Maternal choline availability alters the localization of p15Ink4B and p27Kip1 cyclin-dependent kinase inhibitors in the developing fetal rat brain hippocampus.

Previously we have shown that changes in maternal dietary choline are associated with permanent behavioral changes in offspring. Importantly, in adult male rats, feeding a choline-deficient diet increases the localization of cyclin-dependent kinase inhibitors (CDKIs) in the liver, whereas young adult CDKI knockout mice (p15Ink4B or p27Kip1) exhibit behavioral abnormalities. Thus, maternal dietary choline-CDKI interactions could underlie the changes we observe in fetal hippocampal development and cognitive function in offspring. Here, timed-pregnant rats on embryonic day E12 were fed the AIN-76 diet with varying levels of dietary choline for 6 days, and, on E18, fetal brain sections were collected, and the localization of CDKI proteins was studied using immunohistochemistry and an unbiased image analysis method. In choline-supplemented animals compared to controls, the number of cells with nuclear immunoreactivity for p15Ink4b CDKI protein was decreased 2- to 3-fold in neuroepithelial ventricular zones and adjacent subventricular zones corresponding to the fimbria, primordial dentate gyrus and Ammon's horn regions in the fetal hippocampus. In contrast, maternal dietary choline deficiency significantly decreased nuclear p15Ink4b immunoreactivity in the neuroepithelial layer of the dentate gyrus. Unlike p15Ink4b, the CDKI protein p27Kip1 was observed almost exclusively in the cytoplasm, though the protein was distributed throughout the proliferating and postmitotic zones in the E18 fetal hippocampus. Maternal dietary choline supplementation decreased the cytoplasmic staining intensity for p27Kip1 throughout the fetal hippocampus compared to control animals. Choline deficiency increased the staining intensity of p27Kip1 throughout the hippocampus in association with increased expression of MAP-1 and vimentin proteins. These results link maternal dietary choline availability to CDKI protein immunoreactivity and commitment to differentiation during fetal hippocampal development.

Maternal dietary choline availability alters mitosis, apoptosis and the localization of TOAD-64 protein in the developing fetal rat septum.

Maternal changes in dietary choline availability alter brain biochemistry and hippocampal development in the offspring resulting in lifelong behavioral changes in the offspring. In order to better understand the relationship between maternal diet, brain cytoarchitecture and behavior, we investigated the effects of choline availability on cell proliferation, apoptosis and differentiation in the fetal rat brain septum. Timed-pregnant rats on day E12 were fed AIN-76 diet with varying levels of dietary choline for 6 days. We found that choline deficiency (CD) significantly decreased the rate of mitosis in the progenitor neuroepithelium adjacent to the septum. In addition, we found an increased number of apoptotic cells in the septum of CD animals compared to controls (3.5+/-0.5 vs. 1.7+/-0.5 apoptotic cells per section; p<0.05). However, CD had no effect on apoptosis in the indusium griseum (IG), a region of cortex dorsal to the septum. Using an unbiased image analysis method and a monoclonal antibody we found a decreased expression of the TOAD-64 kDa protein, a marker of commitment to neuronal differentiation during fetal development, in the dorsal lateral septum of CD animals. CD also decreased the expression of TOAD-64 kDa protein in the IG and cortical plate adjacent to the septum. These results show that dietary choline availability during pregnancy alters the timing of mitosis, apoptosis and the early commitment to neuronal differentiation by progenitor cells in regions of the fetal brain septum, as well as hippocampus, two brain regions known to be associated with learning and memory.

Choline availability alters embryonic development of the hippocampus and septum in the rat.

Choline availability in the diet during pregnancy alters fetal brain biochemistry with resulting behavioral changes that persist throughout the lifetime of the offspring. In the present study, the effects of dietary choline on cell proliferation, migration, and apoptosis in neuronal progenitor cells in the hippocampus and septum were analyzed in fetal brains at different stages of embryonic development. Timed-pregnant rats on day E12 were fed AIN-76 diet with varying levels of dietary choline for 6 days, and, on days E18 or E20, fetal brain sections were collected. We found that choline deficiency (CD) significantly decreased the rate of mitosis in the neuroepithelium adjacent to the hippocampus. An increased number of apoptotic cells were found in the region of the dentate gyrus of CD hippocampus compared to controls (5.5+/-0.7 vs. 1.9+/-0.3 apoptotic cells per section; p<0.01). Using a combination of bromodeoxyuridine (BrdU) labeling and an unbiased computer-assisted image analysis method, we found that modulation of dietary choline availability changed the distribution and migration of precursor cells born on E16 in the fimbria, primordial dentate gyrus, and Ammon's horn of the fetal hippocampus. CD also decreased the migration of newly born cells from the neuroepithelium into the lateral septum, thus indicating that the sensitivity of fetal brain to choline availability is not restricted to the hippocampus. We found an increase in the expression of TOAD-64 protein, an early neuronal differentiation marker, in the hippocampus of CD day E18 fetal brains compared to controls. These results show that dietary choline availability alters the timing of the genesis, migration, and commitment to differentiation of progenitor neuronal-type cells in fetal brain hippocampal regions known to be associated with learning and memory processes in adult brain.