ippocampus, between 10.512.5 months of age; plaque accumulation age-dependently progressed in both cortex and hippocampus. While 6E10, 4G8, and 111-3 detected both dense-core and diffuse plaque, 1395 seemed to recognize only dense-core plaques. We quantified burdens of 4G8-immunoreactive plaques in both cerebral cortex and hippocampus of mice between 10.5 and 24.7 months of age. These plaques included both the dense-core and diffuse types. Comparable plaque loads in cortex and hippocampus were found at each of the ages examined. Fig 3. Regional expression pattern of the APP transgene in rTg9191 mice. The regional pattern of APPNLI PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19784385 expression in four distinct anatomical structures, hippocampus, olfactory bulb, and cerebellum ) of brain was analyzed using mouse monoclonal antibody LN27, which specifically recognizes human APP, and 6E10. The APP transgene was expressed in cerebral cortex and hippocampus with a minor portion in olfactory bulb; however, no expression was observed in cerebellum. No immunoreactivity using these human-specific antibodies was seen in non-transgenic littermates. Alpha-tubulin served as the loading control. Representative blots show the APPNLI expression pattern of female mice, and similar results were found in male mice. doi:10.1371/journal.pone.0126317.g003 5 / 26 Characterizing a Model of -Amyloid Toxicity Fig 4. Age-related A plaque progression in rTg9191 mice. A representative sagittal section of brain. Black rectangles indicate the regions of cerebral cortex and hippocampal formation in which A plaques are shown in. Representative photomicrographs showing age-dependent progression of A plaques in female mice, visualized using 6E10; 4G8, directed against a purchase LY341495 mid-region of A; 1395, an Ax-40specific antibody; and 1-11-3, an Ax-42-specific antibody. Upper panels, cerebral cortex; lower panels, hippocampus. Scale bars: 100 m, 200 m. TTA, mice expressing only the tetracycline transactivator. Quantification of 4G8-immunoreactive A plaque load at various ages. doi:10.1371/journal.pone.0126317.g004 At 24.7 months of age, plaques loads reached 19% and 17%, respectively, for cortex and hippocampus. We also revealed dense-core plaques of aged rTg9191 mice using thioflavin S. At 24.7 months of age, plaque loads were 0.41% and 0.37% for cortex and hippocampus, respectively. 6 / 26 Characterizing a Model of -Amyloid Toxicity Age-related A production We determined the levels of A38, A40, and A42 
proteins in brain parenchyma from rTg9191 mice at young, middle, and old ages using an enzyme-linked immunosorbent assay. The levels of A proteins were separately measured in the water-soluble, detergent-soluble, and detergent-insoluble fractions. Overall, we observed an age-dependent increase in the production of A38, A40, and A42 in all three fractions–the one exception being that the level of A40 in the water-soluble fraction of 21-month-old mice was slightly lower than that of 12-month-old mice. This relative reduction in A40 might be caused by the coincidental formation of A40-comprising dense-core plaques. Of particular interest is that in the water-soluble fraction, levels of A40 were higher than A42 prior to plaque formation, but, in aged mice, this relationship between A40 and A42 was reversed; in addition, in 21- and 24-month-old mice with high plaque loads, levels of A42 decreased from water-soluble to detergent-soluble and insoluble PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19786154 fractions, whereas levels of A40 increased. These findings, together with observations con