, mice were housed individually because group housing leads to disturbance of the implant by other mice in the cage.

Mice were allowed 3 weeks to recover from surgery. During this time, mice were housed in the room where video-EEG equipment was placed so that mice could acclimate to the recording environment. To record video-EEG, the pin connector on the head of the mouse was attached to a preamplifier (Cat# 8406, Pinnacle Technology) which was attached to a commutator (Cat# 8408, Mouse Swivel/Commutator, four-channel, Pinnacle Technology) to allow freedom of movement. Signals were acquired at a 500  Hz sampling rate, and band-pass filtered at 1–100  Hz using Sirenia Acquisition software (https://www.pinnaclet.com, RRID:SCR_016183). Video was captured with a high-intensity infrared LED camera (Cat# PE-605EH, Pecham) and was synchronized to the EEG record.

To monitor pilocarpine-induced SE, video-EEG was recorded for an hour before ad 24 hr after the pilocarpine injection. Approximately 5–6 weeks after pilocarpine-induced SE, video-EEG was recorded to measure spontaneous recurrent seizures. Video-EEG was recorded continuously for 3 weeks.

EEG was analyzed offline with Sirenia Seizure Pro, V2.0.7 (Pinnacle Technology, RRID:SCR_016184). A seizure was defined as a period of rhythmic (>3 Hz) deflections that were >2 x the standard deviation of the baseline mean and lasted at least 10 sec (Jain et al., 2019). Seizures were rated as convulsive if an electrographic seizure was accompanied by a behavioral convulsion (observed by video playback), defined as stages 3–5 using the Racine scale (Racine, 1972) where stage 3 is unilateral forelimb clonus, stage 4 is bilateral forelimb clonus with rearing, and stage 5 is bilateral forelimb clonus followed by rearing and falling. A seizure was defined as non-convulsive when there was electrographic evidence of a seizure but there were no stage 3–5 behavior.

SE was defined as continuous seizures for >5 min (Chen and Wasterlain, 2006) and EEG amplitude in all four channels >3 x the baseline mean. For mice without EEG, SE was defined by stage 3–5 seizures that did not stop with a resumption of normal behavior. Often stage 3–5 seizures heralded the onset of SE and then occurred intermittently for hours. In between convulsive behavior mice had twitching of their body, typically in a prone position.

SE duration was defined in light of the fact that the EEG did not return to normal after the initial period of intense activity. Instead, intermittent spiking occurred for at least 24 hr, as we previously described (Jain et al., 2019) and has been described by others (Mazzuferi et al., 2012; Bumanglag and Sloviter, 2018; Smith et al., 2018). We, therefore, chose a definition that captured the initial, intense activity. We defined the end of this time as the point when the amplitude of the EEG deflections were reduced to 50% or less of the peak deflections during the initial hour of SE. Specifically, we selected the time after the onset of SE when the EEG amplitude in at least three channels had dropped to approximately two times the amplitude of the EEG during the first hour of SE, and remained depressed for at least 10 min (Fig S2 in Jain et al., 2019). Thus, the duration of SE was defined as the time between the onset and this definition of the ‘end’ of SE.

To access the severity of chronic seizures, frequency and duration of seizures were measured during the 3 weeks of EEG recording. Inter-cluster interval was defined as the maximum number of days between two clusters.

Mice were perfused after video-EEG recording. To perfuse, mice were deeply anesthetized by isoflurane inhalation followed by urethane (250  mg/kg of 250 mg/ml in 0.9% sodium chloride, intraperitoneal, i.p.; Cat#U2500; Sigma-Aldrich). After loss of a reflex to a tail pinch, and loss of a righting reflex, consistent with deep anesthesia, the heart cavity was opened, and a 25-gauge needle was inserted into the heart, followed by perfusion with 10 ml saline 0.9% sodium chloride in double-distilled water (ddH₂O) using a peristaltic pump (Minipuls 1; Gilson) followed by 30  ml of cold (4 °C) 4% paraformaldehyde (PFA; Cat# 19210, Electron Microscopy Sciences) in 0.1  M phosphate buffer (PB; pH 7.4). The brains were removed immediately, hemisected, and post-fixed for at least 24  hr in 4% PFA at 4 °C. After post-fixation, one hemisphere was cut in the coronal plane and the other in the horizontal plane (50 μm-thick sections) using a vibratome (Cat# TPI-3000, Vibratome Co.). Sections were collected sequentially to select sections that were from similar septotemporal levels. For the dorsal hippocampus, coronal sections were selected every 300 μm starting at the first section where the DG blades are fully formed (between AP −1.94 and −2.06 mm). Horizontal sections were chosen every 300 μm starting from the temporal pole at the place where the GCL is clearly defined (between DV 0.84 and 1.08 mm). This scheme is diagrammed and described in more detail in prior studies (Moretto et al., 2017).

In normal rodent adult brains, Prospero homeobox 1 (Prox1) is expressed in the GCs (Pleasure et al., 2000) and in the hilus (Bermudez-Hernandez et al., 2017). To stain for Prox1, free-floating sections were washed in 0.1 M TB pH 7.4, 3×5 min. Sections were then incubated in 0.1 M TB with 0.25%Triton X-100 for 30 min followed by a 10 min-long wash in 0.1 M TB with 0.1%Triton X-100 (referred to as Tris A). Next, sections were treated with 1% hydrogen peroxide in Tris A for 5 min followed by a 5 min-long wash in Tris A. Sections were blocked in 10% normal horse serum (Cat# S-2000, RRID:AB_2336617, Vector) in Tris A for 1 hr followed by a 10 min-long wash in Tris A and then 0.1 M TB with 0.1% Triton X-100 and 0.005% bovine serum albumin (referred to as Tris B). Next, sections were incubated overnight with primary antibody (goat anti-human Prox1 polyclonal antibody, 1:2000 diluted in Tris B, R, and D systems) rotated on a shaker (described above) at room temperature.

On the next day, sections were washed in Tris A and then in Tris B (5 min each). Sections were then incubated in secondary antibody (biotinylated anti-goat IgG made in horse, 1:500, Vector Laboratories, see Key Resources) for 1 hr in Tris B, followed by a wash with Tris A (5 min) and then Tris B (5 min), blocked in avidin-biotin complex (1:500 in Tris B) for 2 hr, and washed in 0.1 M TB (3×5 min). Sections were reacted in 0.5 mg/ml 3,3′-diaminobenzidine (DAB) with 40 µg/ml ammonium chloride, 3 µg/ml glucose oxidase, 2 mg/ml (D+)-glucose and 5 mM nickel chloride (Cat# N6136, Sigma-Aldrich) in 0.1 M TB. Sections were washed in 0.1 M TB (3×5 min), mounted on 1% gelatin-coated slides and dried overnight at room temperature. On the next day, sections were dehydrated, cleared, and coverslipped (as described above). Sections were viewed and images were captured as DCX above.

Prox1 was quantified in the hilus, defined based on zone 4 of Amaral, 1978. The definition of Amaral was modified to exclude 20 μm below the GCL (Winawer et al., 2007). The GCL boundary was defined as the location where GCs stopped being contiguous. Practically that meant there was no GC with more than a cell body width of cell-free space around it. A cell body width was 10 μm (Claiborne et al., 1990; Amaral et al., 2007).

CA3c was included in the ROI but hilar Prox1 cells have not been detected in the CA3c layer (Scharfman et al., 2000; Winawer et al., 2007). However, there are rare GCs in CA3 according to one study (Szabadics et al., 2010).

In ImageJ, a ROI was traced in the image taken at 20 x magnification and then a threshold was selected where Prox1-immunoreactivity was above the background threshold (Jain et al., 2019). Then Prox1 cells were counted using the Analyzed particle plugin where a particle with an area ≥10 μm² was counted. The following criteria were used to define a hilar Prox1-ir cell (Bermudez-Hernandez et al., 2017): (1) the hilar cell had sufficient Prox1-ir to reach a threshold equal to the average level of Prox1-ir of GCs in the adjacent GC layer, (2) All hilar Prox-ir cells were complete, i.e., not cut at the edge of the ROI. When hilar Prox1-ir cells were in clusters, although not many (typically 2–3 per 50 μm section), cells were counted manually. For each animal, three coronal sections in the dorsal hippocampus and 3–4 horizontal sections in the ventral hippocampus, with sections spaced 300 μm apart were chosen.

Free-floating sections were washed (3×5 min) in 0.1 M TB followed by a 10 min long wash in Tris A and another 10 min-long wash in Tris B. Sections were incubated in blocking solution (5% normal goat serum or donkey serum in Tris B) for 1 hr at room temperature. Next, primary antibodies for anti-rabbit GluR2/3, anti-goat Prox1, anti-rabbit SOM, and anti-mouse parvalbumin (Table 1) were diluted in blocking solution and sections were incubated for 48 hr at 4 °C. For SOM labeling, antigen retrieval was used. Prior to the blocking step, sections were incubated in sodium citrate buffer (2.94 mg/ml in ddH₂O, pH 6.0 adjusted with HCl) in a preheated water bath at 85 °C for 30 min.

Next, sections were washed in Tris A and Tris B (10 min each) followed by 2 hr-long incubation in secondary antibody (1:500 in Tris B, see Table 1). Sections were washed in 0.1 M TB (3×5 min), and coverslipped with Citifluor AF1 mounting solution (Cat# 17970–25, Vector Labs). Images were captured on a confocal microscope (Model LSM 510 Meta; Carl Zeiss Microimaging).

GluR2/3-, SOM-, and parvalbumin- ir cells in the hilus and SGZ were counted from three dorsal and three ventral sections. Sections were viewed at 40 x of the confocal microscope for manual counts. Because ectopic GCs express GluR2/3, sections were co-labeled with Prox1. All co-labeled cells were considered as ectopic and excluded from the GluR2/3- ir cell counting to measure mossy cells.

Fluorojade C (FJ) is a fluorescent dye that is the ‘gold standard’ for stain degenerating neurons (Schmued and Hopkins, 2000; Schmued et al., 2005). First, sections were mounted on gelatin-coated slides (1% porcine gelatin in ddH2O; Cat# G1890, Sigma-Aldrich) and dried on a hot plate at 50–55°C for 1 hr. Then slides were placed in a staining rack and immersed in a 100% ethanol solution for 5 min, then in 70% ethanol for 2 min, followed by a 1 min wash in ddH2O.

Slides were then incubated in 0.06% potassium permanganate (Cat# P-279, Fisher Scientific) solution for 10 min on a shaker (described above) with gentle agitation, followed by washes in ddH2O (2×1 min). Slides were then incubated for 20 min in a 0.0002% solution of FJ in ddH2O with 0.1% acetic acid in the dark. The stock solution of FJ was 0.01% in ddH2O and was stored at 4 °C for up to 3 months. To prepare a working solution, 6 ml of stock solution was added to 294 mL of 0.1% acetic acid (Cat# UN2789, Fisher Scientific) in ddH2O and used within 10 min of preparation. Slides were subsequently protected from direct light. They were washed in ddH2O (3×1 min) and dried overnight at room temperature. On the next day, slides were cleared in Xylene (2×3 min) and coverslipped with DPX mounting medium (Cat# 44581, Sigma-Aldrich). Sections were photographed with an epifluorescence microscope (Model BX51; Olympus of America) and images were captured with a digital camera (Model Infinity3-6URC, Teledyne Lumenera).

We measured the FJ in the cell layers of CA1 and CA3. Manual counting of FJ-positive (FJ+) cells was not possible in these cell layers because there could be so many FJ+ cells that were overlapping. Instead, FJ staining in cell layers was quantified by first outlining the cell layer as a ROI at 10×magnification in ImageJ as before (Jain et al., 2019).

To outline the CA1 cell layer, the border with CA2 was defined as the point where the cell layer changed width, a sudden change that could be appreciated by the background in FJ-stained sections and confirmed by cresyl violet-stained sections. The border of CA1 and the subiculum was defined as the location where the normally compact CA1 cell layer suddenly dispersed. To outline CA3, the border with CA2 and CA3 was defined by the point where the stratum lucidum of CA3 terminated. This location was distinct in its background in FJ-stained sections. The border of CA3 and the hilus was defined according to zone 4 of Amaral, 1978. This location was also possible to detect in FJ-stained sections because the background in the hilus was relatively dark compared to area CA3.

After defining ROIs, a threshold fluorescence level was selected so that all cells that had very bright immunofluorescence were above the threshold but other cells that were similar in fluorescence to background staining were not (Iyengar et al., 2015; Jain et al., 2019). ImageJ was then used to calculate the area within the ROI and this measurement is referred to as the area fraction in the Results and expressed as a percent. For a given animal, the area fraction was determined for three coronal sections in the dorsal hippocampus between AP −1.94 and −2.06 mm and 3–4 horizontal sections in the ventral hippocampus between DV 0.84 and 1.08 mm, with sections spaced 300 μm apart. These area fractions were averaged so that a mean area fraction was defined for each animal.