Differential Sensitivity to Ethanol-Induced Circadian Rhythm Disruption in Adolescent and Adult Mice

Abstract

Background: Growing evidence supports a central role for the circadian system in alcohol use disorders, but few studies have examined this relationship during adolescence. In mammals, circadian rhythms are regulated by the suprachiasmatic nucleus, a biological clock whose timing is synchronized (reset) to the environment primarily by light (photic) input. Alcohol (ethanol [EtOH]) disrupts circadian timing in part by attenuating photic phase-resetting responses in adult rodents. However, circadian rhythms change throughout life and it is not yet known whether EtOH has similar effects on circadian regulation during adolescence.

Methods: General circadian locomotor activity was monitored in male C57BL6/J mice beginning in adolescence (P27) or adulthood (P61) in a 12-hour light, 12-hour dark photocycle for ~2 weeks to establish baseline circadian activity measures. On the day of the experiment, mice received an acute injection of EtOH (1.5 g/kg, i.p.) or equal volume saline 15 minutes prior to a 30-minute light pulse at Zeitgeber Time 14 (2 hours into the dark phase) and then were released into constant darkness (DD) for ~2 weeks to assess phase-resetting responses. Control mice of each age-group received injections but no light pulse prior to DD.

Results: While adults showed the expected decrease in photic phase-delays induced by acute EtOH, this effect was absent in adolescent mice. Adolescents also showed baseline differences in circadian rhythmicity compared to adults, including advanced photocycle entrainment, larger photic phase-delays, a shorter free-running (endogenous) circadian period, and greater circadian rhythm amplitude.

Conclusions: Collectively, our results indicate that adolescent mice are less sensitive to the effect of EtOH on circadian photic phase-resetting and that their daily activity rhythms are markedly different than those of adults.

Keywords: Adolescent; Alcohol; Circadian; Photic Phase-Resetting.

Fig. 1

Experimental procedure. (A) Timeline showing the experimental procedure relative to the ages of mice in each age group (P = postnatal day, LD = light-dark photocycle, Inj = injection, LP = light pulse, DD = constant darkness). (B) Design for the phase-resetting experiments showing the duration of time in each photocycle used in the analyses, the timing of injection (INJ), and timing of the light-pulse (star; control mice received injections at the same time, but no light pulse). Also shown are the periods of time represented by the data in each subsequent figure.

Fig. 2

Age-related differences in photocycle entrainment. (A) Period length did not differ between adolescents (n = 41) and adults (n = 43) in LD, indicating that both were able to entrain normally to a 24-hour photocycle. (B) Circadian rhythm amplitude was higher in adolescents compared to adults. (C) Adolescent mice became active before adults, much closer to the time of lights-off (ZT12). (D) Adolescent mice also became restful before adults, ceasing activity prior to the time of lights-on (ZT0), while adults became inactive after ZT0. (E) The duration of nightly activity (alpha) in adolescents was slightly but significantly shorter than in adults. Data are mean ± SEM; *p < 0.05.

Fig. 3

Differential sensitivity to ethanol and photic input in adolescent versus adult mice. (A) Adolescent mice (n = 10–11/tx) had larger phase-delays to light input at ZT14 than did adults (n = 12–13/tx) regardless of treatment and were unimpaired by acute ethanol (EtOH; 1.5 g/kg) compared to age-matched, saline-treated controls. Adult mice receiving ethanol showed the expected attenuation in photic phase-delays compared to saline-treated adult controls. (B) Neither ethanol (n = 8–10/age group) nor saline (n = 10/age group) had a phase-delaying effect at ZT14 in the absence of a light pulse. Data are mean ± SEM; *p < 0.05.

Fig. 4

Representative, double-plotted actograms of a mouse from each of the four age × treatment groups in the photic phase-resetting experiment (A) and the control phase-resetting experiment (B). The asterisk denotes the time of the light pulse and shading denotes the days spent in DD.

Fig. 5

Age-related changes in endogenous circadian period. (A) Endogenous, or free-running, period (tau in DD) was shorter in adolescents (n = 41) than adults (n = 43). Notably, free-running period in adolescents was shorter than 24 h, whereas it was longer than 24 h in adults. (B) Circadian rhythm amplitude remained higher in adolescents compared to adults in DD. The possibility that acute ethanol affected endogenous period or amplitude in mice from either age group was ruled out prior to this analysis. Data are mean ± SEM; *p < 0.05.