ETHANOL WITHDRAWAL IS CHARACTERIZED BY CIRCADIAN ACTIVITY DISTURBANCES IN THE MOUSE

INTRODUCTION Ethanol withdrawal is associated with various sleep and circadian pathologies; it has disruptive effects on sleep architecture and circadian rhythmicity as well as broad physiological effects on hormonal secretion (e.g. melatonin and cortisol). Also, the severity of these withdrawal-induced disturbances is concentration-dependent. During abstinence, residual disruptions of sleep homeostasis and circadian timing that exacerbate fragmented sleep and wake episodes increase the risk of relapse. Our experimental goals are three-fold: 1) to assess a withdrawal-induced impairment of photic entrainment capacity 2) to quantify an ethanol-induced unconsolidation of circadian locomotor activity, and 3) to illustrate that these withdrawal-induced circadian disturbances are exacerbated at higher ethanol concentrations in C57BL/6J mice.

METHODSEthanol Treatment. Male mice that received 10% or 15% ethanol dissolved in their drinking water for 2 months (n=7, for each treatment) were introduced to water, while controls (n=7, for each treatment) were maintained on water. Consumption of fluid was measured to the nearest 0.25 mL daily at 1030.Photic Entrainment Capacity. The experiment was conducted under a 1 min light pulse (25 lux) skeleton photoperiod . General circadian locomotor activity was measured using a passive infrared motion detector interfaced with a computerized data acquisition system. Rhythm period, alpha, activity onset, and activity offset relative to the 1 min light pulse were analyzed.Circadian Locomotor Activity. An activity bout was defined as a period of locomotor activity separated by at least 10 min of locomotor quiescence. Activity bouts were quantified across the active and rest periods, and across the 24 hr circadian day. Durations of activity bouts were also plotted. Quantities and durations of activity bouts were averaged over the first 3 days of ethanol withdrawal.

RESULTSWithdrawal Unconsolidates Circadian Locomotor Activity. The mean quantities and durations of activity bouts across the active and rest periods and across the 24 hr circadian day did not differ between ethanol drinkers withdrawn from 10% and water controls (p>0.05). Ethanol drinkers withdrawn from 15% had marked unconsolidation of locomotor activity vs. water controls as indicated through more activity bouts across the rest period (11.81 +/- 0.86 vs. 9.29 +/-0.84, respectively; p<0.04) and across the 24 hr circadian day (23.62 +/- 0.81 vs. 19.62 +/- 1.13, respectively; p<0.01) that were subsequently shorter in duration (rest: 12.90 +/- 1.28 min vs. 22.62 +/- 3.85 min, respectively; p<0.04: 24 hr: 24.34 +/- 2.04 min vs. 35.13 +/- 3.25 min, respectively; p<0.02).Withdrawal Increases Sporadic Activity. Frequencies of activity bout durations (bin sizes = 15 min and 60 min) across the 24 hr circadian day revealed that ethanol drinkers withdrawn from 15% had more sporadic activity vs. water controls; ethanol drinkers withdrawn from 15% had more bouts 0-15 min in duration vs. water controls (14.67 +/- 1.17 vs. 10.48 +/- 1.17, respectively; p<0.03) and more bouts <60 min in duration vs. water controls (20.81 +/- 1.31 vs. 16.38 +/- 1.53, respectively; p<0.05).Severity of Circadian Disturbances Is Concentration-Dependent. Ethanol drinkers withdrawn from 15% had more activity bouts vs. ethanol drinkers withdrawn from 10% across the active period (11.81 +/- 0.54 vs. 6.14 +/- 0.60, respectively; p<0.01) and across the 24 hr circadian day (23.62 +/- 0.48 vs. 16.57 +/- 0.81, respectively; p<0.01).

A.J. Brager1, R.A. Prosser2, and J.D. Glass1. 1Dept Biological Sciences, Kent State University, Kent, OH, 2Dept Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, TN.

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Figure 1: Representative actograms from 10% ethanol withdrawal (top), 15% ethanol withdrawal (middle), and a water control (bottom). These analyses are included within a more extensive experiment assessing ethanol concentration-dependent circadian disturbances following chronic ethanol treatment, withdrawal, and reintroduction. Ethanol withdrawal commences at the green arrow. The solid yellow line annotates the presentation of the 1 min (25 lux) skeleton photoperiod.

Figure 2: As shown on the left, mice withdrawn from a 15% ethanol solution had significantly more activity bouts that were subsequently shorter in duration vs. water controls across both the 24 hr circadian day (black) and rest period (dark grey; p>0.05, in all cases). As shown on the right, mice withdrawn from a 15% ethanol solution had more activity bouts across the 24 hr circadian day (black) and across the active period (light grey) vs. mice withdrawn from a 10% ethanol solution (p<0.05, in both cases). Mean duration of an activity bout was independent of concentration (p>0.05).

Figure 3: Mice withdrawn from a 15% ethanol solution had significantly more activity bouts < 60 min in duration vs. water controls as illustrated in the frequencies of durations of activity bouts with 15 min bins (top) and 60 min bins (bottom).

CONCLUSIONS

1. Ethanol withdrawal unconsolidates circadian locomotor activity across the 24 hr circadian day and rest period by increasing the number and reducing the duration of activity bouts.

2. Ethanol withdrawal increases sporadic activity as reflected through greater frequencies of activity bouts relatively short in duration.

3. The severity of ethanol withdrawal is concentration-dependent; there are more marked disturbances of circadian locomotor activity following withdrawal from 15% vs. 10%.

Acknowledgements: NIH grant AA015948 to RAP and JDG