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total RNAs and polypeptides sufficient for global expression analysis. Methacarn fixation resulted in low mRNA expression variability between samples in microarray analysis. The fidelity of polypeptide expression was mostly equivalent in 2-dimensional differential in-gel electrophoresis between methacarn fixed and unfixed tissues.

Extraction of polypeptides during fixation was found to be negligible with methacarn.

These results suggest that whole brain fixation with methacarn retains advantages for global analyses of mRNAs and polypeptides.

In chapter 2, I investigated the neurotoxicity mechanism of glycidol and its effect on developmental hippocampal neurogenesis using rats. Animal study was performed using pregnant SD rats given drinking water containing glycidol at 0 (control), 100, 300, or 1,000 ppm from gestational day 6 until weaning on day 21 after delivery. For

examination of developmental neurotoxicity, I analyzed the distribution of granule cell lineages as well as their proliferation and apoptosis in the SGZ and interneurons in the hilus of the hippocampal dentate gyrus in offspring and also performed a brain

region-specific global gene expression profiling in relation with developmental

neurotoxicity. Four brain regions were selected to cover cerebral and cerebellar tissues,

i.e., the cingulate cortex, corpus callosum, hippocampal dentate gyrus and cerebellar

vermis. As a result, dams revealed gait abnormalities as well as histopathological and

immunohistochemical changes suggestive of axonal injury in the central and peripheral

nervous systems at 1,000 ppm. Global gene expression analysis revealed that glycidol

induced gene expression changes related to axonogenesis, neuritogenesis, myelination

and synaptic transmission in different brain regions. These expression profiles suggested

that developmental exposure to glycidol affected plasticity of neuronal networks in the

broad brain areas. On the other hand, glycidol at 1,000 ppm reversibly decreased the

number of dihydropyrimidinase-like 3 (as known as TUC-4)

immature granule cells in

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the SGZ of offspring. In the dentate hilus, glycidol caused sustained increase of calbindin-2 (Calb2)

or immature reelin

γ-aminobutyric acid (GABA)-ergic interneurons until postnatal day (PND) 77, which suggests a sign of continued aberrations in neurogenesis and migration. Glycidol caused axon injury in adult rats, suggesting that glycidol targets the newly generating nerve terminals of immature granule cells, resulting in the suppression of late-stage hippocampal neurogenesis.

In chapter 3, I investigated the possibility whether similar effect on neurogenesis in the SGZ can be induced by glycidol exposue in a framewok of 28-day tocixity study in rats. Glycidol was orally administered to 5-week-old male rats at 0, 30 or 200 mg/kg in purified water by gavage for 28 days. I examined the distribution of granule cell lineages as well as their proliferation and apoptosis in the SGZ and interneurons in the hilus of the hippocampal dentate gyrus for examination of effects on neurogenesis. I also performed region-specific global gene expression plofiling at the four brain regions, i.e., the cingulate cortex, corpus callosum, hippocampal dentate gyrus and cerebellar vermis, and following immunohistochemical analysis of representative molecules with regard to the distribution changes in immunoreactive cell populations based on the obtained gene expression profiles. At 200 mg/kg, which caused axonal changes in the central and peripheral nervous systems, expression changes of genes related to axonal and synaptic functions were mainly observed in the cingulate cortex, cerebellar vemis and hippocampal dentate gyrus, and these genes were mostly downregulated.

Immunohistochemically, the number of neurons expressing immediate-early genes, i.e.,

Arc, Fos and Jun, were decreased in the dentate granule cell layer, cingulate cortex and

cerebellar vermis. These changes were suggestive of suppression of neuronal plasticity

to maintain axons and denrites in response to glycidol exposure. On the other hand,

glycidol at 200 mg/kg, animals revealed aberrations in neurogenesis at the late-stage

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differentiation as evidenced by decreases of both doublecortin

and TUC-4

type-3 progenitor cells and increases of immature granule cells in the SGZ, as well as the increase of reelin

or Calb2

GABAergic interneurons in the dentate hilus.

In conclusion, whole brain fixation with methacarn is judged to be well suited for analysis of gene expression in anatomically-specific regions in the framework of

toxicity testing in rodents. Using this novel high-throughput tissue sampling method, we could identify the target gene profiles based on the pathological mechanism of

neurotoxicity for both developmental and adult stages. Although there were obvious effects on axons or neurites by glycidol exposure in only cerebellum through

histophathological and immunohistochemical analyses, the analyses focusing on the neurogenesis in the hippocampal dentate SGZ and global gene expression on each brain region revealed the effects on neurogenesis or suppressed neuronal plastisity.

Developmental exposure to glycidol suggested that the mechanism of toxicity of

glycidol has common targets of both mature axon terminals and developing neurites at

the late-stage neurogenesis. Therefore, it was suggested that neurogenesis could be a

common target of developmental neurotoxicants, and adult neurotoxicants could affect

neurogenesis. Glycidol causes aberations in neurogenesis in the SGZ at the late stage

involving the process of neurite extension in a standard regular 28-day toxicity study

similar to the developmental exposure study. Therefore, it is suggested that use of

neuronal developmental markers could be a promising evaluation endpoint for detection

of adult and developmental neurotoxicity in a scheam of 28-day toxicity study.

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