This extra density is also visible in the activated form of the molecule, albeit to a lesser extent

ay be useful to prevent AD or, at least, to delay the appearance, and to reduce the severity, of its symptoms. Materials and Methods Ethics Statement Transgenic CRND8 mice encoding a double-mutant of APP695 and wild type control littermates were used following the 15996703 ECC and National guidelines for animal care. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Italian Ministry of Health. Animals The following 2 age groups of mice were used: 1.5 month-old wt and pre-plaque Tg mice, 4 month-old wt and Tg mice. Different diet treatments were equally given to each age group of wt and Tg mice: 8 weeks with a 5.0% fat diet either alone or containing OLE . We used a modified low-fat AIN-76A diet composed of 50.0% sucrose, 5.0% fat, 20.0% casein, 15.0% corn starch, 5.0% powdered cellulose, 3.5% AIN-76 mineral mix, 1.0% AIN-76A vitamin mix, 0.3% DL-methionine and 0.2% choline bitartrate. the grid floor to which intermittent electric shocks were delivered. On day 1, each mouse was placed on the purchase c-Met inhibitor 2 platform and received an electric shock for 3 s when it stepped down placing all paws on the grid floor. Responsiveness to the punishment in the TT was assessed by animal vocalization; only those mice that vocalized touching the grid were used for retention test. 24 h after TT, each mouse was placed on the platform again. The latencies were measured, considering 180 s as the upper cut-off, during TT and RT. The tests were 12603839 carried out between 10:00 A.M. and 1:00 P.M. For object recognition test, we modified our previously described method using a white box with a grid floor covered by white filter paper. A 75 Watt lamp was suspended 50 cm above the box. Mouse behaviour was recorded by a video-tracking/computer-digitizing system. The day before testing, the mice were allowed to explore the box for 5 min. A session of two trials at 60 min interval was given on the test day. In T1, the time spent by each mouse exploring two identical 8.0 cm side grey cubes presented for 10 min in two opposite corners of the box was recorded. During T2, one of the cubes was replaced by a 8.0 cm side grey cylinder, and the mice were left in the box for 5 min. The time spent for the exploration of the familiar and the new object were recorded and a discrimination score was calculated. A discrimination score above 0.5 indicates the ability of mice to discriminate between the familiar and novel objects while a score below or equal to 0.5, reflecting a novel object exploration time less or equal to the half of the total time spent between the two objects, indicates memory impairment in this task. Animal Tissue Processing After completing the behavioral tests, the mice were sacrificed by cervical dislocation and the brains were rapidly removed and divided sagittally. For protein analysis, cortical and hippocampal samples from one hemibrain were immediately sectioned, snapfrozen and stored at 280uC. The other hemibrain was postfixed in phosphate-buffered 4.0% paraformaldehyde, pH 7.4, at 4uC for 48 h, rinsed in PBS and paraffin embedded for immunohistochemistry and Thioflavin S staining. Oleuropein Deglycosylation Oleuropein deglycosilation was performed according to Konno et al. with minor modifications. Briefly, a 10 mM solution of oleuropein in 0.1 M sodium phosphate buffer, pH 7.0, was incubated with 28.7 I.U./mL of b-glycosidase overnight at room temperature in the dark. The reaction mixture was centrifuged at 10,0006g for 15 min to precipitate th