Prenatal choline deficiency increases choline transporter expression in the septum and hippocampus during postnatal development and in adulthood in rats

Abstract

Supplementation of maternal diet with the essential nutrient, choline, during the second half of pregnancy in rats causes long-lasting improvements in spatial memory in the offspring and protects them from the memory decline characteristic of old age. In contrast, prenatal choline deficiency is associated with poor performance in certain cognitive tasks. The mechanism by which choline influences learning and memory remains unclear; however, it may involve changes to the hippocampal cholinergic system. Previously, we showed that the hippocampi of prenatally [embryonic days (E) 11-17] choline-deficient animals have increased synthesis of acetylcholine (ACh) from choline transported by the high-affinity choline transporter (CHT) and reduced ACh content relative to the control and to the E11-17 choline-supplemented rats. In the current study, we found that, during postnatal period [postnatal days (P) 18-480], prenatal choline deficiency increased the expression of CHT mRNA in the septum and CHT mRNA and protein levels in the hippocampus and altered the pattern of CHT immunoreactivity in the dentate gyrus. CHT immunoreactivity was more prominent in the inner molecular layer in prenatally choline-deficient rats compared to controls and prenatally choline-supplemented animals. In addition, in all groups, we observed a population of hilar interneurons that were CHT-immunoreactive. These neurons are the likely source of the hippocampal CHT mRNA as their number correlated with the levels of this mRNA. The abundance of hippocampal CHT mRNA rose between P1 and P24 and then declined reaching 60% of the P1 value by P90. These data show that prenatal availability of choline alters its own metabolism (i.e., CHT expression). While the upregulated CHT expression during the period of prenatal choline deficiency may be considered as a compensatory mechanism that could enhance ACh synthesis when choline supply is low, the persistent upregulation of CHT expression subsequent to the brief period of prenatal deprivation of choline in utero might be beneficial during choline deficiency in adulthood.

Figure 1. CHT mRNA expression in the medial septum

Septal RNA from P18, P90 and P480 rats was used for RT-PCR of CHT and β-actin. CHT levels were normalized using β-actin levels and are presented as means ± SEM (n=4 per group on P18 and P90, n=3 per group on P480). The levels of CHT were significantly different among the three groups of animals on each day as determined by ANOVA (p<0.005, p<0.05, and p<0.01, respectively). The ANOVA was followed up with Tukey tests at a 5% procedure wise error rate. Prenatally choline-deficient animals had a significantly higher amount of CHT mRNA compared to prenatally choline-supplemented animals on all days and to control animals at P480. On P18, the CHT mRNA levels were significantly higher in the control animals than prenatally choline-supplemented animals. S, prenatally choline-supplemented; C, control; D, prenatally choline-deficient.

Figure 2. CHT protein levels in the hippocampus

Hippocampal lysates from P18 (A) and P480 (B) rats were used for Western blot analysis of CHT. CHT protein levels are presented as means ± SEM, n=3 per group. The levels of CHT were significantly different among the three groups of animals on both days as determined by ANOVA (p<0.005, and p<0.01, respectively). The ANOVA was followed up with Tukey tests at a 5% procedure wise error rate. On both P18 and P480, prenatally choline-deficient animals had a significantly higher amount of CHT protein compared to control animals and to prenatally choline-supplemented animals. S, prenatally choline-supplemented; C, control; D, prenatally choline-deficient.

Figure 3. Developmental changes in CHT expression in the hippocampus of control rats

Hippocampal RNA from P1, 8, 15, 18, 34, 50, 64 and 90 rats was used for RT-PCR of CHT and β-actin. CHT levels were normalized using β-actin levels and are presented as means ± SEM, n=6 per group.

Figure 4. CHT immunocytochemistry in the hippocampus and medial septum of P18 rats

Prenatally choline-supplemented (A, B, C), control (D, E, F), deficient (G, H, I) animals. GC, granular cell layer; SGP, supragranular plexus; IML, inner molecular layer; OML, outer molecular layer; MSN, medial septal nucleus; AC, anterior commissure. The pattern of CHT-immunoreactivity in the molecular layer of the dentate gyrus differed among the three groups and CHT-immunoreactive neurons were generally more prominent in CA4 in the deficient group (G, H) and less prominent in the supplemented group (A, B) compared to controls (D, E) despite similar staining intensities in other brain regions such as the medial septal nucleus (C, F, I). Magnification bars: A, D, G - 500 microns; B, E, H - 50 microns; C, F, I - 500 microns.

Figure 5. Correlation between the number of CHT-positive cells in the hilus and the level of CHT mRNA in the hippocampus at P18

(A) CHT-positive neuronal perikarya in the CA4 regions of the hippocampal formation of P18 animals were quantitated and analyzed using a semi-automated computerized morphometry system. The number of CHT-positive cells was significantly different among the three groups of animals as determined by ANOVA (p<0.005). The ANOVA was followed up with Tukey tests at a 5% procedure wise error rate. Control animals had a significantly higher number of CHT-positive cells compared to prenatally choline-supplemented animals. (B) Hippocampal RNA from P18 rats was used for RT-PCR of CHT and β-actin. CHT levels were normalized using β-actin levels and are presented as means ± SEM (n=6 per group). The levels of CHT were significantly different among the three groups of animals as determined by ANOVA (p<0.05). The ANOVA was followed up with Tukey tests at a 5% procedure wise error rate. Prenatally choline-deficient and control animals had a significantly higher amount of CHT mRNA compared to prenatally choline-supplemented animals. (C) A significant correlation between the number of CHT-positive cells in the hilus and the level of CHT mRNA in the hippocampus was observed (R=0.567 and p<0.05). S, prenatally choline-supplemented; C, control; D, prenatally choline-deficient.

Figure 6. CHT immunocytochemistry in the hippocampus and medial septum of P72 rats

Prenatally choline-supplemented (A, B, C), control (D, E, F), and deficient (G, H, I) animals. GC, granular cell layer; SGP, supragranular plexus; IML, inner molecular layer; OML, outer molecular layer; MSn, medial septal nucleus; LSn, Lateral septal nucleus; NDB, nucleus of the digonal band. The pattern of CHT-immunoreactivity seen at P18 persisted at P72. CHT-immunoreactivity was more prominent in the inner molecular layer of the dentate gyrus and CHT-immunoreactive neurons were generally more prominent in CA4 in the deficient group (G, H) compared to the supplemented (A, B) and control (D, E) groups despite similar staining intensities in other brain regions such as the medial septal nucleus (C, F, I). The prominence of inner molecular layer staining generally paralleled the number CHT-immunoreactive hilar neurons even in the face of case to case variability (B, E, H). Magnification bars: A, D, G - 500 microns; B, E, H - 50 microns; C, F, I - 500 microns.

Figure 7. CHT-positive cells in the hilus at P72

(A) CHT-positive neuronal perikarya in the CA4 regions of the hippocampal formation of P18 animals were quantitated and analyzed using a semi-automated computerized morphometry system. There were no significant differences between the groups. (B) Hippocampal RNA from P64 rats was used for RT-PCR of CHT and β-actin. CHT levels were normalized using β-actin levels and are presented as means ± SEM (n=6 per group). The levels of CHT were significantly different among the three groups of animals as determined by ANOVA (p<0.0005). The ANOVA was followed up with Tukey tests at a 5% procedure wise error rate. Prenatally choline-deficient animals had a significantly higher amount of CHT mRNA compared to prenatally choline-supplemented and control animals.