Water maze experience and prenatal choline supplementation differentially promote long-term hippocampal recovery from seizures in adulthood

Abstract

Status epilepticus (SE) in adulthood dramatically alters the hippocampus and produces spatial learning and memory deficits. Some factors, like environmental enrichment and exercise, may promote functional recovery from SE. Prenatal choline supplementation (SUP) also protects against spatial memory deficits observed shortly after SE in adulthood, and we have previously reported that SUP attenuates the neuropathological response to SE in the adult hippocampus just 16 days after SE. It is unknown whether SUP can ameliorate longer-term cognitive and neuropathological consequences of SE, whether repeatedly engaging the injured hippocampus in a cognitive task might facilitate recovery from SE, and whether our prophylactic prenatal dietary treatment would enable the injured hippocampus to more effectively benefit from cognitive rehabilitation. To address these issues, adult offspring from rat dams that received either a control (CON) or SUP diet on embryonic days 12-17 first received training on a place learning water maze task (WM) and were then administered saline or kainic acid (KA) to induce SE. Rats then either remained in their home cage, or received three additional WM sessions at 3, 6.5, and 10 weeks after SE to test spatial learning and memory retention. Eleven weeks after SE, the brains were analyzed for several hippocampal markers known to be altered by SE. SUP attenuated SE-induced spatial learning deficits and completely rescued spatial memory retention by 10 weeks post-SE. Repeated WM experience prevented SE-induced declines in glutamic acid decarboxylase (GAD) and dentate gyrus neurogenesis, and attenuated increased glial fibrilary acidic protein (GFAP) levels. Remarkably, SUP alone was similarly protective to an even greater extent, and SUP rats that were water maze trained after SE showed reduced hilar migration of newborn neurons. These findings suggest that prophylactic SUP is protective against the long-term cognitive and neuropathological effects of KA-induced SE, and that rehabilitative cognitive enrichment may be partially beneficial.

Figure 1

Timeline of experimental procedures. Adult rat offspring from dams that received either a choline control (CON) or supplemented (SUP) diet during embryonic days 12–17 were all trained in the water maze at postnatal day (P) 56 for 4 days. On P60, CON and SUP rats were given injections of saline or kainic acid (KA) to induce status epilepticus (SE). All rats were then given an injection of BrdU on days 6, 8, 10, 12, and 14 after SE. Saline- and KA-treated CON and SUP rats then either remained in their home cage, or were given three additional water maze testing sessions at 3, 6.5, and 10 weeks after SE, each session lasting 5 days. All rats were then sacrificed one day after the last water maze testing session at approximately 11 weeks after SE.

Figure 2

Water maze performance. (A) During pre-SE training, all CON (grey) and SUP (black) rats learned to locate the hidden platform with similar decreasing total latencies over 4 days of training. (B) Compared to saline-treated rats (solid lines), KA-treated (dashed lines) rats had significant impairments in spatial learning (higher total latencies) across 4 days during each post-SE session, which was attenuated in KA-treated SUP rats. (C) SE also impaired spatial memory over a 3-week retention interval, but KA-treated SUP rats showed a complete recovery of spatial memory retention by 10 weeks post-SE. * significantly different from all other groups at p < 0.05.

Figure 3

Histopathology of the hippocampus 11 weeks following KA-induced SE from representative sections from the dorsal hippocampus of CON (B) and SUP (C) rats. Panel A depicts an intact hippocampus from a saline-treated CON rat. Saline-treated SUP rats also did not show any lesions (histology data not shown). Note significant cell loss in CA1, CA3, and hilus regions (indicated by arrows) while the dentate gyrus granule cell layer remained relatively intact. Patterns of lesion severity were similar across all KA-treated rats. Photomicrographs in each set were taken with a 4x objective. Bars indicate 250 μm. DG, dentate gyrus. H, hilus.

Figure 4

Mean (± SEM) percent of control levels for hippocampal GAD65 and 67 mRNA (A, B) and protein (C, D) for CON and SUP rats that were treated with saline (solid bars) or KA (hatched bars) and that remained in their home cage (grey) or received additional water maze training (black) following treatment. CON rats showed significant SE-induced reductions in GAD67 mRNA, GAD65 protein, and GAD67 protein. There was a trend for an SE-induced decrease in GAD65 mRNA (p = 0.10). Repeated water maze training rescued levels of GAD mRNA in KA-treated CON rats. There was little effect of SE on GAD levels in SUP rats. * significantly different at p < 0.05; main effect of seizure (**) or experience (#) revealed by a within diet 2-way ANOVA (seizure × experience), p < 0.05.

Figure 5

Mean (± SEM) percent of control levels for hippocampal GFAP mRNA (A) and protein (B) for CON and SUP rats that were treated with saline (solid bars) or KA (hatched bars) and that remained in their home cage (grey) or received additional water maze training (black) following treatment. Both CON and SUP rats showed a significant overall SE-induced increase in GFAP mRNA and protein expression, but this increase was attenuated in KA-treated home cage SUP rats. Repeated water maze training attenuated elevated GFAP protein levels in KA-treated CON rats, and further increased GFAP mRNA levels in KA-treated SUP rats. * significantly different at p < 0.05; ** main effect of seizure revealed by a within diet 2-way ANOVA (seizure × experience), p < 0.05; # KA-treated home cage SUP rats are significantly different from KA-treated home cage CON rats (A, B) and KA-treated water maze CON rats (A).

Figure 6

Long-term (9 to 10.5 weeks) survival of BrdU-immunopositive cells (i.e., newly generated cells) born 6 to 14 days after saline or KA treatment. (A–F) Photomicrographs of BrdU+ cells in the SGZ-GCL and hilus in saline treated rats (A, CON; B, SUP) and KA-treated rats who either remained in their home cage (C, CON; D, SUP) or received additional water maze training (E, CON; F, SUP) after SE. Most BrdU+ cells that were found in the GCL had large and rounded BrdU-immunostained nuclei characteristic of mature granule cells (large arrows), but many BrdU+ cells in the SGZ-GCL and hilus had morphological features characteristic of glial or endothelial cells (small arrows). KA-treated SUP rats exhibited more BrdU labeling in the SGZ-GCL than KA-treated CON rats. KA-induced SE increased the number of BrdU+ cells in the hilus of all KA-treated rats. (G, H) Bar graphs show mean (± SEM) numbers of BrdU+ cells detected in the SGZ-GCL (G) and hilus (H) of CON and SUP rats that were treated with saline (solid bars) or KA (hatched bars) and that remained in their home cage (grey) or received additional water maze training (black) following treatment. For the SGZ-GCL (G), CON rats showed a significant decrease in the number of BrdU+ cells, which was rescued by repeated water maze training. SUP rats showed an increase in the number of BrdU+ cells in the SGZ-GCL weeks after SE, regardless of post-SE experience. For the hilus (H), both CON and SUP rats showed a significant overall increase in the number of BrdU+ cells. Note that we did not directly compare numbers of BrdU+ cells between KA-treated CON and SUP rats because we have previously found that the amount of SGZ-GCL and hilar cell proliferation observed shortly after seizures is altered by prenatal choline availability (see text). Bars in photomicrographs indicate 50 μm. Photomicrographs in first and third columns were taken with a 10x objective and photomicrographs in the second and fourth columns were taken with a 40x objective. GCL, granule cell layer. SGZ, subgranular zone. H, hilus. * significantly different at p < 0.05; ** main effect of seizure revealed by a within diet 2-way ANOVA (seizure × experience), p < 0.05.

Figure 7

Confocal images of BrdU+ cells co-labeled with the mature neuronal marker NeuN (arrow heads). Bar indicates 25 μm. Images were taken with a 40x objective. GCL, granule cell layer. H, hilus.

Figure 8

Hippocampal neurogenesis at 11 weeks after saline or KA treatment. (A–F) Photomicrographs of DCX-immunopositive neurons (i.e., newly generated neurons) in the SGZ-GCL and hilus in saline treated rats (A, CON; B, SUP) and KA-treated rats who either remained in their home cage (C, CON; D, SUP) or received additional water maze training (E, CON; F, SUP) after SE. In KA-treated rats of both prenatal diet groups (C–F), DCX+ neurons were aberrantly located in the hilus and exhibited abnormal morphological features, such as horizontally oriented cell bodies and processes (arrows). SUP rats exhibited more overall DCX labeling than CON rats. (G, H) Bar graphs show mean (± SEM) numbers of DCX+ cells detected in the SGZ-GCL (G) and mean percentage of DCX+ found in the hilus (H) of CON and SUP rats that were treated with saline (solid bars) or KA (hatched bars) and that remained in their home cage (grey) or received additional water maze training (black) following treatment. (G) CON rats showed a significant decrease in the number of DCX+ cells in the SGZ-GCL, which was rescued by repeated water maze training. KA-treated SUP rats showed preserved levels of DCX+ cells in the SGZ-GCL, regardless of post-SE experience. Saline-treated SUP rats also had a higher number of DCX+ neurons overall than saline-treated CON rats. (H) Both KA-treated CON and SUP rats generated a proportion of DCX+ cells that migrated to the hilus. KA-treated water maze SUP rats had a significantly lower percentage of DCX+ neurons in the hilus than KA-treated home cage and water maze rats (#, p < 0.05). A–F, Bars in photomicrographs indicate 50 μm. Photomicrographs in first and third columns were taken with a 10x objective and photomicrographs in the second and fourth columns were taken with a 40x objective. GCL, granule cell layer. SGZ, subgranular zone. H, hilus. * significantly different at p < 0.05; ** main effect of seizure revealed by a within diet 2-way ANOVA (seizure × experience), p < 0.05; ## main effect of prenatal diet revealed by a within diet 2-way ANOVA (diet × experience), p < 0.05.

Figure 9

Mean (± SEM) percent of control levels for hippocampal BDNF protein for CON and SUP rats that were treated with saline (solid bars) or KA (hatched bars) and that remained in their home cage (grey) or received additional water maze training (black) following treatment. Both CON and SUP rats showed a significant overall SE-induced increase in BDNF protein. Saline-treated SUP rats also had a higher levels of BDNF protein overall than saline-treated CON rats. * significantly different at p < 0.05; ** main effect of seizure revealed by a within diet 2-way ANOVA (seizure × experience), p < 0.05; # main effect of prenatal diet revealed by a within diet 2-way ANOVA (diet × experience), p < 0.05.