Visual inspection revealed symmetric

clusters of points, and the “ellipse fitting” method was used to quantify short-term variability (SD1) and long-term variability (SD2) (Tulppo et al. 1996; Brennan et al. 2002; Fishman et al. 2012) using Software-R, MatLab (Gonsenhauser et al. 2004). A two-way repeated-measures analysis of variance (ANOVA) was used to test for significance of differences between WT and KO mice. Specific differences were identified by Student-Newman–Keuls test. Results are expressed as means ± SD. Behavioral selleck products assays A number of behavioral Inhibitors,research,lifescience,medical tests were performed using free-fed, gender- and age-matched (8- to 12-week) mice. Motor behavior was assayed using the treadmill assay. WT and KO mice were familiarized with treadmill running prior to measuring exercise endurance using

a rodent Exer3/6 treadmill (Columbus Inhibitors,research,lifescience,medical Instruments, Columbus, OH). On day 1 of habituation, mice were allowed to explore the treadmill freely for 10 min. On day 2, animals were habituated to walking at 10 m/min for 10 min, and on day 3, the speed was increased to 12 m/min. In the testing sessions, Inhibitors,research,lifescience,medical the mice were required to walk at a relatively easy pace of 10 m/min for 10 min before increasing the pace to 20 m/min in 2-min intervals, a standard exercise running test. To encourage the mice to run, the treadmill was equipped with an electrical shock grid at the rear Inhibitors,research,lifescience,medical of the treadmill that delivered a shock (0.15 mA) that did not harm or injure the animals. When the mice reached exhaustion, as defined by their inability to run for 10 sec, testing was discontinued. Home cage activity was measured for 21 h to determine the activity patterns of the WT and KO mice Inhibitors,research,lifescience,medical in their normal habitat without experimenter interference. Locomotor activity was measured by tracking the animals using a high-definition CCD camera (Panasonic, Osaka, Japan) and tracking system software (Ethovision XT, Noldus, Wageningen, NL). Animal activity was tracked during

the light and dark phases and during transitions between the two phases. Anxiety-related behavior in WT and KO mice was compared using an automated elevated plus-maze (Med Associates, VT). The maze consists of a platform and four arms, two of which are enclosed. The animals can see their high elevation in the open, but not the closed arms. The animals were placed in the center facing an enclosed arm and Carfilzomib allowed to explore freely. Animal activity was tracked for 5 min; the number of explorations (defined as entrance of only the front paws), entries (defined as entrance with all four paws), and time spent in the open arms, the closed arms, or in the central platform were recorded. RNA preparation and quantitative real-time PCR Brains were rapidly removed from euthanized mice and frozen at −80°C until use.