Abstract
Study Objectives Alcohol abuse is a significant public health problem, particularly in populations in which sleep deprivation is common as such as shift workers and aged individuals. Although research demonstrates the effect of alcohol on sleep, little is known about the role of sleep in alcohol sensitivity and toxicity. We investigated sleep as a factor modulating alcohol toxicity using Drosophila melanogaster, a model system ideal for studies of sleep, alcohol and aging.
Methods Following 24 hours of sleep deprivation using mechanical stimulation, Drosophila were exposed to binge-like alcohol exposures. Behavioral sensitivity, tolerance, and mortality were assessed. The effects of chronic sleep deprivation on alcohol toxicity were investigated using a short sleep mutant insomniac. Pharmacological induction of sleep for prior to alcohol exposure was accomplished using a GABAA-receptor agonist, 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol (THIP) to determine if increased sleep mitigated the effects of alcohol toxicity on middle-aged flies and flies with environmentally disrupted circadian clocks mimicking groups more vulnerable to the effects of alcohol.
Results Acute sleep deprivation increased alcohol-induced mortality following alcohol exposure. However, sleep deprivation had no effect on alcohol absorbance or clearance. Sleep deprivation also abolished functional tolerance measured 24 hours after the initial alcohol exposure, although tolerance at 4 h was observed. Pharmacologically increasing sleep prior to alcohol exposure decreased alcohol-induced mortality.
Conclusions Sleep quantity prior to alcohol exposure affects alcohol toxicity with decreased sleep increasing alcohol toxicity and dampened 24-hour alcohol tolerance. In contrast, increased sleep mitigated alcohol-induced mortality even in vulnerable groups such as aging flies and those with circadian dysfunction.
Statement of significance With the growing incidence of sleep deprivation and sleep disorders across adolescents and adults, it is important to understand the role of sleep in alcohol toxicity to develop future therapies for prevention and treatment of alcohol-induced pathologies. Using Drosophila melanogaster, an established model for both sleep and alcohol research, we found that acute and chronic sleep deprivation increased alcohol toxicity and eliminated long-term functional alcohol tolerance. In contrast, increased sleep prior to binge-like alcohol exposure mitigated alcohol-induced mortality even in vulnerable groups with higher susceptibility to alcohol toxicity.
Introduction
Alcohol abuse and its associated pathologies is a pervasive societal problem with serious negative impacts on individual health, family structure and the economy1–8. In the United States alcohol use disorders account for 79% of all diagnoses of substance use disorders9 and the economic impact of alcohol misuse is estimated at $249 billion annually7, 10. Alcohol abuse and alcohol pathologies appear higher in populations in which sleep deprivation is common including teenagers, young adults, shift workers and aged individuals11–18. Although considerable behavioral research has demonstrated the effects of alcohol on sleep homeostasis19–21, surprisingly little is known about the role of sleep in modulating alcohol sensitivity and toxicity at the physiological level. Sleep impairments are traditionally viewed as symptoms of alcohol use disorders; however, sleep disorders increase the incidence and risk of relapse in recovering alcoholics22–25. Sleep deprivation represents a significant rising public health problem in the United States and the world26–29. The pervasiveness of factors contributing to sleep disruptions including artificial light at night, the use of personal electronics and the increase in shiftwork and extended work days30, 31, combined with the increased risk of substance abuse associated with sleep deprivation, makes understanding how sleep deprivation affects alcohol-induced behaviors and toxicity imperative to identify and optimize therapies for future prevention and treatment of alcohol-induced pathologies.
The high degree of physiological, molecular and neurological conservation between the fruit fly Drosophila melanogaster and mammals makes Drosophila an ideal model for the investigation of sleep and alcohol interactions32–34. Sleep in Drosophila occurs in stages, varying in intensity during the night with observable sex and age dependent differences35–44. As in other species, circadian and homeostatic processes regulate sleep in flies with waking activity affecting sleep need38, 45. Moreover, alcohol physiology is remarkably conserved from flies to humans with parallels in behaviors as well as the underlying molecular mechanisms46, 47. When exposed to alcohol vapor, initially flies exhibit hyperactivity with increased locomotor activity, followed by a loss of motor control and eventually sedation48–51. Flies also develop functional alcohol tolerance dependent upon changes in neural plasticity50, 52–55 and addiction-like behaviors56–58 with a preference for alcohol following previous exposure59, 60.
We investigated the role of decreased and increased sleep in modulating alcohol toxicity. We found that acute sleep deprivation increased behavioral sensitivity and mortality following acute and repeated exposure to alcohol. These effects were independent of alcohol metabolism as no differences were observed in alcohol absorption and clearance between sleep deprived and non-sleep deprived flies. Sleep deprivation also inhibited the induction of long-term functional alcohol tolerance observed 24 h following the first alcohol exposure, although short-term tolerance measured 4 h following the first alcohol exposure was not affected. Chronic sleep restriction also increased alcohol-induced mortality. Encouragingly, we found that pharmacologically increasing sleep had the opposite effect of sleep deprivation, ameliorating alcohol mortality in middle-aged flies and flies with a disrupted circadian clock. This research highlights the critical role of sleep as a factor in alcohol toxicity.
Methods
Fly Maintenance
All flies were maintained on standard cornmeal-molasses food at 25°C and 60-70% relative humidity in 12:12 light-dark (LD) cycles. Insomniac (inc) mutants and the background w1118 line were generously provided by Dr. Nicholas Stavropoulos, New York University. Adult flies (∼30 per vial) were transferred approximately every 3 days to maintain stress-free cultures. All experiments were carried out in an environmentally controlled dark room at 25°C and 60-70% relative humidity under dim red light. Zeitgeber time (ZT) 0 represents lights on and ZT 12 corresponds with lights off. For experiments performed in constant light (LL) conditions, flies were transferred to LL on the day of eclosion.
Alcohol Exposure
Alcohol vapor exposure was performed as previously described51, 61, 62. Four tubes, each containing ∼30 flies, received a steady flow of ethanol vapor at a pre-determined percentage. Precise alcohol percentages were achieved by mixing air bubbled through deionized water and 95% ethanol (Koptec, Declon Labs, Inc.). Air flow rates were monitored throughout the experiment to ensure consistency of alcohol concentration. Water vapor controls were run simultaneously with 100% water vapor. Alcohol exposures were performed at ZT 9 to avoid circadian variation in responses unless otherwise stated for a specific protocol.
Sleep Deprivation
Consistent sleep deprivation was achieved using gentle mechanical stimulation on the GyroMini Nutating Mixer (Labnet International, Inc.). Vials containing ∼30 flies were placed at an angled position in a larger beaker with a raised block at a fixed position inside the beaker. Mixer rotation caused the vials to rotate within the beaker and then gently jump over the raised block, providing the flies with a startle movement every 2.5 seconds. The constant motion of the vials combined with the startle ensured consistent sleep deprivation with no apparent injuries or increased mortality observed after 24 hours of sleep deprivation. Sleep deprivation was performed in an incubator under 25°C, 60-70% relative humidity and 12:12 LD conditions. Non-sleep deprived controls were housed in the same incubator.
Sedation
Alcohol-induced sedation was performed as previously described48. Briefly, flies were exposed to 50% alcohol vapor for one hour with observations of behavioral state made every five minutes following a gentle tap of the vial. Flies were scored as sedated when immobile and lacking coordinated leg movements except for spontaneous twitching52. The mean time to 50% sedation was calculated using a linear extrapolation.
Tolerance
Tolerance was determined as previously described51. Flies received a pre-exposure of 50% alcohol for 30 minutes at ZT4.5 following a one-hour dark room acclimation period. Sedation was assessed during the pre-exposure. Flies were then returned to food vials to allow time for recovery and complete metabolism of the alcohol before testing. Testing occurred 4 hours later at ZT9 for short-term rapid tolerance or 24 hours later for long-term rapid tolerance with all experimental groups represented at each test. Tolerance was defined as an increase in average time to reach 50% sedation from the pre-exposure with the difference in sedation time between naïve and pre-exposed flies used for quantification. For tolerance experiments, sleep deprivation took place from ZT3.5 – ZT3.5.
Mortality
Following each alcohol exposure, flies were returned to food vials placed horizontally for approximately 2 h to allow recovery of postural control. Immediate mortality was assessed 24 hours following the last alcohol exposure and then daily for 6 days. Delayed mortality refers to the cumulative mortality within seven days of the final alcohol exposure.
Gaboxadol Treatment
Sleep was pharmacologically increased with the GABA-A agonist, 4,5,6,7-tetrahydroisoxazolo [5,4-c]pyridin-3-ol (THIP or Gaboxadol). 10 d old (in constant light) or 20 d old (in LD) flies were transferred to Gaboxadol-containing food (0.05 mg/mL) for either 48 or 24 h respectively prior to repeated alcohol exposure. A repetitive alcohol exposure protocol was used to assess alcohol-induced mortality as described previously61. Flies were exposed for 3 days to 1 h alcohol vapor at ZT 9 (exposures separated by 24 h).
Alcohol Absorbance
Following 24 h sleep deprivation, batches of 20 flies were exposed to 50% alcohol vapor for 30 minutes at ZT 9, after which they were frozen at 0, 0.5, 1, 2, or 4 h following alcohol exposure. Alcohol absorbance was measured using an enzymatic alcohol dehydrogenase assay (ADH-NAD kit; Sigma-Aldrich) per the manufacturer’s directions and as described previously49, 51. Briefly, flies were homogenized in 200 uL refrigerated Tris-HCl (pH 7.5) buffer. Homogenate was spun at 15,000 x g for 20 min at 4°C. 250 uL NAD-ADH reagent was added to a 5-µL aliquot of supernatant. Absorbance was measured at 340 nm within 20 min using a 96-well plate format and a Versa-Max plate reader (brand). Alcohol absorbance was normalized to total protein to eliminate the effect of body size variation between batches of flies.
Locomotor Activity Rhythms
Locomotor activity of adult male flies was monitored using Drosophila activity monitors (Trikinetics, Waltham, MA) as described previously63. Sleep activity of flies was recorded following entrainment either in LL or LD cycles at 25°C for 4 days, after which the flies were transferred without using anesthetic to Gaboxadol-containing media for 48 hours for further measurements. Sleep data were analyzed using the ClockLab Suite.
Statistics
Statistics were performed using GraphPad Prism Version 6.0. Experimental groups were compared using analysis of variance (ANOVA). Post-hoc analyses in multiple comparisons were performed using the Bonferroni correction.
Results
Sleep deprivation increases sensitivity to alcohol-induced sedation
Sleep deprivation appears to be a contributing factor to the increased use of alcohol as suggested in studies of shift workers and young adults64–69. However, relatively little is known about the effects of sleep deprivation on alcohol toxicity and alcohol pathologies. As a first step in exploring the modulatory role of sleep on alcohol sensitivity, we evaluated the effect of 24 hours sleep loss on alcohol sensitivity. Drosophila (mixed sex, 10 d old) were sleep deprived using mechanical sleep deprivation for 24 hours (ZT 8 – ZT 8) and then exposed to 50% alcohol vapor (1 h exposure; Figure 1A). As predicted given the sedative effects of alcohol, sleep deprived flies were sedated significantly faster than non-sleep deprived age-matched controls indicating that sleep loss increases behavioral sensitivity to alcohol (Figure 1B – 1C; t(14) = 13.46, p = 0.0002).
Potentially, the alteration in alcohol sensitivity between the groups may have arisen from the mechanical procedure used for sleep deprivation rather than sleep deprivation itself. To test whether sleep deprivation or mechanically induced stress was the underlying cause of the observed difference in alcohol sensitivity, we took advantage of the sex-specific difference in daytime sleep in flies38. Male flies exhibit a daytime “siesta sleep” period in which they sleep significantly more during the daytime as compared to mated females70. If the increased behavioral alcohol sensitivity observed in sleep deprived flies was attributed to sleep loss, sleep deprivation during the daytime should have a greater impact on males. However, if the increased sensitivity to alcohol were due to mechanically induced stress, the effects of daytime sleep deprivation would be similar in males and females. Mated 10-d old female and male flies were separately sleep deprived for the first eight hours of the subjective day (ZT 0 – ZT 8) and exposed to 50% alcohol vapor for 1 h at ZT 9 with sedation assayed at 5 min intervals. Sleep deprived males were significantly more sensitive to alcohol than non-sleep deprived males with shorter exposure times inducing sedation (Figure 1D – 1E; ANOVA: F3,28 = 29.24, p < 0.0001). In contrast, sleep deprivation appeared to have little effect on female flies with sleep deprived females showing similar alcohol responses to non-sleep deprived flies (Figure 1D – 1E; ANOVA: F3,28 = 29.24, p < 0.0001). These results suggest that sleep deprivation increases the behavioral sensitivity to alcohol and these effects are independent of any stress from mechanical perturbation.
Sleep deprivation increases acute and chronic alcohol toxicity
Excessive binge drinking escalates the incidence of alcohol-poisoning deaths71, 72. Therefore, it is important to understand the potential confounding effects of sleep loss on alcohol toxicity. To determine whether sleep deprivation alters alcohol toxicity, we tested the effect of a single exposure to 50% alcohol vapor on mortality. Flies (10 d, mixed sex) were sleep deprived for 24 h (ZT 8 – ZT 8) and exposed to 50% alcohol vapor for one h at ZT 9 (Figure 2A). Nearly 100% of the flies become sedated under this protocol. Mortality was assessed 24 h and 7 days following alcohol exposure. When exposed to alcohol vapor following sleep deprivation, flies showed significant mortality 24 h after alcohol exposure compared to non-sleep deprived flies exposed to alcohol vapor or flies that were sleep deprived and exposed to water vapor (Figure 2B, ANOVA: F3,28 = 22.50, p < 0.0001 and 2C; ANOVA: F3,28 = 14.01, p < 0.0001). However, there was no significant further increase in alcohol induced mortality when cumulative delayed mortality was measured 7 days following the exposure to alcohol suggesting that the effects of sleep deprivation on alcohol toxicity occurred within the first 24 hours. Non-sleep deprived and sleep deprived flies exposed to water vapor alone had negligible levels of mortality at either 24 h or 7 days suggesting that 24 h sleep deprivation itself does not result in mortal injury to the flies (Figure 2B and 2C). Exposure to alcohol vapor caused a noticeable but not significant rise in immediate and delayed mortality compared to water vapor controls (Figure 2B and C). These results suggest that sleep deprivation exacerbates the acute toxicity of alcohol with primary mortality observed within 24 hours of alcohol exposure (Figure 2B). We next investigated the effects sleep deprivation prior to a repeat binge alcohol exposure paradigm. As previously, flies were sleep deprived for 24 h (ZT 8 – ZT 8) and then exposed to 40% alcohol vapor for 1 h (ZT 9) on 3 consecutive days (Figure 2D). Perhaps not surprisingly, the first alcohol exposure after sleep deprivation induced a significant increase in mortality (Figure 2E, ANOVA: F3,76 = 15.42, p < 0.0001). Alcohol-induced mortality was not significantly higher following the 2nd and 3rd alcohol exposures (Figure 2F), potentially due to the opportunity for recovery sleep following the 1st exposure to alcohol. The degree of mortality observed 7 days following the last alcohol exposure was similar between the acute and repeated binge alcohol paradigms (Figure 2G, ANOVA: F3,76 = 19.91, p < 0.0001).
Sleep deprivation does not affect the rate of alcohol clearance
It is possible that the increases in alcohol sensitivity and mortality observed following sleep deprivation were due to increased alcohol absorption or a decline in the rate of alcohol clearance resulting in greater alcohol exposure and subsequent toxicity. To investigate this possibility, flies were sleep deprived as previously described and exposed to 50% alcohol vapor for 30 min at ZT 9 and alcohol absorbance was measured (Figure 3A). There were no significant differences in alcohol absorbance or clearance between sleep deprived flies and non-sleep deprived flies (Figure 3B). These results suggest that potential metabolic changes due to sleep deprivation do not account for the observed increased sensitivity to alcohol with the more likely possibilities including sleep deprivation induced changes in neuroadaptation at the molecular or cellular levels.
Chronic sleep deprivation induces increased alcohol-induced mortality
Chronic sleep deprivation with multiple short sleep nights may also be a predisposing factor for increased alcohol consumption and other recreational drug use18, 73. In the United States, approximately 70 million Americans suffer from chronic sleep loss with serious consequences for health and longevity as well as economic productivity29, 74, 75. To investigate the effects of chronic sleep restriction on alcohol neurobiology, we used a genetic approach rather than a mechanical system to induce sleep deprivation to avoid the possibility of stress arising from long-term mechanical stimulation. Numerous mutants with short sleep phenotypes have been identified in Drosophila. However, the circadian clock also regulates aspects of sleep and sleep timing, and many sleep mutants have circadian phenotypes. Given previous research demonstrating circadian modulation of alcohol sensitivity and increased alcohol-induced mortality with circadian disruption48, 61, we used the mutant insomniac that has normal circadian rhythms but exhibits a short sleep phenotype76 to investigate the effects of chronic sleep restriction on alcohol toxicity. Insomniac (inc) is a mutation in a putative adaptor protein for the Cullin-3 ubiquitin ligase complex76. We used two inc mutant lines, inc1 and inc2 (generous gifts of Nicholas Stavropoulos at NYU), to test the effects of chronic sleep restriction on alcohol sensitivity and alcohol-induced mortality. Both inc1and inc2 mutant lines have a 90% reduction in inc transcript mRNA levels with no detectable protein produced76. Confirming previously published results, we found that that inc1 and inc2 flies (Figure 4A – C) exhibit considerable reductions in total sleep time with inc1 flies sleeping a little over 300 minutes per day and inc2 flies sleeping approximately 600 min per day (Figure 4A, ANOVA: F2,67 = 81.13, p < 0.0001). These mutants exhibit significantly shortened sleep bouts (Figure 4B, ANOVA: F2,67 = 17.52, p < 0.0001) reflecting a decrease in sleep consolidation, although they do have a greater number of sleep bouts (Figure 4C, ANOVA: F2,67 = 8.64, p < 0.0001).
To investigate the effects of chronic sleep restriction on alcohol sensitivity, we exposed 10 d old inc1 and inc2 flies to 50% alcohol for 1 h at ZT 9 with sedation assessed at 5-minute intervals (Figure 4D). Surprisingly, inc1 and inc2 flies did not exhibit increased sensitivity to alcohol; indeed, these mutants were more resistant to the sedating effects of alcohol with significantly longer times to reach 50% sedation than the w1118 control flies (Figure 4D and 4E, ANOVA: F2,34 = 47.28, p < 0.0001 with post-hoc analysis identifying significant differences between w1118 vs. inc1 and w1118 vs inc2). These results suggest that either compensatory mechanisms exist to buffer against increased sensitivity to alcohol in these mutants or chronic sleep loss associated with the disruption of the Cullin-3 ubiquitin ligase complex does not increase alcohol sensitivity.
While the chronic sleep deficit associated with the disruption of the Cullin-3 ubiquitin ligase complex in the inc mutants did not increase alcohol sensitivity, we hypothesized that it would still increase alcohol toxicity as alcohol affects multiple signaling pathways both in the central nervous system and in peripheral tissues. To test this, we gave 10 d inc1 and inc2 flies a single exposure to 50% alcohol vapor for 1 h at ZT 9 and assessed mortality 24 h and 7 d following the alcohol exposure. Both inc1 and inc2 flies exhibited significantly higher mortality immediately (24 h) and 7 d following the exposure compared to w1118 background controls (Figure 5B, ANOVA: F2,29 = 16.46, p < 0.0001 and Figure 5C, ANOVA: F2,29 = 20.87, p < 0.0001). Given that the inc mutants are postulated to have defects in ubiquitination that may affect many target proteins and signaling pathways, it is possible that the observed mortality was due to other consequences of the mutation and not to the effects of chronic sleep restriction on alcohol toxicity. Presumably as inc flies age, the short sleep phenotype results in an accumulated sleep debt. If this is the case, we hypothesized that younger flies (3 d) would show lower alcohol-induced mortality at levels similar to that seen with acute sleep deprivation. To test this hypothesis, we exposed 3 d inc1 and inc2flies to alcohol and assessed mortality 24 h and 7 d following exposure. While there was higher mortality observed in 3 d inc1 and inc2 flies following alcohol exposure than 3 d w1118 control flies (Figure 5E, ANOVA: F2,34 = 15.03, p < 0.0001 and Figure 5F, ANOVA: F2,34 = 25.01, p < 0.0001), the 3 d inc1 and inc2 flies exhibited significantly lower mortality following a single exposure to alcohol than 10 d inc1 and inc2 flies (Figure 5E - F). No significant differences were observed in alcohol-induced mortality between 3 d and 10 d w1118 flies. These results suggest that the increase in alcohol-induced mortality in the 10 d inc mutants was due to the accrued sleep debt in the older flies rather than a non-sleep related consequence of the mutation. Together, these results suggest that separate mechanisms mediate the behavioral sensitivity to alcohol and the alcohol’s toxic effects whereby insomniac is necessary for the resistance to alcohol-induced mortality but not alcohol behavioral sensitivity.
Pharmacologically increasing sleep ameliorates alcohol-induced mortality in populations with sleep phenotypes
Previously we found that circadian arrhythmia and aging significantly increase alcohol-induced mortality61 mirroring human populations such as shift-workers and the elderly with sleep disturbances. If accrued sleep loss is the driving force for the observed alcohol-induced mortality in inc mutants, we hypothesized that increasing sleep in the inc1 and inc2 mutants should decrease mortality following exposure to alcohol. To pharmacologically increase sleep, inc1 and inc2 mutant flies were raised on standard Drosophila media for 9 d and then transferred to media containing the GABAAagonist THIP which has previously been shown to pharmacologically increase sleep in Drosophila 77–79. Following THIP exposure, inc1 and inc2 flies were given a single 1 h exposure to alcohol (Figure 6A). THIP exposure significantly reduced mortality 24 h and 7 d following alcohol exposure in both inc1 and inc2 mutant flies compared to non-THIP exposed inc1 and inc2 mutants (Figure 6B, ANOVA: F3,44 = 13.27, p < 0.0001 and Figure 6C, ANOVA: F2,44 = 12.83, p < 0.0001 respectively). However, THIP has dual effects as an analgesic and anxiolytic, and has been tested as a treatment for both alcohol use disorders as well as insomnia80. Potentially, as an agonist for GABAAreceptors, THIP may be affecting alcohol-receptor interactions to affect mortality rather than through its pharmacological induction of sleep. To determine whether acute THIP interactions decreased alcohol-induced mortality by altering alcohol-receptor interactions rather than through increased sleep prior to alcohol exposure, we fed 10 d inc1, inc2 and w1118 flies 0.1 mg/mL THIP for 1 h at ZT 7-8 and then exposed them to 50% alcohol vapor. There were no differences in mortality between THIP-fed inc1 and inc2 flies and non-THIP fed flies following alcohol exposure (Figure 6E, ANOVA: F5,46 = 50.88, p < 0.0001 and Figure 6F, ANOVA: F5,46 = 55.83, p < 0.0001). These results are consistent with the hypothesis that increased sleep prior to binge-like alcohol exposure buffers the toxic effects of alcohol.
Pharmacologically increasing sleep, independent of circadian rhythmicity, decreases alcohol-induced mortality
In Drosophila, the circadian clock can be rendered non-functional using environmental manipulation by housing the flies in constant light. Constant light (LL) is sufficient to dampen molecular oscillations and abolish circadian rhythms in locomotor activity, memory formation and the rhythm in alcohol-induced loss-of-righting reflex 51, 63, 81–85. We have previously shown environmental disruption of circadian function exacerbates alcohol sensitivity and mortality48, 61. Along with a disrupted circadian clock, we found that 10 d CS flies in LL have significantly lower total sleep, specifically less sleep during the subjective night compared to 10 d CS flies in LD (Figure 7A; t(236) = 4.46, p < 0.0001) and Figure 7B, ANOVA: F3,442 = 27.31, p < 0.0001), consistent with mis-timed sleep due to circadian dysfunction. Flies housed in LL had significantly higher number of sleep bouts in both the subjective day and night (Figure 7C, ANOVA: F3,442 = 98.99, p < 0.0001), although the sleep bout length was significantly shorter than flies housed in LD resulting in the decrease in total sleep (Figure 7D, ANOVA: F3,442 = 78.45, p < 0.0001). As a first step to separate the effects of sleep from the effects of circadian disruption on alcohol toxicity, we characterized the effects of THIP on sleep for flies housed in LL. As expected, flies housed on THIP containing food in constant light slept significantly more than flies on regular Drosophila food in LL (Figure 7E – H; Mean sleep time per day: t(233) = 29.43, p < 0.0001; Mean sleep time, day vs night, ANOVA: F3,463 = 437.1, p < 0.0001; number of sleep bouts, ANOVA: F3,463 = 358.1, p < 0.0001; Mean sleep bout length, ANOVA: F3,463 = 277.6, p < 0.0001). To separate the role of sleep from circadian regulation in mediating alcohol toxicity following a repeat binge-like alcohol exposure, we increased sleep in flies in LL as they remained under conditions of circadian disruption. 10 d LL flies were maintained on medium containing THIP for 48 hours prior to exposure to the first of three exposures of 40% alcohol vapor (Figure 7I). LL flies housed on THIP containing food prior to alcohol exposure had a significantly lower mortality rate than those exposed to alcohol vapor alone (Figure 7J, ANOVA: F3,36 = 132.6, p < 0.0001). However, LL flies given a short exposure to THIP followed by alcohol exposure exhibited no differences in mortality compared to LL flies exposed to alcohol alone (Figure 7K, ANOVA: F3,36 = 92.26, p < 0.0001). These results suggest that increased sleep is sufficient to ameliorate mortality following repeated binge-like alcohol exposure even under conditions of circadian disruption.
Increasing sleep buffers age-related susceptibility to alcohol-induced mortality
Aging is accompanied by the breakdown of circadian rhythmicity at the cellular, metabolic and physiological levels as well as disruptions in sleep architecture61, 86–90. In recent years, chronic and binge alcohol consumption in middle-aged and older adults has significantly increased91, 92 with more than 75% of the alcohol-induced poisoning deaths occurring in these age groups11, 14. More than 10% of older adults engage in binge drinking behavior91. Given that the aging population is expected to double by 205093, it is necessary to identify ways to treat or ameliorate alcohol toxicity in middle-aged and older individuals. In previous studies, we have shown that aging exacerbates alcohol sensitivity and mortality61. Middle-aged flies (20 d) exhibit shorter sleep times compared to younger flies (40; Figure 8A, (t(147) = 6.54, p < 0.0001) and Figure 8B, ANOVA: F3,294 = 38.12, p < 0.0001). While we found no differences in total sleep amount during the night between 10 and 20 d flies, 20 d flies had a significantly greater number of sleep bouts with shorter duration reflecting decreases in sleep consolidation (Figure 8C, ANOVA: F3,294 = 19.21, p < 0.0001 and Figure 8D; ANOVA: F3,294 = 38.12, p < 0.0001). We tested whether pharmacologically increasing sleep in middle-age was sufficient to overcome the age-related increase in mortality following repeated binge-like exposures to alcohol. We pharmacologically induced sleep in 20 d CS flies by housing them on 0.1 mg/mL THIP for 24 h after which they were given a 1 h alcohol exposure for 3 consecutive days (Figure 8I). 20 d THIP-fed flies slept significantly more than control 20 d flies (Figure 8E – 8H; Avg total sleep/day: t(89) = 13.05, p < 0.0001; Avg sleep time, day vs night, ANOVA: F3,178 = 91.72, p < 0.0001; number of sleep bouts, ANOVA: F3,178 = 60.24, p < 0.0001; Sleep bout length, ANOVA: F3,178 = 113.0, p < 0.0001). Middle-aged flies housed on THIP containing food prior to the repeated binge-like alcohol exposures had significantly lower rates of mortality than those exposed to alcohol alone (Figure 8J, ANOVA: F3,44 = 243.8, p < 0.0001). The decreased mortality observed in THIP- fed flies was not due to increased alcohol tolerance from THIP interactions as 20 d flies given THIP for 1 h at ZT 7 followed by alcohol exposure at ZT 9 show mortality rates similar to 20 d flies exposed to alcohol alone (Figure 8K, ANOVA: F3,30 = 23.09, p < 0.0001). These results suggest that increased sleep is sufficient to ameliorate mortality following repeated alcohol exposures in middle-aged flies that have both circadian and sleep disruption.
Sleep deprivation inhibits long-term but not short-term tolerance
Drosophila exhibit drug tolerance with repeat alcohol exposures in which the behavioral response to subsequent exposures of alcohol is lessened similar to that observed in rodent models and humans. At the behavioral level, functional tolerance results in a decreased sensitivity to alcohol during subsequent exposures with increased alcohol concentrations or longer alcohol exposures necessary to induce sedation33, 94, 95. In flies, rapid tolerance develops after a single alcohol exposure and can be observed during a second alcohol exposure 4 h or 24 h later50, 96. The development of functional alcohol tolerance is dependent upon changes in neural plasticity rather than changes in the metabolism or clearance of alcohol50, 53–55, 96, 97. Changes in neural plasticity associated with drug and alcohol tolerance share features in common with synaptic plasticity observed in learning and memory98–100. Potentially, sleep loss affects the development of alcohol tolerance as sleep deprivation disturbs memory formation as seen across invertebrate and vertebrate species101–103. To investigate the effect of sleep deprivation on tolerance formed after a single alcohol exposure, 10 d wild-type flies were sleep deprived for 24 hours (ZT 3.5 – ZT 3.5) and given a pre-exposure of 50% alcohol vapor for 30 minutes (ZT 4.5; Figure 9A). Pre-exposed sleep deprived flies and sleep deprived naïve flies were exposed to alcohol 4 h later at ZT 9 during which sedation was measured (Figure 9A). Similarly, non-sleep deprived flies were pre-exposed to alcohol with responses compared during a second alcohol exposure to naïve flies. Non-sleep deprived flies demonstrated robust 4 h alcohol tolerance with significant increases observed in the time necessary for 50% of the flies to reach sedation compared to naïve flies (Figure 9B, ANOVA: F3,20 = 49.62, p < 0.0001 and Figure 9C). Surprisingly, sleep deprived flies also demonstrated robust 4 h alcohol tolerance (Figure 9B and C) suggesting that sleep disruption does not affect the cellular signaling mechanisms necessary for the formation of 4 h tolerance.
To determine the effect of sleep deprivation on the formation of long-term alcohol tolerance, flies were sleep deprived for 24 hours (ZT 7.5 – ZT 7.5) and given a pre-exposure of 50% alcohol vapor for 30 minutes (ZT 8.5) and tested 24 hours later at ZT 9 (Figure 9D). Groups of non-sleep deprived flies were handled concurrently. When flies were tested 24 h after the initial alcohol exposure, sleep-deprived flies demonstrated significantly less tolerance to alcohol with the time to sedation similar to sleep-deprived naïve flies while non-sleep deprived flies demonstrated robust long-term tolerance with response times significantly different than naïve flies (Figure 9E, ANOVA: F3,26 = 125.7, p < 0.0001 and Figure 9F). Although our previous research found that tolerance was not modulated by the circadian clock, we verified the effect on long-term tolerance by exposing flies to alcohol at the same time that we observed the formation of 4 h tolerance in sleep-deprived flies (Figure 9G). Sleep-deprived flies pre-exposed to alcohol at ZT 8.5 and then subsequently exposed to alcohol at ZT 9 the following day also exhibited little or no alcohol tolerance, while non-sleep deprived flies exhibited significant long-term tolerance (Figure 9H, ANOVA: F3,20 = 0.92, p = 0.4488 and Figure 9I). Thus, acute sleep deprivation prior to alcohol exposure inhibits the expression of alcohol tolerance 24 h following the initial alcohol pre-exposure while no effect is observed on the development of short-term tolerance expressed 4 h after the initial exposure. These results are consistent with the hypothesis that different molecular mechanisms underlie the development of short-term and long-term rapid alcohol tolerance similar to the differences in formation of short and long-term memory.
Discussion
Research from our lab and others suggests a bidirectional relationship between clock dysfunction and the onset and severity of alcohol-related pathologies18, 48, 51, 104, 105. Social jet lag, large shifts in sleep timing between the weekday and the weekend is observed in numerous populations, including individuals on shift and rotating schedules106, 107 and is strongly correlated with increased alcohol use108, 109. Due to the long working hours, rotating schedules and work-associated stress, many individuals report using alcohol as a sleep aid64, 65, 110, 111 which can eventually lead to an increased number of binge drinking episodes and other detrimental effects associated with alcohol abuse64, 65, 67, 112. Previous studies from our lab found that the circadian clock modulates alcohol sensitivity and toxicity and that circadian dysfunction significantly increases the behavioral sensitivity to alcohol and mortality following acute and repeated alcohol exposures48, 51. In humans, differences in individual chronotype also appears to modulate alcohol use and its associated pathologies. Individuals expressing an “evening chronotype” report significantly increased alcohol use113–124125. Interestingly, individuals with an evening chronotype also have lower quality of sleep and increased greater daytime fatigue126, 127. However, it has been difficult to detangle the effects of circadian dysfunction from the effects of altered sleep on alcohol use.
Sleep disorders and sleep disturbances have become increasingly prevalent in modern society with longer working hours, irregular work schedules and the prevalence of electronics, affecting more than 35% of adults and 70% of teenagers27, 74, 128–131. Insufficient sleep exacerbates the risk of developing chronic diseases and health problems including cancer, diabetes, neurodegenerative and psychiatric disorders132–136. Consequently, we investigated the effects of sleep loss on the alcohol sensitivity and toxicity using Drosophila melanogaster to dissect the interactions between sleep deprivation and alcohol sensitivity and mortality.
We found that acute (24 h) sleep deprivation significantly increased sensitivity and mortality in young flies following a single binge-like exposure to alcohol. Most of the observed increase in mortality following alcohol exposure occurred within 24 h following alcohol exposure. These effects were independent of stress or injury as 48 h recovery sleep prior to alcohol minimized alcohol-induced mortality. The increases in sensitivity and mortality were also independent of changes in metabolic tolerance as there were no differences between sleep deprived and non-sleep deprived flies in alcohol absorbance or clearance. Thus, sleep deprivation changes both immediate alcohol sensitivity and acute alcohol toxicity after a single binge-like alcohol exposure. Our data underlines the phylogenetic conservation across species showing a correlation between sleep loss and alcohol behaviors. Studies from rodents and humans outline a correlation between sleep loss and increased severity of alcohol behavioral responses including increased alcohol intake, accelerated development of alcohol abuse, dependence and relapse following alcohol abstinence137–140. In mice, alcohol dose dependently increases hyperactive locomotor activity in open-field tests with acute sleep deprivation for 48 h abolishing these stimulatory effects141. Insufficient sleep (< 8 h per night) is correlated with increased number of drinking sessions in adolescents and young adults142–144. College-aged students are considered a vulnerable population for risk-taking behaviors and multiple studies show a strong correlation between poor sleep quality and excessive alcohol intake and the accompanying consequences for mental health and academic performance including increased rates of depression, anxiety, psychological stress and academic issues in these students66, 145, 146. Insufficient and poor quality sleep also appear to predict the onset of alcohol abuse and its adverse consequences68, 147–150. Sleep disturbances observed in children 3-5 years of age predicted the early onset of alcohol use at ages 12-14151. This is particularly harmful because recovering alcoholics who used alcohol as a sleep aid are three times more likely to relapse in 12 months22, 23, 152. Altogether, these studies emphasize disturbed sleep as a potent risk factor for the initiation of alcohol use, escalation of problems associated with alcohol abuse and hindrance of recovery from alcohol-use disorders. With the genetic tools and mutants available, Drosophila provides a suitable model system to test the relationship between chronic sleep disturbances and alcohol induced pathologies. Using flies with mutations in the insomniac gene (inc) that provide a model mirroring chronic sleep restriction, we found that inc mutants have significantly increased mortality following alcohol exposure than background controls. Moreover, we found that alcohol exposure is more lethal in 10 d old inc-mutant flies compared to 3 d old flies, although wild type flies in either age groups show little alcohol-induced mortality. Inc mutant flies were surprisingly less sensitive to the sedative effects of alcohol compared to their background controls supporting previous research that the different physiological consequences of alcohol can be regulated separately. Although, the mechanism through which sleep buffers alcohol toxicity is unknown, it is possible that the changes in oxidative stress in the inc mutant flies may contribute to the change in alcohol toxicity. The inc gene seems necessary for mediating the oxidative stress response as reducing inc both globally and neuronally significantly increases mortality following a single injection to paraquat, a common inducer of oxidative stress76, 153. Support for this hypothesis is found in previous research demonstrating that pharmacologically increasing sleep in inc-mutant flies using gaboxadol significantly decreased the sensitivity to paraquat-induced oxidative stress153. Sleep loss has also been shown to increase reactive oxygen species in the gut154 raising the possibility that peripheral mechanisms also contribute to increased alcohol toxicity. As changes in sleep potentially impact multiple physiological processes in the central nervous system as well as in peripheral organs, the precise mechanism(s) through which sleep buffers alcohol toxicity will undoubtedly be the focus of future studies.
Pharmacologically increasing sleep alone in circadianly disrupted and middle-aged wild-type flies was sufficient to significantly reduce alcohol-induced mortality. Gaboxadol increased total sleep duration as well as significantly increasing sleep bout length suggesting a greater consolidation of sleep. Both increased total sleep and increased sleep consolidation suggest that improved sleep quality could aid in mitigating alcohol-induced pathologies. Although there have been few studies examining the relationship between sleep health and alcohol toxicity, sleep loss or decreased sleep consolidation has been shown to reduce reproductive output, accelerate aging and increase the accumulation of reactive oxygen species and death in flies154, 155. In humans, increasing sleep in adolescents is correlated with decreased risk of emotional and cognitive disruption as well as lowered risk of obesity156. Also, increasing sleep by 30 minutes for 3 days over the weekend in healthy industrial workers and individuals susceptible to obesity significantly increased insulin sensitivity and had a restorative effect of sleep on metabolic homeostasis157, 158. Finally, increasing sleep in older adults significantly improves performance on visual tasks and stabilizes memory recall159. Although more specific research needs to be done assessing the direct effects of increased sleep on alcohol toxicity in vulnerable groups, these data suggest a role for sleep as a buffer to protect against the toxic effects of alcohol in populations vulnerable to chronic sleep loss as aged adults and shift workers.
The development of acute tolerance to alcohol is a distinct and critical behavioral metric used to gauge the propensity for alcohol dependence and abuse160, that can be separated from alcohol sensitivity and alcohol toxicity. Similar to mammals, an acute exposure to a high concentration of alcohol induces functional tolerance in Drosophila at the behavioral50, 96, 161 and the molecular levels97, 162–165. Functional alcohol tolerance is dependent on changes in neuronal strength and connectivity or synaptic plasticity97, 162, 163. Consistent with previous findings, we observed tolerance 4 hours and 24 hours following a short pre-exposure to alcohol vapor50, 51. We found that sleep deprivation abolished the development of tolerance at 24 hours but had no effect on tolerance at 4 hours. Potentially, acute sleep deprivation selectively impairs the cellular and molecular processes necessary for encoding long-term rapid tolerance to alcohol without severe disruption of those mechanisms necessary for the development of 4 hour tolerance. In fact, previous studies demonstrate altered expression of rapid tolerance in flies with mutations in genes necessary for learning and memory46, 96, 166. For example, the gene dunce (dnc) encodes a phosophodiesterase required for cAMP degradation and is necessary for behavioral and synaptic plasticity167, 168. Originally identified as a learning mutant169, 170, dnc-mutant flies exhibit significant sleep deficits171 and are incapable of forming rapid tolerance172, 173. Time-dependent differences in the effects of sleep deprivation can also be seen for memory with acute sleep deprivation affecting the consolidation of long-term but not short-term hippocampal dependent memory in mice174, 175. Together with support from existing research, the results from our studies suggest that sleep deprivation selectively impacts processes underlying synaptic plasticity to affect the development of long-term rapid tolerance. In conclusion, the results from our study start to dissociate the role of sleep in modulating alcohol toxicity from the regulation of alcohol neurobiology by the circadian clock. These results lay the groundwork for future studies and treatments considering sleep quality and sleep duration as an important component of alcohol use disorder and alcohol-induced pathologies.
Disclosure Statement
Financial Disclosure: None
Non-financial Disclosure: None
An early version of this manuscript (the authors’ original version) prior to peer review may be found in bioRxiv.
Acknowledgements
This work was supported by the National Institutes of Health, National Institute on Alcohol Abuse and Alcoholism grant R21AA021233.
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