2.1. Animals

Male C57BL/6J mice (3 weeks old, Jackson Laboratories, Bar Harbor, ME) were grouped housed (4-5 mice per group) in standard Plexiglas cages under a 12 h light (>500 lux)/dark (<0.5 lux) cycle with lights on at 6:00 AM. Food and water were provided ad libitum for 1 week. Mice were singly housed when they reached 4 weeks of age, at which time caffeine exposed mice (CAF) were provided with a caffeine solution, while control animals continued ad libitum water access. Male mice only were used as male and female mice respond differently to adolescent social isolation for behaviors we tested, including acoustic startle and anxiety [19]. Food was provided ad libitum unless otherwise stated. Behavioral testing began at 18 days of caffeine drinking and two cohorts, one for light cycle (9AM) and one for dark cycle (8PM) experiments, were used sequentially for: open field test (OFT), forced swim test (FST), home cage activity, prepulse inhibition (PPI), elevated plus maze (EPM), and sucrose reward seeking. As previous work has demonstrated a connection between caffeine and ethanol consumption, caffeine use was discontinued while we assessed ethanol consumption, allowing us to determine how a history of caffeine use during adolescence effects adult ethanol consumption without interference from acute caffeine effects [20]. Taste preference was assessed after ethanol consumption assessment and both CAF and control animals were provided with ad libitum water without caffeine (Fig. 1). Mice were brought to the test room 30 minutes before testing to allow them to acclimate; this acclimation occurred in the light for animals tested during the light cycle and in the dark for animals tested during the dark cycle. A second cohort of mice was used to measure plasma corticosterone. Animal care and handling procedures were approved by the Mayo Clinic Institutional Animal Care and Use Committees in accordance with NIH guidelines.

Increased home cage locomotor activity and reduced sleep during the dark cycle in caffeine drinking mice, (a) Activity per minute for 12 days in the home cage, (b) Representative single day activity; CAF treated mice (light brown = activity counts per minute; dark brown = activity counts per minute averaged every hour) were hyperactive during the dark cycle compared with control mice (light blue = activity counts per minute; dark blue = activity counts per minute averaged every hour), particularly between zeitgeber time 13 and 17. (^#P <0.05 for main effect of treatment by two-way repeated measures ANOVA). (c) Total activity counts for dark cycle and light cycle. CAF treated mice exhibited significantly increased activity counts during the dark cycle compared to controls (^#P <0.05 for main effect of treatment by two-way ANOVA; *P < 0.05 compared to control by Tukey’s post hoc test). (d) Percent time immobile was reduced in CAF treated mice (*P < 0.05 by student’s t-test). (n = 9 per treatment group; all data are presented as mean ± standard error of the mean (SEM).