Intergenerational Transmission of Enhanced Seizure Susceptibility after Febrile Seizures

Abstract

Environmental exposure early in development plays a role in susceptibility to disease in later life. Here, we demonstrate that prolonged febrile seizures induced by exposure of rat pups to a hyperthermic environment enhance seizure susceptibility not only in these hyperthermia-treated rats but also in their future offspring, even if the offspring never experience febrile seizures. This transgenerational transmission was intensity-dependent and was mainly from mothers to their offspring. The transmission was associated with DNA methylation. Thus, our study supports a "Lamarckian"-like mechanism of pathogenesis and the crucial role of epigenetic factors in neurological conditions.

Keywords: DNA methylation; Febrile seizures; Seizure susceptibility; Transgeneration.

Fig. 1

Induction of febrile seizures and long-lasting effects of prolonged hyperthermic seizures. (a) Timeline of the experiment. (b) A typical febrile seizure occurred when the core temperature reached threshold. Left, rat before seizure onset; right, behavioral seizure after core temperature reached threshold. (c) Upper trace, EEG before the onset of seizures, showing non-rhythmic waves; middle trace, the onset of a behavioral seizure, with rhythmic discharges of increasing amplitude in the amygdala; lower trace, post-ictal state after behavioral seizures stopped. (d) Percentage of hindlimb seizures and (e) seizure scores induced by MES (40 mA, 0.2 s, 50 Hz) in P90 rats (n = 9 each for the FS and control groups). (f) Percentage of hindlimb seizures and (g) seizure scores induced by MES (65 mA, 0.2 s, 50 Hz) in P360 rats (n = 8 for the FS group, n = 7 for the control group). (h) FS rats (both 10 FSs and 4*2 FSs) showed enhanced susceptibility to MES-(65 mA, 0.2 s, 50 Hz) induced seizures (n = 6 for each group), whereas hyperthermic control group, in which rat pups were exposed to hyperthermia but seizures were suppressed with pentobarbital, and control group showed similar seizures scores to MES. Seizure susceptibility was tested in 360 days old rats. CON: control group; HT: hyperthermic control group; FS:FS group. *P < 0.05 compared with control group; #P < 0.05 compared with hyperthermic control group. Error bars indicate SEM.

Fig. 2

Effect of hyperthermic seizures was transmitted transgenerationally. (a) and (b) MES induced higher scores in female (n = 8 for the FS group and n = 15 for the control group) and male offspring, respectively (n = 7 for the FS group and n = 8 for the control group). (c) The developmental weight curve after FS. The FS group and control group did not show obvious differences. (d) Susceptibility to MES in F2 offspring of control and FS groups (n = 16 for the FS group, n = 12 for the control group). (e) Susceptibility to MES in 90-day-old offspring whose parents experienced 10 seizures or 4 × 2 seizures was compared with age-matched offspring of control rats (n = 15 for the 10 FS group, n = 18 for the 4 × 2 FS group, and n = 23 for the control group). (f) MES-induced seizure scores were assessed in the 90-day-old offspring of 10-FS female and non-FS male rats (n = 13) or in the offspring of non-FS female and 10-FS male rats (n = 8). MES-induced seizure scores in the offspring of non-FS parents are also shown for comparison (n = 13). (g) Maternal care did not affect the transgenerational transmission of seizure susceptibility. Offspring of FS rats, either raised by FS or non-FS rats (n = 6 for each group), displayed higher susceptibility to MES-induced seizures compared with control rats (n = 6); offspring of non-FS rats, reared by FSs foster mothers (n = 6) displayed normal seizure susceptibility compared with control rats. C(F1)-C(F0): offspring of control rats reared by control mothers; C(F1)-FS(F0): offspring of control rats reared by FS mothers; FS(F1)-C(F0): offspring of FS rats reared by control mothers; FS(F1)-FS(F0): offspring of FS rats reared by FS mothers; Error bars indicated SEM. ***P < 0.001, *P < 0.05 compared with control group reared by control mothers; #P < 0.05 compared with control group reared by FS mothers.

Fig. 3

Transgenerational transmission of enhanced susceptibility to kainate (KA)-induced seizures. (a) Percentage of stage 4 and 5 seizures induced by KA injection in the offspring of control and FS rats. (b) Average seizure stage after intrahippocampal KA injection in the 90-day-old offspring of control and FS rats (n = 10 for the FS group, n = 6 for the control group). (c) Average seizure stage after intrahippocampal KA injection in 90-day-old F2 generation rats (n = 6 for the FS group, n = 6 for the control group). (d) Representative EEG traces in amygdala and CA3 of offspring of control and FS rats. Error bars indicate SEM. ***P < 0.001.

Fig. 4

DNA methylation participated in the transgenerational transmission. (a) DNMT 1 mRNA levels were up-regulated in the hippocampus of FS rats and their F1offspring. DNMT 1 mRNA level did not change in F2 rats, the F0 generation of FS rats treated with zebularine, and FS rat offspring treated with zebularine. Zebualrine treatment did not change DNMT 1 mRNA levels in control rats. DNMT 3A and 3B mRNA levels did not differ among these groups (n = 6 for each group). (b) DNMT 1 protein expression was up-regulated in the hippocampus of FS rats and their future offspring but not in the F2 generation. Zebualrine treatment inhibited the up-regulation of DNMT 1 protein levels in F0 FS rats and the F1 offspring of FS rats but not in control and F2 rats (n = 6 for each group). DNMT 3A and 3B protein levels did not differ among them (n = 6 for each group, P > 0.05). (c) MES-induced seizure scores in adult FS rats treated with DNMT inhibitors (zebularine or 5-AZA) or saline immediately after FSs and once daily for the following 5 days in infant (n = 8 for each group). (d) MES-induced seizure scores in offspring of FS rats treated with DNMT inhibitors or saline in infant (n = 6 for each group). (e) MES-induced seizure scores in FS rats treated with DNMT inhibitors or saline in adult. C, control group; FS, 10 FS group; F1, F1 generation of FS group; F2, F2 generation of FS group; Con + Veh: control group treated with vehicle; FS + Veh: FS group treated with vehicle; (FS + Veh)-F1:offspring of FS group treated with vehicle; Error bars indicate SEM. *P < 0.05.

Fig. 5

Transgenerational transmission of DNA methylation was through the mother. (a) DNMT1 mRNA level was up-regulated in the hippocampus of FS rats. DNMT1 mRNA was not upregulated in offspring of 10-FS male rats and control female rats (n = 6 for F0 and F1 group, n = 6 for F2 group). DNMT 3A and 3B did not show differences in these groups (n = 6 for each group, P > 0.05). (b) DNMT1 protein expression was up-regulated in the hippocampus of FS rats. DNMT1 protein was not upregulated in male offspring of 10-FS male rats and control female rats (n = 6 for F0 and F1 group, n = 6 for F2 group). DNMT 3A and 3B protein levels were similar in these groups (n = 6 for each group, P > 0.05). C: control male rats; FS:10-FS male rats; F1: F1 male generation of 10-FS father and control mother; F2: Offspring of F1 generation; Error bars indicated SEM. *P < 0.05.

Fig. 6

Generation and validation of genome-wide CpG methylation maps of normal, hyperthermia-only (H), FS, F1, and F2 rats. Distribution of differentially-mediated Regions (DMRs) in different subgenomic compartments. (a) IGV (Integrative Genomics Viewer) view of sequenced DNA methylation tracks of 5 rats from different groups in the 41,848,235–47,512,328 region of chromosome 16. (b), (c) and (d) Odds ratios (fraction of experimentally observed DMRs divided by relative size of subgenomic compartment) of hyper- and hypo-methylation within intergenic, genebody, promoters (all) and promoters that contain or do not contain CpG island (CGI) regions. Dashed lines demarcate over- versus under-representation. Comparisons between FS and control groups (b), F1 generation of FS and control groups (c), and F2 generation of FS and control groups (d).

Fig. 7

GO and Pathway Analysis for RNA down-regulation and DNA methylation up-regulation in the transgenerational transmission of enhanced seizure susceptibility. (a) Venn diagram showing the overlapped transcriptionally down-regulated genes of F0 and F1 generations but not F2 generation comparing to control group. (b) and (c) GO and pathways with the overlapped transcriptionally down-regulated genes. (d), Venn diagram showing overlapped hypermethylated genes of F0 and F1 generations but not F2 generation comparing to control group by DNA methylation analysis. (e) and (f) GO and pathway analysis of the overlapped hypermethylated genes. (g) and (h) Heat maps of the differentially methylation analysis among groups. (g) Heat map of the differential DNA methylation among the F0 and F1 generations of FS and the F2 generation of the FS and control groups. (h) Heat map of the differential DNA methylation among the F0 and F1 generations of FS, and the F2 generation of FS and control groups only when log₁₀LR ≥ 150. A, control group; B, hyperthermia only group; C, 10FS group; D, F1 group; E, F2 group.