Prenatal choline deficiency does not enhance hippocampal vulnerability after kainic acid-induced seizures in adulthood

Abstract

Choline is a vital nutrient needed during early development for both humans and rodents. Severe dietary choline deficiency during pregnancy leads to birth defects, while more limited deficiency during mid- to late pregnancy causes deficits in hippocampal plasticity in adult rodent offspring that are accompanied by cognitive deficits only when task demands are high. Because prenatal choline supplementation confers neuroprotection of the adult hippocampus against a variety of neural insults and aids memory, we hypothesized that prenatal choline deficiency may enhance vulnerability to neural injury. To examine this, adult offspring of rat dams either fed a control diet (CON) or one deficient in choline (DEF) during embryonic days 12-17 were given multiple injections (i.p.) of saline (control) or kainic acid to induce seizures and were euthanized 16 days later. Perhaps somewhat surprisingly, DEF rats were not more susceptible to seizure induction and showed similar levels of seizure-induced hippocampal histopathology, GAD expression loss, upregulated hippocampal GFAP and growth factor expression, and increased dentate cell and neuronal proliferation as that seen in CON rats. Although prenatal choline deficiency compromises adult hippocampal plasticity in the intact brain, it does not appear to exacerbate the neuropathological response to seizures in the adult hippocampus at least shortly after excitotoxic injury.

Figure 1

Hippocampal Histopathology of CON and DEF Rats 16 Days After KA-Induced SE. Histopathology of the hippocampus in CON (B) and DEF (C) rats. Panel A depicts representative sections of CA1 (A1), CA3 (A2), and dentate gyrus (A3) regions from an intact hippocampus from a saline-treated CON rat. Saline-treated DEF rats also did not show any lesions (histology data not shown). Areas of damage (cell loss, disruption of cytoarchitecture, and gliosis) are indicated by arrows. KA-treated CON and KA-treated DEF rats exhibited similar severity of hippocampal neurodegeneration. Photomicrographs in each set were taken with a 10x objective. Bars indicate 50 μm. GCL, granule cell layer. SGZ, subgranular zone. H, hilus.

Figure 2

Hippocampal GAD Expression 16 Days After Saline or KA Treatment in CON and DEF Rats. Comparison between CON (white bars) and DEF rats (grey bars) in GAD65 and GAD67 mRNA levels (A) and protein levels (B) (mean ± SEM percent of control levels) in the intact hippocampus (open bars) and 16 days following KA-induced SE (hatched bars). mRNA levels were quantifed using RT-PCR and protein levels were quantified using Western blot analysis. An overall effect of Seizure was evident for GAD67 mRNA, GAD65 protein, and GAD67 protein (ps < 0.05) with a strong trend for GAD65 mRNA (p = 0.08) where KA-induced SE similarly reduced hippocampal GAD expression in CON and DEF rats. # main effect of Seizure at p < 0.05. * statistically different from within-diet saline-treated group at p < 0.05.

Figure 3

BrdU Immunolabeling 16 Days After Saline or KA Treatment in CON and DEF rats. BrdU-immunopositive cells (i.e., newly generated cells) in the SGZ-GCL and hilus 16 days following saline treatment (A, CON; B, DEF) or KA-induced SE (C, CON; D, DEF). The number of BrdU-labeled cells significantly increased 16 days after SE for both diet groups. Photomicrographs in the left column were taken with a 10x objective and images in the right column were taken with a 40x objective. Bars indicate 50 μm. GCL, granule cell layer. SGZ, subgranular zone. H, hilus.

Figure 4

Quantification of BrdU+ Cells in the Dentate Gyrus 16 Days After Saline or KA Treatment in CON and DEF Rats. Mean (±SEM) numbers of BrdU+ cells detected in the SGZ-GCL and hilus of CON (white bars) and DEF (grey bars) rats 16 days following saline treatment (open bars) or KA-induced SE (hatched bars). SE significantly increased the number of BrdU+ cells in both CON and DEF rats (ps < 0.05). * statistically different from within-diet saline-treated group.

Figure 5

DCX Immunolabeling 16 Days After Saline or KA Treatment in CON and DEF Rats. DCX-immunopositive neurons (i.e., newly generated neurons) in the SGZ-GCL and hilus 16 days following saline treatment (A, CON; B, DEF) or KA-induced SE (C, CON; D, DEF). The number of DCX-labeled cells significantly increased 16 days after SE for both CON (A, C) and DEF (B, D) rats. Note that in KA-treated rats of both diet groups (C, D), DCX-positive neurons were aberrantly located in the hilus and exhibited abnormal morphological features, such as horizontally oriented cell bodies and processes (arrows). Photomicrographs in the left column were taken with a 10x objective and images in the right column were taken with a 40x objective. Bars indicate 50 μm. GCL, granule cell layer. SGZ, subgranular zone. H, hilus.

Figure 6

Quantification of DCX+ Cells in the Dentate Gyrus 16 Days After Saline or KA Treatment in CON and DEF Rats. Mean (±SEM) numbers of DCX+ cells detected in the SGZ-GCL and hilus of CON (white bars) and DEF (grey bars) rats 16 days following saline treatment (open bars) or KA-induced SE (hatched bars). KA-induced SE significantly increased the number of DCX+ cells in both CON and DEF rats (ps < 0.05). * statistically different from within-diet saline-treated group.

Figure 7

Hippocampal GFAP Expression 16 Days After Saline or KA Treatment in CON and DEF Rats. Comparison between CON (white bars) and DEF rats (grey bars) in GFAP mRNA levels (A) and protein levels (B) (mean ± SEM percent of control levels) in the intact hippocampus (open bars) and 16 days after KA-induced SE (hatched bars). mRNA levels were quantifed using RT-PCR and protein levels were quantified using Western blot analysis. SE significantly similarly increased GFAP mRNA and protein levels in both CON and DEF rats (ps < 0.05). # main effect of Seizure at p < 0.05. * statistically different from within-diet saline-treated group. + different from within-diet saline-treated group at p = 0.07.

Figure 8

Hippocampal Growth Factor Expression 16 Days After Saline or KA Treatment in CON and DEF Rats. Comparison between CON (white bars) and DEF (grey bars) rats in growth factor protein levels (mean ± SEM percent of control levels) in the intact hippocampus (open bars) and 16 days following KA-induced SE (hatched bars). Protein levels were quantified using ELISA. A main effect of Diet was present for IGF-1, where DEF rats had lower levels of hippocampal IGF-1 protein than CON rats across both saline- and KA-treated groups (p < 0.05). An overall effect of Seizure was evident for BDNF and FGF-2 protein (ps < 0.05) with a strong trend for IGF-1 protein (p = 0.06) where KA-induced SE similarly increased hippocampal growth factor expression in CON and DEF rats. Saline-treated DEF rats had significantly lower expression levels of IGF-1 and significantly higher expression levels of FGF-2 compared to saline-treated CON rats. * statistically different from within-diet saline-treated group at p < 0.05. + statistically different from saline-treated CON rats at p < 0.05.