1. Figure 1. Schematic of anatomical connections between reward centers (orange) and circadian timing centers (green). The ventral tegmental area (VTA) and pedunculopontine tegmentum (PPT) send cholinergic input to the intergeniculate nucleus (IGL), which regulates the timing of the suprachiasmatic (SCN) clock via release of neuropeptide Y and other transmitters. The SCN sends information back to the tegmentum and also to the nucleus accumbens (NA) via multisynaptic connections. The NA receives dopaminergic input from the tegmentum and send information to the prefrontal cortex (PFC). NEW CIRCADIAN-GENETIC PERSPECTIVES ON ALCOHOLISM: MPER2 CLOCK GENE DELETION POTENTIATES BEHAVIORAL DISTURBANCE AND ENHANCES DRINKING IN MICE UNDERGOING CHRONIC ETHANOL TREATMENT C.L. Ruby 1, A.J. Brager 1, R.A. Prosser 2, and J.D. Glass 1 1 Dept. of Biological Sciences, Kent State Univ., Kent, OH 2 Dept. of Biochemistry and Cellular and Molecular Biology, Univ. of Tennessee, Knoxville, TN BACKGROUND Central reward pathways implicated in alcoholism are closely and reciprocally tied to those regulating circadian timing (Fig.1). People who experience chronic circadian rhythm disruptions such as shift work and sleep disturbances have a higher risk of developing alcohol problems (Crum et al., 2004; Poole et al., 1992; Smart, 1979). Mice lacking the clock gene mPer2 show altered circadian rhythm regulation (shortened free-running period, lack of phase-delays to light, loss of rhythmicity in constant darkness) and higher alcohol consumption than wildtypes (Albrecht et al., 2001; Spanagel et al., 2005; Steinlechner et al., 2002). Photic phase-advances in hamsters and photic phase- delays in mice are attenuated by chronic EtOH drinking (Ruby et al., 2009a&b). Hypotheses: 1) EtOH consumption disrupts circadian activity patterns in mice. 2) Simulated jetlag increases EtOH consumption and/or preference in mPer2 knockout mice. 3) mPer2 knockout mice have longer reentrainment rate to a shifted photocycle than wildtypes; EtOH exacerbates this disturbance. METHODS Activity bout analysis.Adult, male mPer2 knockout mice (MPER2; n=6) and wildtype controls (WT; n=8) maintained under a 12L:12D photocycle were individually-caged and general circadian locomotor activity was monitored for 3 wk prior to experimentation using overhead infrared sensors linked to a computerized data acquisition system. Mice in the experimental group then received free-choice between 10% ethanol (EtOH) and water (n=4/genotype), while controls received two bottles of water. Water and EtOH consumption was measured daily. Activity measurements were averaged over a 3 day period after EtOH introduction. Activity duration was defined as the number of activity bouts (periods of activity separated by at least 18 min of quiescence) X (the average duration of one bout). Activity bout analysis. Adult, male mPer2 knockout mice (MPER2; n=6) and wildtype controls (WT; n=8) maintained under a 12L:12D photocycle were individually-caged and general circadian locomotor activity was monitored for 3 wk prior to experimentation using overhead infrared sensors linked to a computerized data acquisition system. Mice in the experimental group then received free-choice between 10% ethanol (EtOH) and water (n=4/genotype), while controls received two bottles of water. Water and EtOH consumption was measured daily. Activity measurements were averaged over a 3 day period after EtOH introduction. Activity duration was defined as the number of activity bouts (periods of activity separated by at least 18 min of quiescence) X (the average duration of one bout). Simulated jetlag. WT and MPER2 mice under a 12L:12D photocycle were maintained on free-choice 10% EtOH for 2 wks. Their general circadian locomotor rhythms were monitored as described. Following the EtOH acclimation period, the photocycle was delayed by 6 h. Mice remained under the new LD cycle for 4 wks. EtOH and water consumption was monitored throughout the duration of the experiment. Statistical analyses. Differences in activity duration, EtOH consumption, and EtOH preference were assessed by ANOVA and Newman-Keuls post hoc mean comparison test. The level of significance was set at p<0.05. CONCLUSIONS Experiment 1: All EtOH-drinking mice showed less nocturnal activity than their water-drinking counterparts. WT mice appear more active than MPER2 mice. MPER2 mice drank nearly twice as much as WTs, yet show less activity suppression. Thus, MPER2 mice may be less sensitive to the sedative effects of EtOH than WTs. Experiment 2: EtOH consumption and preference during simulated jetlag was unaffected in MPER2 mice, but significantly decreased in WTs. This supports the idea that WTs may be more sensitive to the sedative effects of EtOH, and suggests a potential “ceiling” in EtOH drinking among MPER2 mice. These results underscore the importance of proper genetic controls in revealing an experimental effect, particularly regarding alcohol self-administration. Experiment 3: All mice showed rapid reentrainment to the 6 h delayed photocycle. It is possible that MPER2 mice did so via phase-advances. EtOH drinking did not appear to disrupt reentrainment in either WT or MPER2 mice. A more detailed analysis of the actogram data will clarify the results of this experiment. Figure 2. EtOH effect on activityBoth WT (n=3) and MPER2 (n=3) given free-choice EtOH showed marked decreases in activity during their active phase relative to water-drinking controls of their respective genotypes (p=0.0014). WT water-drinking controls (n=2) showed higher baseline activity (815.5+47.5 min) versus the water-drinking MPER2 mice (n=3; p<0.05), showing 545.0+49.7 min duration. No difference in activity duration was seen between the WT and MPER2 mice given free-choice EtOH, showing 270.3+35.8 min and 314.7+75.8 min durations, respectively. However, MPER2 mice (n=4) drank more EtOH (17.0+1.6 g/kg/day) than WT mice (n=4; 10.4+1.7 g/kg/day; p=0.0131). Figure 2. EtOH effect on activity. Both WT (n=3) and MPER2 (n=3) given free-choice EtOH showed marked decreases in activity during their active phase relative to water-drinking controls of their respective genotypes (p=0.0014). WT water-drinking controls (n=2) showed higher baseline activity (815.5+47.5 min) versus the water-drinking MPER2 mice (n=3; p<0.05), showing 545.0+49.7 min duration. No difference in activity duration was seen between the WT and MPER2 mice given free-choice EtOH, showing 270.3+35.8 min and 314.7+75.8 min durations, respectively. However, MPER2 mice (n=4) drank more EtOH (17.0+1.6 g/kg/day) than WT mice (n=4; 10.4+1.7 g/kg/day; p=0.0131). Figure 3. Effect of 6 h delayed LD photocycle on free-choice EtOH consumption and preferenceThe 6 h delayed LD cycle affected subsequent free-choice EtOH drinking differentially in MPER2 vs. WT mice. EtOH consumption by MPER2 mice (n=4) remained high during the 2 wks after the photocycle was delayed (16.6+1.0 g/kg/day vs. 14.9+1.0 g/kg/day baseline). In contrast, WT mice (n=4) showed a sustained decrease in EtOH consumption in comparison to their baseline consumption (5.9+0.7 g/kg/day vs. 9.4+1.0 g/kg/day baseline, respectively; p<0.01). A similar pattern was seen for EtOH preference, with that of MPER2 mice remaining high after the photocycle was changed (71.1+3.6% baseline vs. 77.3+2.9% after delay), while the preference of WT mice for the 10% EtOH solution decreased from 53.7+4.6% baseline to 41.4+4.4% (p<0.05). Figure 3. Effect of 6 h delayed LD photocycle on free-choice EtOH consumption and preference. The 6 h delayed LD cycle affected subsequent free-choice EtOH drinking differentially in MPER2 vs. WT mice. EtOH consumption by MPER2 mice (n=4) remained high during the 2 wks after the photocycle was delayed (16.6+1.0 g/kg/day vs. 14.9+1.0 g/kg/day baseline). In contrast, WT mice (n=4) showed a sustained decrease in EtOH consumption in comparison to their baseline consumption (5.9+0.7 g/kg/day vs. 9.4+1.0 g/kg/day baseline, respectively; p<0.01). A similar pattern was seen for EtOH preference, with that of MPER2 mice remaining high after the photocycle was changed (71.1+3.6% baseline vs. 77.3+2.9% after delay), while the preference of WT mice for the 10% EtOH solution decreased from 53.7+4.6% baseline to 41.4+4.4% (p<0.05). REFERENCES Albrecht U, Zheng B, Larkin D, Sun ZS, Lee CC (2001). mPer1 and mPer2 are essential components for normal resetting of the circadian clock. J Biol Rhythms 16: 100-104. Crum RM, Storr CL, Chan YF, Ford DE (2004). Sleep disturbance and risk for alcohol-related problems. Am J Psychiatry 161: 1197-1203. Poole CJ, Evans GR, Spurgeon A, Bridges KW (1992). Effects of a change in shift work on health. Occup Med (Lond) 42: 193-199. Ruby CL, Brager AJ, DePaul MA, Prosser RA, Glass JD (2009a). Chronic ethanol attenuates circadian photic phase resetting and alters nocturnal activity patterns in the hamster. Am J Physiol Regul Integr Comp Physiol 297: R729-R737. Ruby CL, Prosser RA, Brager AJ, Glass JD (2009b). From bottle to brain: ethanol drinking patterns, pharmacokinetics, and long-term consumption on circadian photic phase-resetting in vivo. RSA Abst # 585 Smart RG (1979). Drinking problems among employed, unemployed and shiftworkers. J Occup Med 21:731-736. Spanagel R, Pendyala G, Abarca C, Zghoul T, Sanchis-Segura C, Magnone MC, Lascorz J, Depner M, Holzberg D, Soyka M, Schreiber S, Matsuda F, Lathrop M, Schumann G, Albrecht U (2005). The clock gene Per2 influences the glutamatergic system and modulates alcohol consumption. Nat Med 11: 35-42. Steinlechner S, Jacobmeier B, Scherbarth F, Dernbach H, Kruze F, Albrecht U (2002). Robust circadian rhythmicity of per1 and per2 mutant mice in constant light, and dynamics of per1 and per2 gene expression under long and short photoperiods. J Biol Rhythms 17: 202-209. Figure 4. Representative actograms. Left panel: WT control (top) and free-choice drinker (bottom); right panel: MPER2 control (top) and free-choice drinker (bottom). Blue arrows denote the onset of the 6 h delayed photocycle. Yellow arrows indicate introduction to 10% EtOH. The change in activity levels is visible for both WT and MPER2 mice given free-choice EtOH. Note the rapid reentrainment exhibited by all animals. Supported by NIH/NIAAA grant AA-150948 to RAP & JDG.