To investigate the neurotoxicity profile of glycidol and its effect on developmental hippocampal neurogenesis, pregnant Sprague Dawley rats were given drinking water containing 0, 100, 300, or 1000 ppm glycidol from gestational day 6 until weaning on day 21 after delivery. At 1000 ppm, dams showed progressively worsening gait abnormalities, and histopathological examination showed generation of neurofilament-L(+) spheroids in the cerebellar granule layer and dorsal funiculus of the medulla oblongata, central chromatolysis in the trigeminal nerve ganglion cells, and axonal degeneration in the sciatic nerves. Decreased dihydropyrimidinase-like 3(+) immature granule cells in the subgranular zone (SGZ) and increased immature reelin(+) or calbindin-2(+) γ-aminobutyric acid-ergic interneurons and neuron-specific nuclear protein (NeuN)(+) mature neurons were found in the dentate hilus of the offspring of the 1000 ppm group on weaning. Hilar changes remained until postnatal day 77, with the increases in reelin(+) and NeuN(+) cells being present at ≥ 300 ppm, although the SGZ change disappeared. Thus, glycidol caused axon injury in the central and peripheral nervous systems of adult rats, suggesting that glycidol targets the newly generating nerve terminals of immature granule cells, resulting in the suppression of late-stage hippocampal neurogenesis. The sustained hilar changes may be a sign of continued aberrations in neurogenesis and migration. The no-observed-adverse-effect level was determined to be 300 ppm (48.8mg/kg body weight/day) for dams and 100 ppm (18.5mg/kg body weight/day) for offspring. The sustained developmental exposure effect on offspring neurogenesis was more sensitive than the adult axonal injury.

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Autoimmunity is prevented by the function of the autoimmune regulator [AIRE (Aire in mice)], which promotes the expression of a wide variety of tissue-restricted antigens (TRAs) from medullary thymic epithelial cells (mTECs) and from a subset of peripheral antigen-presenting cells (APCs). We examined the effect of additive expression of human AIRE (huAIRE) in a model of autoimmune diabetes in NOD mice. Unexpectedly, we observed that mice expressing augmented AIRE/Aire developed muscle-specific autoimmunity associated with incomplete maturation of mTECs together with impaired expression of Aire-dependent TRAs. This led to failure of deletion of autoreactive T cells together with dramatically reduced production of regulatory T cells in the thymus. In peripheral APCs, expression of costimulatory molecules was augmented. We suggest that levels of Aire expression need to be tightly controlled for maintenance of immunological tolerance. Our results also highlight the importance of coordinated action between central tolerance and peripheral tolerance under the common control of Aire.

We have shown that maternal manganese (Mn) exposure caused sustained disruption of hippocampal neurogenesis of mouse offspring. To clarify the effects of maternal Mn exposure on epigenetic gene regulation contributing to the sustained disruption of hippocampal neurogenesis, we treated pregnant ICR mice with MnCl₂ in diet from gestational day 10 through day 21 after delivery on weaning and searched epigenetically downregulated genes by global promoter methylation analysis in the hippocampal dentate gyrus of male offspring on postnatal day (PND) 21 and PND 77. By CpG promoter microarray analysis on PND 21 following 800-ppm Mn exposure, sustained promoter hypermethylation and transcript downregulation through PND 77 were confirmed with Mid1, Atp1a3, and Nr2f1, whereas Pvalb showed a transient hypermethylation only on weaning. The numbers of Pvalb⁺ and ATP1a3⁺ neurons suggestive of γ-aminobutyric acid (GABA)ergic interneurons, Mid1⁺ cells suggestive of late-stage granule cell lineage and GABAergic interneurons, and COUP-TF1⁺ cells suggestive of early-stage granule cell lineage were all reduced on PND 21, and reductions were sustained on PND 77 except for no change in Pvalb⁺ cells. Mid1⁺ cells showed asymmetric distribution with right-side predominance, and Mn exposure abolished it by promoter hypermethylation of the right side. These findings indicate epigenetic mechanisms as mediators, through which Mn exposure modulates neurogenesis involving both granule cell lineage and GABAergic interneurons with long-lasting and stable repercussions. Disruption of asymmetric cellular distribution of Mid1 suggests that higher brain functions specialized in the left or right side of the brain were affected.

-For molecular analysis in anatomically-specific brain regions for rodent studies, it is necessary to establish a fast and accurate procedure for tissue sampling to achieve high integrity and expression fidelity of extracted molecules. The present study was performed to examine suitability of whole brain fixation with methacarn and subsequent tissue sampling using punch-biopsy devices for gene expression analysis in rats. After fixation, each specific region, i.e., hippocampal dentate gyrus, corpus callosum, cingulate cortex or cerebellar vermis was collected, and the integrity and variability of expression data of extracted total RNAs and polypeptides were examined. Methacarn fixation, acetone fixation, and unfixed tissues were compared. Methacarn fixation resulted in high integrity of total RNAs sufficient for conducting global expression analysis and superior in terms of uniformity in the integrity among brain regions to that of acetone fixation. Extracted polypeptide after methacarn fixation revealed similar integrity to that without fixation or with acetone fixation. Methacarn fixation resulted in lower mRNA expression variability between samples than acetone fixation in microarray analysis. The fidelity of polypeptide expression was mostly equivalent between methacarn and acetone fixation in 2-dimensional differential in-gel electrophoresis, although the expression levels of a small number of polypeptides from acetonefixed tissues were affected. These results suggest that whole brain fixation with methacarn retains advantages for global analyses of mRNAs and polypeptides in rodent studies. Original ArticleThe Journal of Toxicological Sciences (J. Toxicol. Sci.) Vol.38, No.3, 431-443, 2013 Vol. 38 No. 3 431 throughput and use of fixed brain samples may be preferable for this purpose.Methacarn is a protein-precipitating and non-crosslinking organic solvent fixative (Mitchell et al., 1985;Puchtler et al., 1970), and fixation with this solution enables preservation of tissue morphology for histopathological assessment in paraffin-embedded tissues (Delfour et al., 2006;Srinivasan et al., 2002). Methacarn generally gives superior immunohistochemical results over aldehyde-based cross-linking fixatives (Banks, 1979;Orstavik et al., 1981;Rognum et al., 1980) because antigenicity is usually maintained (Mitchell et al., 1985). We previously found that methacarn fixation yields high quality DNA, RNA and protein even in paraffin-embedded sections for analyses of genomic DNA, expression microarrays, real-time RT-PCR and western blotting (Shibutani et al., 2000;Shibutani and Uneyama, 2002;Takagi et al., 2004;.For global assessment of genes and proteins in anatomically-specific brain regions in animal studies using rodents, both high integrity of extracted molecules and minimal inter-animal variability in the expression of extracted macromolecules are essential. Therefore, establishment of a procedure for quick and accurate tissue sampling and processing which can overcome these hurdles is necessary. For th...

The effect of exogenously administered manganese (Mn) on developmental neurogenesis in the hippocampal dentate gyrus was examined in male mice after maternal exposure to MnCl(2) (0, 32, 160, or 800 ppm as Mn in diet) from gestational day 10 to day 21 after delivery on weaning. Immunohistochemistry was performed to monitor neurogenesis and interneuron subpopulations on postnatal days (PNDs) 21 and 77 (adult stage). Reelin-synthesizing γ-aminobutyric acid (GABA)ergic interneurons increased in the hilus with ≥ 160 ppm on weaning to sustain to PND 77 at 800 ppm. Apoptosis in the neuroblast-producing subgranular zone increased with 800 ppm and TUC4-expressing immature granule cells decreased with 800 ppm on weaning, whereas at the adult stage, immature granule cells increased. On PND 21, transcript levels increased with Reln and its receptor gene Lrp8 and decreased with Dpysl3 coding TUC4 in the dentate gyrus, confirming immunohistochemical results. Double immunohistochemistry revealed a sustained increase of reelin-expressing and NeuN-lacking or weakly positive immature interneurons and NeuN-expressing mature neurons in the hilus through to the adult stage as examined at 800 ppm. Brain Mn concentrations increased at both PNDs 21 and 77 in all MnCl(2)-exposed groups. These results suggest that Mn targets immature granule cells causing apoptosis and neuronal mismigration. Sustained increases in immature reelin-synthesizing GABAergic interneurons may represent continued aberration in neurogenesis and following migration to cause an excessive response for overproduction of immature granule cells through to the adult stage. Sustained high concentration of Mn in the brain may be responsible for these changes.

Hypothyroidism affects neurogenesis. The present study was performed to clarify the sensitivity of neurogenesis-related cellular responses in the hippocampal dentate gyrus between developmental and adult-stage hypothyroidism. An exposure study of methimazole (MMI) as an anti-thyroid agent at 0, 50, 200 ppm in the drinking water was performed using pregnant rats from gestation day 10 to postnatal day (PND) 21 (developmental hypothyroidism) and adult male rats by setting an identical exposure period from PND 46 through to PND 77 (adult-stage hypothyroidism). Offspring with developmental hypothyroidism were killed at PND 21 or PND 77, and animals with adult-stage hypothyroidism were killed at PND 77. Proliferation and apoptosis were unchanged in the dentate subgranular zone by either developmental or adult-stage hypothyroidism. With regard to precursor granule cells, a sustained reduction of paired box 6-positive stem or early progenitor cells and a transient reduction of doublecortin-positive late-stage progenitor cells were observed after developmental hypothyroidism with MMI at 50 and 200 ppm. These cells were unchanged by adult-stage hypothyroidism. With regard to γ-aminobutyric acid (GABA) ergic interneuron subpopulations in the dentate hilus, the number of parvalbumin-positive cells was decreased and the number of calretinin-positive cells was increased after both developmental and adult-stage hypothyroidism with MMI at 50 and 200 ppm. Fluctuations in GABAergic interneuron numbers with developmental hypothyroidism continued through to PND 77 with 200 ppm MMI. Considering the roles of GABAergic interneuron subpopulations in neurogenesis and neuronal differentiation, subpopulation changes in GABAergic interneurons by hypothyroidism may be the signature of aberrant neurogenesis even at the adult stage.