Sex Differences in Photic Entrainment and Sensitivity to Ethanol-Induced Chronodisruption in Adult Mice After Adolescent Intermittent Ethanol Exposure

Abstract

Background: Evidence supports a role for the circadian system in alcohol use disorders, but the impact of adolescent alcohol exposure on circadian timing later in life is unknown. Acute ethanol (EtOH) attenuates circadian photic phase-resetting in adult, but not adolescent, rodents. However, nearly all studies have focused on males and it is unknown whether this adolescent-typical insensitivity to EtOH persists into adulthood after adolescent drinking.

Methods: Circadian activity was monitored in C57BL/6J mice receiving adolescent intermittent EtOH (AIE) exposure (15% EtOH and water every other day throughout adolescence) or water alone followed by 24 days wherein EtOH was not available (washout). Mice then received a challenge dose of EtOH (1.5 g/kg, intraperitoneal) or saline 15 minutes prior to a 30-minute phase-delaying light pulse and then were released into constant darkness (DD). To control for possible phase-shifting by EtOH challenge alone, a separate group of mice underwent AIE exposure (or water-only) and washout and then received an EtOH or saline injection, but did not receive a light pulse prior to DD.

Results: Striking sex differences in nearly all measures of circadian photic entrainment were observed during adolescence but AIE effects were subtle and few. Only EtOH-naïve adult male mice showed attenuated photic phase-shifts with EtOH challenge, while all other groups showed normal phase-resetting responses to light. AIE-exposed females showed a persistent delay in activity offset.

Conclusions: Adult male AIE-exposed mice retained adolescent-like insensitivity to EtOH-induced suppression of photic phase-resetting, suggesting AIE-induced "lock-in" of an adolescent behavioral phenotype. Adult AIE-exposed females showed delayed initiation of the rest phase. Our results also indicate that intermittent EtOH drinking has subtle effects on circadian activity in mice during adolescence that differ from previously reported effects on adult males. The observed sex differences in circadian activity, EtOH consumption and preference, and responses to EtOH challenge merit future mechanistic study.

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

Fig. 1.

Experimental procedure. (A) Timeline showing the experimental procedure relative to the ages of mice (AIE = adolescent intermittent ethanol, LD = light-dark photocycle, P = postnatal day, Inj = injection, 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 subsequent figures.

Fig. 2.

Sex differences in ethanol consumption during AIE. (A) On average, females (n = 18) consumed more ethanol than males (n = 19) during AIE, but preference did not differ (B). Data are mean ± SEM; *p < 0.05.

Fig. 3.

Sex differences in photocycle entrainment among adolescent mice during AIE. (A) Circadian rhythm amplitude was greater in female mice (n = 32) compared to males (n = 36) regardless of exposure. (B) Active-phase duration (alpha) was longer in female mice compared to males. (C) Activity onsets were earlier in females than in males, while (D) activity offsets tended (p = 0.0753) to be later in females than in males. Compared to their male counterparts, female mice had (E) fewer nightly bouts of activity, (F) more time spent active at night, and (G) more counts of nighttime activity. Compared to males, female mice had (H) more bouts of daytime activity, (I) more time spent active during the day, and (J) more counts of daily activity. No differences between AIE-exposed and control mice were noted when data were averaged over the entire 24-day exposure period. Data are mean ± SEM; *p < 0.05.

Fig. 4.

Changes in photocycle entrainment during ethanol availability in AIE-exposed mice. Pairwise analyses of circadian entrainment measures in AIE-exposed mice on days when ethanol was available (On EtOH) versus those when ethanol was not available (Off EtOH) revealed that (A) both males (n = 18) and females (n = 17) were active for longer (alpha) when ethanol was available, that (B) females became active earlier when ethanol was available, and that (C) activity offsets were delayed during ethanol availability in both sexes. Female mice had (D) more bouts during ethanol availability, while (E) both males and females spent less time active at night when ethanol was available, and (F) adolescent mice were less active at night when drinking ethanol. Compared to males, female mice had (G) more bouts of daytime activity, (H) more time spent active during the day, and (I) more counts of daily activity, whether ethanol was available or not. Data are mean ± SEM; *p < 0.05.

Fig. 5.

Sex differences in photocycle entrainment during Washout in adult mice. (A) Circadian rhythm amplitude remained greater in adult female mice (n = 32) compared to males (n = 36) regardless of whether or not they had prior AIE exposure. (B) Active-phase duration (alpha) remained longer in adult female mice compared to males, and (C) activity onsets continued to occur earlier in females than in males during adulthood, with neither measure affected by prior AIE exposure. (D) Activity offsets were later in adult females than in males and prior exposure to AIE delayed offsets even further in females only. Compared to their adult male counterparts, female mice continued to have (E) fewer nightly bouts of activity, (F) more time spent active at night, and (G) more counts of nighttime activity. Compared to males, female mice still showed (H) more bouts of daytime activity, (I) more time spent active during the day, and (J) more counts of daily activity during adulthood regardless of AIE exposure. No differences in nightly or daily activity were seen due to previous AIE exposure. Data are mean ± SEM; *p < 0.05.

Fig. 6.

Lasting insensitivity to ethanol-induced attenuation of photic phase-resetting in male, but not female, adult mice with prior AIE exposure. (A) Only ethanol-naïve (control) male mice (n = 5) showed attenuated photic phase-shifts when given an ethanol challenge dose (1.5 g/kg, i.p.), while AIE-exposed males (n = 6) had normal phase-shifts after ethanol challenge compared to saline-treated groups (n = 7-8/exposure). (B) Female mice showed robust phase-shifts to light regardless of prior AIE exposure or ethanol challenge. (C) Neither AIE nor ethanol challenge had phase-resetting effects on their own (or in combination) in the absence of a light pulse. Data are mean ± SEM; *p < 0.05.

Fig. 7.

Representative, double-plotted actograms of male mice in each exposure X treatment group. Actograms of two mice from each group are shown, with Water X Saline at the top left, AIE X Saline at the top right, Water X EtOH Challenge at the bottom left, and AIE X EtOH Challenge at the bottom right. Lightly shaded portions at the top of each actogram shows the 24-day AIE (or control) exposure period. Unshaded middle regions of the actograms show the 24-day washout period; mice received a challenge injection and light pulse 2.5 hours into the dark phase on the final day of this period. Darkly shaded portions at the bottom of each actogram show the time of constant darkness (DD) after the light pulse.

Fig. 8.

Representative, double-plotted actograms of female mice in each exposure X treatment group. Actograms of two mice from each group are shown, with Water X Saline at the top left, AIE X Saline at the top right, Water X EtOH Challenge at the bottom left, and AIE X EtOH Challenge at the bottom right. Lightly shaded portions at the top of each actogram shows the 24-day AIE (or control) exposure period. Unshaded middle regions of the actograms show the 24-day washout period; mice received a challenge injection and light pulse 2.5 hours into the dark phase on the final day of this period. Darkly shaded portions at the bottom of each actogram show the time of constant darkness (DD) after the light pulse.