Rhythms in locomotor activity

Methods employed were as previously reported [36,43]. Mice were individually housed in cages equipped with running wheels, and their voluntary wheel-running activity was recorded as revolutions (rev) per 3 minute intervals using a data acquisition system obtained from Mini Mitter Co. (Bend, OR). Diurnal rhythms in running wheel activity were assessed over 14 days while animals were exposed to a 12:12 LD cycle, and circadian rhythms were assessed in constant darkness (DD) over the subsequent 10 days. To determine the effects of a phase delaying light treatment, a pulse of white light (100 lux, 10 min) was applied at circadian time (CT) 16 after 10–14 days in DD. The resulting phase shift was calculated from the 10 days subsequent to the light-pulse as previously described [43]. As is the convention, CT 12 was defined as the time of activity onset. All handling of mice in DD was performed with the aid of night vision goggles (FJW Industries, Palantine, IL).

Analysis of locomotor activity rhythms was as described previously [43, 44]. Briefly, we determined the period (hr) and power (%V, rhythm strength) by χ² periodogram analysis. Periodogram-derived period estimates were confirmed using the slope of an eye-fitted line through behavioral onsets. Alpha was defined as the duration of the main activity bout from 10 days of activity, corrected for free-running period in DD. Fragmentation (bouts/day) and precision of day-to-day activity onset were determined using Clocklab (Actimetrics, Wilmette, IL). Fragmentation was defined by bouts/day, where a bout was determined as 21 consecutive minutes of activity (maxgap setting of 21 min). Imprecision was determined by calculating the inverse of daily variation in onset from a best-fit regression line drawn across 10 days of activity. DD imprecision was corrected for free-running period. Phase shift magnitude following the phase-delaying light treatment was determined by measuring the phase difference between best-fit regression lines drawn through the 10 days preceding and 10 days subsequent to the light treatment. Investigators masked as to the experimental group made measurements, and reported values are the average of two independent determinations.

Statistically significant effects of genotype, sex, and age on activity rhythm parameters were tested using Three-Way ANOVA, with P < 0.05. When main or interaction effects were identified, significant genotype differences within sex/age, significant sex differences within genotype/age, and significant age differences within genotype/sex were identified post-hoc using the Holm-Sidak method for multiple pairwise comparisons, with P < 0.05.