Ltd.). This procedure was repeated four times with 15-s intervals. Cued and contextual tests were carried out 1 day after fear conditioning. For the cued test, the freezing response was measured in the neutral cage for 1 min in the presence of a continuous-tone stimulus identical to the conditioned stimulus. For the contextual test, mice were placed in the conditioning cage, and the freezing response was measured for 2 min in the absence of the conditioned stimulus. All results were expressed as the mean ± S.E.M. for each group. The difference

among groups was analyzed with a one-way, two-way, or repeated ANOVA, followed by the Student–Newman–Keuls DNA Damage inhibitor multiple range-test. The Student’s t-test was used to compare two sets of data. IgG antibodies to Aβ were detected in the serum of nasally treated Tg2576 mice with rSev-Aβ at 4 weeks and less amount at 8 weeks after vaccination (Fig. 2a). However, intramuscularly treated mice showed poor antibody response (not shown). The immune sera from nasally vaccinated mice stained the senile plaque amyloid in the tissue. Nasal vaccination with rSeV-Aβ resulted in marked reduction of Aβ burden in the ON-01910 manufacturer frontal cortex, parietal association cortex and hippocampus compared to the control (Fig. 2b and c). Thioflavin S-positive senile

plaques were also significantly reduced in vaccinated mice. However, intramuscular injection of rSeV-Aβ had little effects on Aβ clearance (Fig. 2d and e). Quantitative analyses showed a marked reduction of Aβ deposition in nasally vaccinated mice compared to the control (Fig. 2f), but intramuscular injection showed no difference in Aβ clearance (Fig. 2g). To investigate the expression of Aβ43 in the olfactory bulb and brain stem through trafficking of rSeV via the olfactory or trigeminal nerves, we stained the brain tissue with anti-Aβ43 antibody. Although Tg2576

mice expressed very little endogenous Aβ43, we could not find any Aβ43 depositions after the nasal administration of rSeV-Aβ (data not shown). Soluble/insoluble Aβ40 and Aβ42 in brain homogenate fractions extracted with TBS or 2% SDS and 70% formic acid were quantified using the sandwich ELISA. Nasal vaccination of rSeV-Aβ significantly reduced the contents of soluble and insoluble Aβ40 and Aβ42 compared to the control PD184352 (CI-1040) (Aβ40 in TBS, p = 0.04; 2% SDS, p = 0.027; formic acid, p = 0.001. Aβ42 in TBS, p = 0.008; 2% SDS, p = 0.01; formic acid, p = 0.045.) ( Fig. 3A), but again intramuscular injection of rSeV-Aβ was ineffective (Aβ40 in TBS, p = 0.3; 2% SDS, p = 0.45; formic acid, p = 0.41. Aβ42 in TBS, p = 0.15; 2% SDS, p = 0.27; formic acid, p = 0.48) ( Fig. 3B). The trimeric, tetrameric, nonameric and dodecameric (Aβ*56) Aβ oligomers in soluble fraction of Tg2576 mice were detected by using Western blotting. Nasal vaccination with rSeV-Aβ in Tg2576 mice resulted in a marked reduction in the contents of Aβ*56 (dodecamer) but not in soluble sAPPα (Fig. 3C).