Physiology & Behavior 147 (2015) 342–347
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Photoperiodic modulation of voluntary ethanol intake in C57BL/6 mice A.M. Rosenwasser a,b,c,⁎, M.C. Fixaris a, W.D. McCulley III a b c
a
Department of Psychology, University of Maine, Orono, ME 04469, United States School of Biology and Ecology, University of Maine, Orono, ME 04469, United States Graduate School of Biomedical Science and Engineering, University of Maine, Orono, ME 04469, United States
H I G H L I G H T S • • • • •
Mice consumed more ethanol under short-day relative to long-day photoperiods. Similar effects were seen in standard light–dark cycles and skeleton photoperiods. These effects are mediated by circadian adaptation rather than by light exposure. Access to running wheels increased water intake but did not affect ethanol intake. Effects may be related to seasonal and geographic variations in human alcohol use.
a r t i c l e
i n f o
Article history: Received 21 May 2014 Received in revised form 13 May 2015 Accepted 15 May 2015 Available online 17 May 2015 Keywords: Ethanol Preference Running wheel Circadian Photoperiod Inbred mice
a b s t r a c t Seasonal and geographic variations in light exposure influence human mood and behavior, including alcohol consumption. Similarly, manipulation of the environmental lighting regimen modulates voluntary ethanol intake in experimental animals. Nevertheless, previous studies in rats and hamsters have been somewhat inconsistent, and little is known concerning such effects in mice. In the present study, we maintained male C57Bl/6 mice in running-wheel cages under either short- or long-photoperiod light–dark cycles (LD 6:18 vs. LD 18:6); subsequently, the same animals were maintained under short or long “skeleton photoperiods”, consisting of two daily 15-min light pulses signaling dusk and dawn (SP 6:18 vs. SP 18:6). Running wheels were locked mechanically for half the animals under each photoperiod. Analysis of running wheel patterns showed that mice displayed stable circadian adaptation to both standard LD cycles and skeleton photoperiods. Mice consumed more ethanol and less water, and thus showed higher ethanol preference, under LD 6:18 and SP 6:18 relative to the corresponding long-photoperiod regimens. While running-wheel access increased water intake, ethanol intake was unaffected by this manipulation. These effects are consistent with previous studies showing that short photoperiods or constant darkness increases ethanol intake in rodents. Further, the similarity of the effects of complete and skeleton photoperiods suggests that these effects are mediated by photoperiod-induced alterations in the circadian entrainment pattern, rather than by light exposure per se. © 2015 Published by Elsevier Inc.
1. Introduction Reciprocal interactions between the circadian system and alcohol (ethanol) intake have been detected at both physiological and molecular-genetic levels of analysis, and in both human populations and experimental animals [11,16,17,28,42,51]. The primary environmental factor influencing the circadian system is light, and indeed, seasonal and latitudinal variations in environmental photoperiod exert powerful effects on circadian physiology and behavior. Epidemiological studies indicate that alcohol use and abuse varies with both season [7, 12,32,35,40,53] and latitude [33,34,39], and exhibits co-morbidity and ⁎ Corresponding author at: Department of Psychology, University of Maine, Orono, ME 04469, United States. E-mail address: [email protected] (A.M. Rosenwasser).
http://dx.doi.org/10.1016/j.physbeh.2015.05.011 0031-9384/© 2015 Published by Elsevier Inc.
genetic overlap with Seasonal Affective Disorder (SAD) [2,35,47,48]. While seasonal patterns in human alcohol consumption may reflect the combined influence of both geophysical and sociocultural factors [49], these observations suggest a significant role for photoperiod in the modulation of human alcohol consumption. Previous animal experiments examining the effects of the lighting regimen on voluntary (2-bottle, free-choice) ethanol intake have yielded somewhat inconsistent results. Thus, while housing in constant darkness (DD) has been reported to increase ethanol intake in both hamsters and rats relative to standard light–dark (LD 12:12) conditions [4,21,22,41], Goodwin et al. [24] found reduced ethanol intake in rats under both DD and constant light (LL). Similarly, ethanol intake has been reported to be increased [20], decreased [8,43] or unchanged [44] under so-called “shift-lag” lighting schedules, in which light–dark cycles are repeatedly phase-shifted in order to simulate the effects of
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chronic shift-work or jet-lag. While all of these studies examined the effects of repeated phase-advances, which are known to be more disruptive to circadian synchronization than phase-delays, it should be noted that Gauvin et al. [20] used a somewhat different shift schedule than did the other studies, and also that the animals in Gauvin et al. [20] had limited daily access to ethanol, rather than continuous 24-hour access. Like photoperiodic effects on reproduction, immune status and other functions [23], the effects of environmental lighting on ethanol intake could be mediated in part by photoperiod-induced alterations in the pattern of pineal melatonin secretion. Thus, pinealectomy reduces ethanol intake in both rats and hamsters [4,41], and exogenous melatonin treatment increases ethanol intake in rats [6,10,21,50]. To date, possible effects of the lighting regimen on voluntary ethanol intake in mice are largely unexplored. In an earlier study, Millard and Dole [37], found that C57BL/6 (B6) inbred mice displayed reduced ethanol intake under LD 6:6 relative to standard LD 12:12 conditions, despite the fact that both lighting regimens provided the same total daily light exposure. Importantly, analysis of circadian drinking patterns indicated that mice “interpreted” the LD 6:6 schedule as though it was a long-photoperiod (LD 18:6) regimen, suggesting that the effects on ethanol intake were mediated by photoperiodic reorganization of the circadian system. In addition, these results are also noteworthy given that B6 mice, like many other inbred mouse strains, are known to be “melatonin deficient”, releasing little or no endogenous melatonin and expressing a degenerate nocturnal secretory pattern [9,13,26,30,54]. More recently, however, we failed to detect significant differences in ethanol intake between B6 mice maintained in LD 18:6 or LD 6:18, while “melatonin-proficient” C3H/He (C3) mice actually consumed more ethanol under the long photoperiod [45]. In another recent study, B6 mice consumed less ethanol under both LL and DD relative to standard LD 12:12 cycles, while DBA/2 (D2) mice showed reduced intake only in DD [44]. Finally, neither B6 nor D2 mice displayed alterations in ethanol intake under a shift-lag lighting regimen [44]. The purpose of the present study was to investigate further the effects of photoperiod on voluntary ethanol intake in B6 mice. As already mentioned, while Millard and Dole [37] reported photoperioddependent alterations in ethanol intake in this strain, we failed to replicate this effect in a recent study [45], albeit using different lighting schedules. Since the animals in our study were housed in runningwheel cages, and since access to running wheels has been shown to influence ethanol intake [5,15,27,36,38], in the present study we maintained half the animals with continuous access to running wheels and half with locked running wheels. In addition to testing for possible photoperiod by running wheel interactions on ethanol intake, this also allowed assessment of circadian adaptation to photoperiod in the wheel-access animals. Finally, in order to separate the effects of circadian adaptation from light exposure per se, we tested animals under both complete and “skeleton” photoperiods.
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2.2. Procedures After a two-week adaptation period during which tap water was the only drinking fluid, animals were offered concurrent free-choice access to both 10% (v/v) ethanol solution and tap water via separate drinking tubes throughout the remainder of the experiment, and ethanol consumption and water consumption were determined at weekly intervals. Ethanol drinking was quantified as intake in g/kg/day and as ethanol preference ratios (i.e., the volume of 10% ethanol divided by the total volume of fluid consumed); water intake was quantified as ml/day, since correcting water intake for body weight did not affect the statistical results. After six weeks of maintenance under “complete” photoperiods (i.e., standard LD cycles), the same animals were exposed subsequently to long- or short-day “skeleton photoperiods” (SP 18:6, SP 6:18), in which the daily light-to-dark and dark-to-light transitions were replaced by brief, 15-minute light pulses, and fluid intakes were recorded for an additional six weeks. Light pulses were timed such that the “dusk” pulse was terminated at the time of the previous light-to-dark transition, while the “dawn” pulse was initiated at the time of the previous dark-to-light transition. For wheel-access animals, wheel turns were monitored continuously via microswitches, stored in 6-minute bins using the ClockLab interface system (Coulbourn Instruments, Whitehall, PA), and subsequently rebinned into 30-minute epochs for inspection of daily activity patterns. Fluid intake data were analyzed by a three-factor mixed ANOVA, with photoperiod duration and running-wheel access as between-groups factors and photoperiod type (complete vs. skeleton) as a repeatedmeasures factor. 3. Results 3.1. Circadian activity patterns Circadian activity patterns are shown for one representative animal in each group in raster-style “actogram” format in Fig. 1 and as groupaveraged waveforms in Fig. 2. These data reveal dramatic differences in activity patterning as a function of photoperiod duration (long vs. short), but only relatively minor differences as a function of photoperiod type (regular vs. skeleton). Under complete photoperiods, mice displayed clear activity peaks at around the lights-off and lights-on transitions, and generally low, sporadic activity throughout most of the light phase of the LD cycle. Stable entrainment was maintained in all animals following transfer to skeleton photoperiods, and despite slight modifications, activity patterns clearly retained their characteristic “short” and “long” photoperiod waveforms, despite the fact that SP 6:18 and SP 18:6 are essentially identical lighting regimens. Indeed, the most consistent differences between activity patterns under complete and skeleton photoperiods are that (1) “pre-dusk” activity emerged under SP 18:6 that was generally absent under LD 18:6 and (2) the “dusk” light pulse resulted in suppression of locomotor activity under both SP 18:6 and SP 6:18 that lasted for about an hour after the pulse.
2. Methods 3.2. Ethanol intake 2.1. Animals and apparatus Male C57BL/6J (B6) mice (N = 30) were obtained from the Jackson Laboratory (Bar Harbor, ME) at about 6 weeks of age. Immediately upon arrival in the laboratory, mice were housed individually in runningwheel cages (Coulbourn Instruments, Whitehall, PA; model ACT 551; wheel diameter = 11.5 cm), but wheels were mechanically locked for half the animals. Running-wheel cages were placed three per shelf within sound-attenuating ventilated cabinets equipped with computerprogrammable lighting via 15-watt fluorescent tubes located above each shelf. Separate cabinets were maintained under either long-day (LD 18:6) or short-day (LD 6:18) photoperiods.
Ethanol intake showed significant effects of photoperiod duration (F(1,26) = 25.00, p b 0.001) and photoperiod type (F(1,26) = 65.78, p b 0.001), as well as a photoperiod duration by type interaction (F(1,26) = 9.29, p = 0.005) (Fig. 3). These main effects indicate that mice consumed more ethanol under short photoperiods than under long photoperiods and also consumed more ethanol under complete than under skeleton photoperiods, while the photoperiod durationby-type interaction was due to the fact that exposure to skeleton photoperiods reduced ethanol intake more substantially under short than under long photoperiods. Access to running wheels did not significantly affect ethanol intake.
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Fig. 1. Daily activity patterns for one representative mouse from each group (long-photoperiod, left; short-photoperiod, right), presented in double-plotted (48-hour span) actogram format. Animals were initially maintained under complete light–dark (LD) cycles, and subsequently transferred to a skeleton photoperiod (SP) comprising two daily 30-minute light pulses, one at the former light-to-dark transition, and one at the former dark-to-light transition. Yellow areas indicate lights-on while gray areas indicate lights-off periods. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. Group-averaged daily activity patterns for wheel-housed mice under each of the four experimental lighting regimens (LD 6:18, LD 18:6, SP 6:18, SP 18:6). Wheel-turns were collapsed into 30-min bins, averaged across the six weeks of data collection, and normalized according to each individual animal's overall daily mean. Data are expressed as mean +/− SEM for each time bin. Lighting regimens are represented by the black and white horizontal bars above each panel.
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Fig. 3. Mean (+SEM) daily ethanol intake (top panels), water intake (middle panels) and ethanol preference (bottom panels) in mice housed under short or long complete photoperiods (LD 6:18 and LD 18:6; left) or skeleton photoperiods (SP 6:18 and SP 18:6; right). All animals were housed in running-wheel cages, but the wheels were mechanically locked (“no wheel”) in approximately half the cages in each group; the remaining animals (“wheel”) had free access to a functional wheel.
3.3. Ethanol preference Ethanol preference was significantly higher under short relative to long photoperiods (F(1,26) = 14.24, p = 0.001; Fig. 3). There were no significant effects of either photoperiod type or wheel access on ethanol preference.
seen for ethanol intake, mice consumed more water under complete than under skeleton photoperiods, but opposite to the results for ethanol intake, water consumption was higher under long relative to short photoperiods. In addition, there was also a significant main effect of wheel access (F(1,26) = 13.49, p = 0.001), and a wheel-access by photoperiod duration interaction (F(1,26) = 6.64, p = 0.016), indicating that running-wheel access increased water intake, but mainly under long photoperiods.
3.4. Water intake 3.5. Daily activity levels Water intake also showed significant main effects of photoperiod duration (F(1,26) = 14.00, p = 0.001) and type (F(1,26) = 22.17, p b 0.001), but no photoperiod duration by type interaction (Fig. 3). As
Running-wheel activity was higher under complete than under skeleton photoperiods (F(1,12) = 10.36, p = 0.007) and higher under long
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relative to short photoperiods (F(1,12) = 6.60, p = 0.025), but there was no photoperiod duration by photoperiod type interaction (Fig. 4). 4. Discussion In the present study, B6 mice displayed lower levels of ethanol intake and ethanol preference under a summer-like, long-photoperiod light–dark cycle relative to a winter-like, short-photoperiod cycle. Importantly, differences in both ethanol intake and daily activity patterns persisted when animals were transferred from complete to skeleton photoperiods, suggesting that this effect is related to photoperioddependent differences in circadian entrainment patterns rather than to the overall amount of light exposure per se. While ethanol intake was somewhat lower under skeleton photoperiods relative to complete photoperiods, water intake displayed a similar effect, and there were no differences in ethanol preference between complete and skeleton photoperiods. Since the sequence of exposure to complete and skeleton photoperiods was not counterbalanced, the reduced fluid intake seen under skeleton photoperiods may have been due simply to the passage of time, and unrelated to the change in the lighting schedule. Nevertheless, since long and short photoperiods were studied in parallel groups, the effects of photoperiod duration were not confounded by sequence. Our results are consistent with those of Millard and Dole [37], who reported that B6 mice consumed less ethanol under LD 6:6 relative to LD 12:12. In that study, analysis of circadian drinking patterns revealed that mice housed under LD 6:6 were active during only one of the two daily 6-hour dark periods, and thus showed a circadian drinking pattern characteristic of entrainment to long photoperiods [37]. Together, these results suggest that mice may consume less ethanol under long photoperiods than under either short or standard 12-hour photoperiods. On the other hand, we did not include a standard LD 12:12 condition in the present experiment, and thus cannot determine whether short or long photoperiods (or both) alter ethanol intake relative to LD 12:12. In a previous study, we failed to detect a photoperiodic effect on ethanol intake in B6 mice, although there was a non-significant trend in the same direction as seen here [45]. In marked contrast, however, C3 mice actually consumed more ethanol under LD 18:6 than LD 6:18, opposite to the photoperiodic effect found in B6 mice in the present study [45]. While strain-dependent effects of photoperiod on ethanol intake have not been explored previously, numerous studies have reported lower levels of anxiety- and depression-like behavior under long photoperiods, or higher levels under short photoperiods, in diverse nocturnal and diurnal rodent species [52,56,57]. Nevertheless, affective responses to photoperiod appear to be far less consistent in mice [19,31], and indeed, C3 mice apparently display increased anxiety and depression under long photoperiods [3,31]. While these strain differences could
Fig. 4. Mean (+SEM) number of daily wheel-turns under short (6:18) and long (18:6) complete and skeleton photoperiods.
be related to strain differences in melatonin secretion [31], it is peculiar that the melatonin-deficient B6 mouse responds similarly to rats and hamsters, while the melatonin-proficient C3 strain shows opposite responses. Unfortunately, very little is known concerning possible photoperiod-related changes in melatonin secretion patterns, even in melatonin-proficient strains like C3, presumably because laboratory mice are generally viewed as a non-photoperiodic species. Nevertheless, von Gall et al. [55] found that C3 mice display virtually identical melatonin secretion patterns under both LD 12:12 and LD 8:16, suggesting that the photoperiodic control of melatonin secretion in melatoninproficient mice could differ from the more well-studied rats and hamsters. An alternative possibility that cannot presently be ruled out is that species and strain differences in photoperiodic effects on ethanol intake could be related to differences in overall intake levels. Thus, B6 mice, hamsters, and many strains of rats display generally higher ethanol preference than C3 mice, which could affect their responses to environmental lighting manipulations. Clearly, much more work will be required to understand strain differences in photoperiod effects on ethanol intake in inbred mice, including the possible role of melatonin in these effects. Since constant darkness mimics the effects of short-day LD cycles on reproductive physiology [18,23], it would not be surprising if this was also true for other, less well-studied photoperiodic responses. On the other hand, multiple photoperiodic responses may display differences in both environmental photoperiod-dependence and underlying neuroendocrine mechanisms [25]. Early studies with rats and hamsters reported higher ethanol intake under DD, relative to LD entrainment [4, 21,22,41]. In contrast, however, Goodwin et al. [24] found reduced levels of ethanol intake in both DD and LL, and we have recently replicated this finding in B6 mice (Rosenwasser et al., unpublished). While similar effects of DD and LL are not likely to reflect a classical photoperiodic mechanism, non-monotonic responses to photoperiod have been observed [29,46]. For example, Hong et al. [29] found that Turkish hamsters display gonadal regression when housed under photoperiods providing either less than 15 or more than 17 h each day, a response that was not correlated with photoperiodic effects on melatonin secretion. Nevertheless, it seems likely that the effects of DD and LL and the effects of LD photoperiod duration may be mediated by partially distinct mechanisms. A number of previous studies reported that access to running wheels can reduce ethanol consumption in several rodent species [5,15,27,36, 38]. In contrast, we did not detect a significant effect of wheel access on either ethanol intake or ethanol preference. On the other hand, wheel-housed animals consumed significantly more water than did mice housed with locked running wheels, leading to a non-significant reduction in ethanol preference. This result is consistent with previous findings that access to running wheels increases water intake when water is the only drinking fluid [1,14], and that alterations in water intake contribute substantially to the effects of running-wheel access on ethanol preference in B6 mice [15]. Finally, it should be noted that the present experiment compared mice housed with either locked or unlocked running wheels, while previous work indicates that even locked wheels can have significant effects on fluid intake when compared to standard caging [15]. In conclusion, male B6 mice consumed less ethanol and showed lower levels of ethanol preference when housed under a long photoperiod (LD 18:6) than under a short photoperiod (LD 6:18). This effect persisted when the animals were subsequently maintained under skeleton photoperiods, suggesting that it is mediated by photoperioddependent alterations in the circadian entrainment pattern, rather than by differential light exposure per se. While these effects are consistent with the findings of Millard and Dole [37], who also studied B6 mice, they are opposite in direction to our previous results for C3 mice, which showed lower ethanol intake under the short photoperiod [45]. Future studies comparing the effects of a wider range of photoperiod manipulations and additional genetic backgrounds may help to
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Photoperiodic modulation of voluntary ethanol intake in C57BL/6 mice A.M. Rosenwasser a,b,c,⁎, M.C. Fixaris a, W.D. McCulley III a b c
a
Department of Psychology, University of Maine, Orono, ME 04469, United States School of Biology and Ecology, University of Maine, Orono, ME 04469, United States Graduate School of Biomedical Science and Engineering, University of Maine, Orono, ME 04469, United States
H I G H L I G H T S • • • • •
Mice consumed more ethanol under short-day relative to long-day photoperiods. Similar effects were seen in standard light–dark cycles and skeleton photoperiods. These effects are mediated by circadian adaptation rather than by light exposure. Access to running wheels increased water intake but did not affect ethanol intake. Effects may be related to seasonal and geographic variations in human alcohol use.
a r t i c l e
i n f o
Article history: Received 21 May 2014 Received in revised form 13 May 2015 Accepted 15 May 2015 Available online 17 May 2015 Keywords: Ethanol Preference Running wheel Circadian Photoperiod Inbred mice
a b s t r a c t Seasonal and geographic variations in light exposure influence human mood and behavior, including alcohol consumption. Similarly, manipulation of the environmental lighting regimen modulates voluntary ethanol intake in experimental animals. Nevertheless, previous studies in rats and hamsters have been somewhat inconsistent, and little is known concerning such effects in mice. In the present study, we maintained male C57Bl/6 mice in running-wheel cages under either short- or long-photoperiod light–dark cycles (LD 6:18 vs. LD 18:6); subsequently, the same animals were maintained under short or long “skeleton photoperiods”, consisting of two daily 15-min light pulses signaling dusk and dawn (SP 6:18 vs. SP 18:6). Running wheels were locked mechanically for half the animals under each photoperiod. Analysis of running wheel patterns showed that mice displayed stable circadian adaptation to both standard LD cycles and skeleton photoperiods. Mice consumed more ethanol and less water, and thus showed higher ethanol preference, under LD 6:18 and SP 6:18 relative to the corresponding long-photoperiod regimens. While running-wheel access increased water intake, ethanol intake was unaffected by this manipulation. These effects are consistent with previous studies showing that short photoperiods or constant darkness increases ethanol intake in rodents. Further, the similarity of the effects of complete and skeleton photoperiods suggests that these effects are mediated by photoperiod-induced alterations in the circadian entrainment pattern, rather than by light exposure per se. © 2015 Published by Elsevier Inc.
1. Introduction Reciprocal interactions between the circadian system and alcohol (ethanol) intake have been detected at both physiological and molecular-genetic levels of analysis, and in both human populations and experimental animals [11,16,17,28,42,51]. The primary environmental factor influencing the circadian system is light, and indeed, seasonal and latitudinal variations in environmental photoperiod exert powerful effects on circadian physiology and behavior. Epidemiological studies indicate that alcohol use and abuse varies with both season [7, 12,32,35,40,53] and latitude [33,34,39], and exhibits co-morbidity and ⁎ Corresponding author at: Department of Psychology, University of Maine, Orono, ME 04469, United States. E-mail address: [email protected] (A.M. Rosenwasser).
http://dx.doi.org/10.1016/j.physbeh.2015.05.011 0031-9384/© 2015 Published by Elsevier Inc.
genetic overlap with Seasonal Affective Disorder (SAD) [2,35,47,48]. While seasonal patterns in human alcohol consumption may reflect the combined influence of both geophysical and sociocultural factors [49], these observations suggest a significant role for photoperiod in the modulation of human alcohol consumption. Previous animal experiments examining the effects of the lighting regimen on voluntary (2-bottle, free-choice) ethanol intake have yielded somewhat inconsistent results. Thus, while housing in constant darkness (DD) has been reported to increase ethanol intake in both hamsters and rats relative to standard light–dark (LD 12:12) conditions [4,21,22,41], Goodwin et al. [24] found reduced ethanol intake in rats under both DD and constant light (LL). Similarly, ethanol intake has been reported to be increased [20], decreased [8,43] or unchanged [44] under so-called “shift-lag” lighting schedules, in which light–dark cycles are repeatedly phase-shifted in order to simulate the effects of
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chronic shift-work or jet-lag. While all of these studies examined the effects of repeated phase-advances, which are known to be more disruptive to circadian synchronization than phase-delays, it should be noted that Gauvin et al. [20] used a somewhat different shift schedule than did the other studies, and also that the animals in Gauvin et al. [20] had limited daily access to ethanol, rather than continuous 24-hour access. Like photoperiodic effects on reproduction, immune status and other functions [23], the effects of environmental lighting on ethanol intake could be mediated in part by photoperiod-induced alterations in the pattern of pineal melatonin secretion. Thus, pinealectomy reduces ethanol intake in both rats and hamsters [4,41], and exogenous melatonin treatment increases ethanol intake in rats [6,10,21,50]. To date, possible effects of the lighting regimen on voluntary ethanol intake in mice are largely unexplored. In an earlier study, Millard and Dole [37], found that C57BL/6 (B6) inbred mice displayed reduced ethanol intake under LD 6:6 relative to standard LD 12:12 conditions, despite the fact that both lighting regimens provided the same total daily light exposure. Importantly, analysis of circadian drinking patterns indicated that mice “interpreted” the LD 6:6 schedule as though it was a long-photoperiod (LD 18:6) regimen, suggesting that the effects on ethanol intake were mediated by photoperiodic reorganization of the circadian system. In addition, these results are also noteworthy given that B6 mice, like many other inbred mouse strains, are known to be “melatonin deficient”, releasing little or no endogenous melatonin and expressing a degenerate nocturnal secretory pattern [9,13,26,30,54]. More recently, however, we failed to detect significant differences in ethanol intake between B6 mice maintained in LD 18:6 or LD 6:18, while “melatonin-proficient” C3H/He (C3) mice actually consumed more ethanol under the long photoperiod [45]. In another recent study, B6 mice consumed less ethanol under both LL and DD relative to standard LD 12:12 cycles, while DBA/2 (D2) mice showed reduced intake only in DD [44]. Finally, neither B6 nor D2 mice displayed alterations in ethanol intake under a shift-lag lighting regimen [44]. The purpose of the present study was to investigate further the effects of photoperiod on voluntary ethanol intake in B6 mice. As already mentioned, while Millard and Dole [37] reported photoperioddependent alterations in ethanol intake in this strain, we failed to replicate this effect in a recent study [45], albeit using different lighting schedules. Since the animals in our study were housed in runningwheel cages, and since access to running wheels has been shown to influence ethanol intake [5,15,27,36,38], in the present study we maintained half the animals with continuous access to running wheels and half with locked running wheels. In addition to testing for possible photoperiod by running wheel interactions on ethanol intake, this also allowed assessment of circadian adaptation to photoperiod in the wheel-access animals. Finally, in order to separate the effects of circadian adaptation from light exposure per se, we tested animals under both complete and “skeleton” photoperiods.
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2.2. Procedures After a two-week adaptation period during which tap water was the only drinking fluid, animals were offered concurrent free-choice access to both 10% (v/v) ethanol solution and tap water via separate drinking tubes throughout the remainder of the experiment, and ethanol consumption and water consumption were determined at weekly intervals. Ethanol drinking was quantified as intake in g/kg/day and as ethanol preference ratios (i.e., the volume of 10% ethanol divided by the total volume of fluid consumed); water intake was quantified as ml/day, since correcting water intake for body weight did not affect the statistical results. After six weeks of maintenance under “complete” photoperiods (i.e., standard LD cycles), the same animals were exposed subsequently to long- or short-day “skeleton photoperiods” (SP 18:6, SP 6:18), in which the daily light-to-dark and dark-to-light transitions were replaced by brief, 15-minute light pulses, and fluid intakes were recorded for an additional six weeks. Light pulses were timed such that the “dusk” pulse was terminated at the time of the previous light-to-dark transition, while the “dawn” pulse was initiated at the time of the previous dark-to-light transition. For wheel-access animals, wheel turns were monitored continuously via microswitches, stored in 6-minute bins using the ClockLab interface system (Coulbourn Instruments, Whitehall, PA), and subsequently rebinned into 30-minute epochs for inspection of daily activity patterns. Fluid intake data were analyzed by a three-factor mixed ANOVA, with photoperiod duration and running-wheel access as between-groups factors and photoperiod type (complete vs. skeleton) as a repeatedmeasures factor. 3. Results 3.1. Circadian activity patterns Circadian activity patterns are shown for one representative animal in each group in raster-style “actogram” format in Fig. 1 and as groupaveraged waveforms in Fig. 2. These data reveal dramatic differences in activity patterning as a function of photoperiod duration (long vs. short), but only relatively minor differences as a function of photoperiod type (regular vs. skeleton). Under complete photoperiods, mice displayed clear activity peaks at around the lights-off and lights-on transitions, and generally low, sporadic activity throughout most of the light phase of the LD cycle. Stable entrainment was maintained in all animals following transfer to skeleton photoperiods, and despite slight modifications, activity patterns clearly retained their characteristic “short” and “long” photoperiod waveforms, despite the fact that SP 6:18 and SP 18:6 are essentially identical lighting regimens. Indeed, the most consistent differences between activity patterns under complete and skeleton photoperiods are that (1) “pre-dusk” activity emerged under SP 18:6 that was generally absent under LD 18:6 and (2) the “dusk” light pulse resulted in suppression of locomotor activity under both SP 18:6 and SP 6:18 that lasted for about an hour after the pulse.
2. Methods 3.2. Ethanol intake 2.1. Animals and apparatus Male C57BL/6J (B6) mice (N = 30) were obtained from the Jackson Laboratory (Bar Harbor, ME) at about 6 weeks of age. Immediately upon arrival in the laboratory, mice were housed individually in runningwheel cages (Coulbourn Instruments, Whitehall, PA; model ACT 551; wheel diameter = 11.5 cm), but wheels were mechanically locked for half the animals. Running-wheel cages were placed three per shelf within sound-attenuating ventilated cabinets equipped with computerprogrammable lighting via 15-watt fluorescent tubes located above each shelf. Separate cabinets were maintained under either long-day (LD 18:6) or short-day (LD 6:18) photoperiods.
Ethanol intake showed significant effects of photoperiod duration (F(1,26) = 25.00, p b 0.001) and photoperiod type (F(1,26) = 65.78, p b 0.001), as well as a photoperiod duration by type interaction (F(1,26) = 9.29, p = 0.005) (Fig. 3). These main effects indicate that mice consumed more ethanol under short photoperiods than under long photoperiods and also consumed more ethanol under complete than under skeleton photoperiods, while the photoperiod durationby-type interaction was due to the fact that exposure to skeleton photoperiods reduced ethanol intake more substantially under short than under long photoperiods. Access to running wheels did not significantly affect ethanol intake.
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Fig. 1. Daily activity patterns for one representative mouse from each group (long-photoperiod, left; short-photoperiod, right), presented in double-plotted (48-hour span) actogram format. Animals were initially maintained under complete light–dark (LD) cycles, and subsequently transferred to a skeleton photoperiod (SP) comprising two daily 30-minute light pulses, one at the former light-to-dark transition, and one at the former dark-to-light transition. Yellow areas indicate lights-on while gray areas indicate lights-off periods. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. Mean (+SEM) daily ethanol intake (top panels), water intake (middle panels) and ethanol preference (bottom panels) in mice housed under short or long complete photoperiods (LD 6:18 and LD 18:6; left) or skeleton photoperiods (SP 6:18 and SP 18:6; right). All animals were housed in running-wheel cages, but the wheels were mechanically locked (“no wheel”) in approximately half the cages in each group; the remaining animals (“wheel”) had free access to a functional wheel.
3.3. Ethanol preference Ethanol preference was significantly higher under short relative to long photoperiods (F(1,26) = 14.24, p = 0.001; Fig. 3). There were no significant effects of either photoperiod type or wheel access on ethanol preference.
seen for ethanol intake, mice consumed more water under complete than under skeleton photoperiods, but opposite to the results for ethanol intake, water consumption was higher under long relative to short photoperiods. In addition, there was also a significant main effect of wheel access (F(1,26) = 13.49, p = 0.001), and a wheel-access by photoperiod duration interaction (F(1,26) = 6.64, p = 0.016), indicating that running-wheel access increased water intake, but mainly under long photoperiods.
3.4. Water intake 3.5. Daily activity levels Water intake also showed significant main effects of photoperiod duration (F(1,26) = 14.00, p = 0.001) and type (F(1,26) = 22.17, p b 0.001), but no photoperiod duration by type interaction (Fig. 3). As
Running-wheel activity was higher under complete than under skeleton photoperiods (F(1,12) = 10.36, p = 0.007) and higher under long
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relative to short photoperiods (F(1,12) = 6.60, p = 0.025), but there was no photoperiod duration by photoperiod type interaction (Fig. 4). 4. Discussion In the present study, B6 mice displayed lower levels of ethanol intake and ethanol preference under a summer-like, long-photoperiod light–dark cycle relative to a winter-like, short-photoperiod cycle. Importantly, differences in both ethanol intake and daily activity patterns persisted when animals were transferred from complete to skeleton photoperiods, suggesting that this effect is related to photoperioddependent differences in circadian entrainment patterns rather than to the overall amount of light exposure per se. While ethanol intake was somewhat lower under skeleton photoperiods relative to complete photoperiods, water intake displayed a similar effect, and there were no differences in ethanol preference between complete and skeleton photoperiods. Since the sequence of exposure to complete and skeleton photoperiods was not counterbalanced, the reduced fluid intake seen under skeleton photoperiods may have been due simply to the passage of time, and unrelated to the change in the lighting schedule. Nevertheless, since long and short photoperiods were studied in parallel groups, the effects of photoperiod duration were not confounded by sequence. Our results are consistent with those of Millard and Dole [37], who reported that B6 mice consumed less ethanol under LD 6:6 relative to LD 12:12. In that study, analysis of circadian drinking patterns revealed that mice housed under LD 6:6 were active during only one of the two daily 6-hour dark periods, and thus showed a circadian drinking pattern characteristic of entrainment to long photoperiods [37]. Together, these results suggest that mice may consume less ethanol under long photoperiods than under either short or standard 12-hour photoperiods. On the other hand, we did not include a standard LD 12:12 condition in the present experiment, and thus cannot determine whether short or long photoperiods (or both) alter ethanol intake relative to LD 12:12. In a previous study, we failed to detect a photoperiodic effect on ethanol intake in B6 mice, although there was a non-significant trend in the same direction as seen here [45]. In marked contrast, however, C3 mice actually consumed more ethanol under LD 18:6 than LD 6:18, opposite to the photoperiodic effect found in B6 mice in the present study [45]. While strain-dependent effects of photoperiod on ethanol intake have not been explored previously, numerous studies have reported lower levels of anxiety- and depression-like behavior under long photoperiods, or higher levels under short photoperiods, in diverse nocturnal and diurnal rodent species [52,56,57]. Nevertheless, affective responses to photoperiod appear to be far less consistent in mice [19,31], and indeed, C3 mice apparently display increased anxiety and depression under long photoperiods [3,31]. While these strain differences could
Fig. 4. Mean (+SEM) number of daily wheel-turns under short (6:18) and long (18:6) complete and skeleton photoperiods.
be related to strain differences in melatonin secretion [31], it is peculiar that the melatonin-deficient B6 mouse responds similarly to rats and hamsters, while the melatonin-proficient C3 strain shows opposite responses. Unfortunately, very little is known concerning possible photoperiod-related changes in melatonin secretion patterns, even in melatonin-proficient strains like C3, presumably because laboratory mice are generally viewed as a non-photoperiodic species. Nevertheless, von Gall et al. [55] found that C3 mice display virtually identical melatonin secretion patterns under both LD 12:12 and LD 8:16, suggesting that the photoperiodic control of melatonin secretion in melatoninproficient mice could differ from the more well-studied rats and hamsters. An alternative possibility that cannot presently be ruled out is that species and strain differences in photoperiodic effects on ethanol intake could be related to differences in overall intake levels. Thus, B6 mice, hamsters, and many strains of rats display generally higher ethanol preference than C3 mice, which could affect their responses to environmental lighting manipulations. Clearly, much more work will be required to understand strain differences in photoperiod effects on ethanol intake in inbred mice, including the possible role of melatonin in these effects. Since constant darkness mimics the effects of short-day LD cycles on reproductive physiology [18,23], it would not be surprising if this was also true for other, less well-studied photoperiodic responses. On the other hand, multiple photoperiodic responses may display differences in both environmental photoperiod-dependence and underlying neuroendocrine mechanisms [25]. Early studies with rats and hamsters reported higher ethanol intake under DD, relative to LD entrainment [4, 21,22,41]. In contrast, however, Goodwin et al. [24] found reduced levels of ethanol intake in both DD and LL, and we have recently replicated this finding in B6 mice (Rosenwasser et al., unpublished). While similar effects of DD and LL are not likely to reflect a classical photoperiodic mechanism, non-monotonic responses to photoperiod have been observed [29,46]. For example, Hong et al. [29] found that Turkish hamsters display gonadal regression when housed under photoperiods providing either less than 15 or more than 17 h each day, a response that was not correlated with photoperiodic effects on melatonin secretion. Nevertheless, it seems likely that the effects of DD and LL and the effects of LD photoperiod duration may be mediated by partially distinct mechanisms. A number of previous studies reported that access to running wheels can reduce ethanol consumption in several rodent species [5,15,27,36, 38]. In contrast, we did not detect a significant effect of wheel access on either ethanol intake or ethanol preference. On the other hand, wheel-housed animals consumed significantly more water than did mice housed with locked running wheels, leading to a non-significant reduction in ethanol preference. This result is consistent with previous findings that access to running wheels increases water intake when water is the only drinking fluid [1,14], and that alterations in water intake contribute substantially to the effects of running-wheel access on ethanol preference in B6 mice [15]. Finally, it should be noted that the present experiment compared mice housed with either locked or unlocked running wheels, while previous work indicates that even locked wheels can have significant effects on fluid intake when compared to standard caging [15]. In conclusion, male B6 mice consumed less ethanol and showed lower levels of ethanol preference when housed under a long photoperiod (LD 18:6) than under a short photoperiod (LD 6:18). This effect persisted when the animals were subsequently maintained under skeleton photoperiods, suggesting that it is mediated by photoperioddependent alterations in the circadian entrainment pattern, rather than by differential light exposure per se. While these effects are consistent with the findings of Millard and Dole [37], who also studied B6 mice, they are opposite in direction to our previous results for C3 mice, which showed lower ethanol intake under the short photoperiod [45]. Future studies comparing the effects of a wider range of photoperiod manipulations and additional genetic backgrounds may help to
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