Phenomaster software

To stimulate neuronal activation-induced cFos expression in the hippocampus, mice were subjected to a complex running wheel (CRW) set-up for 4 h [43 (link)]. Mice were single housed in type III cages (42 × 26 × 18 cm, Tecniplast), equipped with CRW (TSE-Systems) characterized by randomized omitted bars [26 (link), 27 (link)]. Mice were habituated to the experimental room and CRW for 2 h prior to dark phase. After dark phase onset, voluntary running was automatically tracked for 4 h via Phenomaster software (TSE-Systems) and the total running distance per mouse calculated. Mice were perfused with Ringer and 4% formaldehyde/PBS, 30 min after removal from the CRW set-up.

Mice were run on an indirect colorimetry treadmill (TSE Systems) before and 6 wk after TAC/DOCA procedure. Mice underwent a graded exercise tolerance test until exhaustion as follows (speed, duration, incline grade): 0 m/min, 3 min, 0°; 6 m/min, 2 min, 0°; 9 m/min, 2 min, 5°; 12 m/min, 2 min, 10°; 15 m/min, 2 min, 15°; 18 m/min, 1 min, 15°; 21 m/min, 1 min, 15°; 23 m/min, 1 min, 15°; and +1 m/min, each 1 min thereafter, 15°. Exhaustion was defined as the point at which mice maintained continuous contact with the shock grid for 5 s or 20 visits to the shock grid in a 1-min period. During testing, gas was collected continuously and analyzed every 5 s. PhenoMaster software (TSE Systems) recorded and calculated oxygen consumption (VO₂), carbon dioxide consumption (VCO₂), the respiratory exchange ratio (RER), distance run, and visits to the shock grid. The VO_2max was determined by the peak VO₂ reached during this test when RER was >1.0. Maximum running speed was defined as the treadmill speed at which VO_2max was achieved. Mice failing to reach an RER >1.0 were excluded.

Mice were single-housed in two-hopper feeding chambers (TSE Systems, Chesterfield, MO, USA) for five days to acclimate, and received ad-libitum access to SD and water throughout behavioral testing. At the time of testing, mice were given access for the first time to the hopper containing Western Diet (WD; Research Diets D12079B, New Brunswick, NJ, USA; 40% kcal from fat, 17% kcal from protein, 43% kcal from carbohydrates as mostly sucrose). Food weights were measured in real time and recorded every minute using Phenomaster software (TSE Systems). Preferences for WD versus SD (% total kcals from WD), total caloric intake of each diet (kcals), average meal size of each diet (kcals), and meal frequency were calculated from recorded data, beginning one hour before the dark cycle (1700 h). The criteria for a meal was consumption of a minimum of 0.1 g of food with an inter-meal interval less than 30 min.

During the running wheel sessions, mice were single-housed and a running wheel (TSE Systems, Bad Homburg, Germany) was placed in each home cage (Type II long, Ebeco, Germany) in a dedicated room with the same light/dark cycle and environmental conditions as in the housing room. Data of 3 consecutive weeks of wheel running activity were automatically collected and processed with PhenoMaster software (TSE Systems). In week 1 and week 2, mice were exposed to a standard wheel, while during week 3 a “complex” wheel was introduced. This wheel has a fewer number of rods which are irregularly spaced, though with a predictable pattern (Liebetanz and Merkler, 2006 (link); Mandillo et al., 2014 (link)). Food and water were available ad libitum, and nestpaper was provided. Cages were changed every week. Wheel running activity for the 12 h dark phase was included in this study. Parameters measured were weekly values of: (1) total distance (m); (2) average run duration (s), where “runs” are running episodes at a velocity exceeding 30 rpm (~0.18 m/s); (3) average maximum speed (m/s) from daily values calculated as average of maximum speed values sampled every 5 min during the 12 h dark period; (4) total time on wheels (s).