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Chronic swim stress alters sensitivity to acute behavioral effects of ethanol in mice
Physiology & Behavior 91 (2007) 77 – 86
Chronic swim stress alters sensitivity to acute behavioral effects of ethanol in mice Janel M. Boyce-Rustay a,⁎, Heather A. Cameron b , Andrew Holmes a a
Section on Behavioral Science and Genetics, Laboratory for Integrative Neuroscience, National Institute on Alcohol Abuse and Alcoholism, Rockville, MD, United States b Unit on Neuroplasticity, Mood and Anxiety Disorders Program, National Institute of Mental Health, Bethesda, MD, United States Received 18 August 2006; received in revised form 9 January 2007; accepted 31 January 2007
Abstract Epidemiological data support a strong link between stress, stress-related disorders and risk for alcoholism. However, precisely how stress might impact sensitivity to the intoxicating effects of ethanol or the willingness to voluntary consume ethanol remains unclear. The present study assessed the effects of daily exposure to forced swim stress on subsequent sensitivity to the sedative/hypnotic, hypothermic, ataxic (measured using accelerating rotarod), and anxiolytic-like (measured using elevated plus-maze) effects of ethanol, and ethanol consumption and preference in a two-bottle choice paradigm, in male C57BL/6J mice. Stress effects on the sedative/hypnotic effects of the barbiturate pentobarbital were also tested. Results showed that chronic (fourteen days) but not acute (one or three days) swim stress significantly potentiated the sedative/hypnotic and hypothermic effects of 4 g/kg, but not 3 g/kg, ethanol. The sedative/hypnotic effects of pentobarbital were attenuated by chronic swim stress. Irrespective of chronicity, swim stress did not alter the ataxic or anxiolytic-like effects of ethanol, or alter ethanol self-administration either during or after stress. These data provide further evidence that stress alters the intoxicating effects of high doses of ethanol in a behaviorally selective manner. Published by Elsevier Inc. Keywords: Ethanol; Stress; Drinking; Anxiety; Sedation; Mouse; Hypothermia; Ataxia; Forced swim; Depression; Alcohol; Pentobarbital
1. Introduction There is considerable comorbidity between mood disorders and alcoholism [1,2]. Individuals with a history of stress and mood disorders such as anxiety and depression have approximately three times the risk for developing an alcohol-related disorder, and alcoholics with a co-morbid mood disorder tend to drink more heavily and have a poorer prognosis [3–6]. These epidemiological data have lent support to the notion that mood abnormalities represent a major risk factor for alcoholism, possibly because individuals abuse alcohol (ethanol) for its ‘anti-stress’ or ‘anti-tension’ properties [2,7,8]. The association between stress, mood disturbances and alcoholism appears to be complex. For example, while a history of adverse life events positively correlates with increased rates of alcoholism, a ⁎ Corresponding author. R4N5, AP9A/L018, Abbott Laboratories, 100 Abbott Park Rd, Abbott Park, IL 60064-6126. Tel.: +1 847 937 2559; fax: +1 847 938 1656. E-mail address: [email protected] (J.M. Boyce-Rustay). 0031-9384/$ - see front matter. Published by Elsevier Inc. doi:10.1016/j.physbeh.2007.01.024
proportion of individuals actually exhibit higher than normal rates of abstinence following stress [9–16]. These data suggest that individual differences, possibly at the levels of modifying psychosocial, biological and genetic factors, determine the relationship between stress and alcoholism. In this context, a number of studies have shown that individuals with a family history of alcoholism (family history positive, FHP) are less sensitive to certain behavioral effects of ethanol than family history negatives (FHN), apparently due to increased acute functional tolerance to the drug's effects [17,18]. Moreover, some studies show that FHP's also exhibit greater hypothalamic–pituitary–adrenal (HPA)-axis activation than FHN's following stress or ethanol challenge [19–25]. In addition, FHP's show more pronounced neuroendocrine and autonomic responses to ethanol itself than FHN's [26–28]. While these findings suggest that decreased sensitivity to ethanol and increased risk for alcoholism is associated with abnormal stress responsivity, how stress might impact sensitivity to ethanol intoxication has not been fully elucidated. Previous studies have shown that non-alcoholics exposed to laboratory
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stressors report increased feelings of sedation in response to ethanol challenge [29,30]. In rodents, while stress reinstates ethanol-seeking behavior after periods of abstinence [31,32], the effects of various types of stress on voluntary self-administration have been inconsistent [33–44]. Moreover, to-date there have been relatively few studies examining the effects of stress on sensitivity to the acute intoxicating effects of ethanol in rodents, with existing studies again report conflicting results [45–52]. The aim of the present study was to further examine the effects of stress on sensitivity to ethanol-related behaviors including self-administration in mice. Forced swim stress was employed as a stressor. Swim stress was used because it is a simple and readily replicable procedure and variants of this test are commonly used to assay depression-related behaviors in rats and mice [53]. Previous studies have also shown that swim stress can increase ethanol self-administration in mice [54–56]. In the present study, C57BL/6J mice were subjected to either acute or chronic swim stress and subsequently tested for ethanol-induced sedation/hypnosis, hypothermia, ataxia in the accelerating rotarod, and anxiety-like behavior in the elevated plus-maze. The effects of stress on ethanol consumption were measured using a standard two-bottle free choice paradigm. 2. Animals, materials and methods 2.1. Ethics All experimental procedures were approved by the National Institute on Alcohol Abuse and Alcoholism Animal Care and Use Committee, and followed the National Institute of Health guidelines outlined in ‘Using Animals in Intramural Research.’ 2.2. Animals Subjects were male C57BL/6J mice obtained from The Jackson Laboratory (Bar Harbor, ME) and aged 10–15 weeks
at the time of testing. Mice were housed in groups of 3–5 (except for the voluntary ethanol consumption experiment) in a temperature- and humidity-controlled vivarium under a 12 h light/dark cycle (lights on 0600 h). Separate cohorts of experimentally-naïve mice were used for each behavioral (and neuroendocrine) assay. The number of animals used in each experiment is shown in the corresponding table and figures legends. Ethanol doses were chosen to elicit an appropriate behavioral response based on previous studies [57]. With the exception of the voluntary ethanol consumption experiment, mice were given a 1 h acclimation period in the test room prior to testing, and apparatuses were cleaned with 70% v/v ethanol and dried between subjects. 2.3. Forced swim stress Mice were stressed by being placed into a transparent Plexiglas cylinder (20 cm diameter) filled halfway with water for 10 min, as previously described [58]. Water temperature was 24 ± 1 °C; in line with that typically used for studies employing the forced swim test as an assay for depressionrelated behavior and which likely produces a mild hypothermia [53]. A schematic representation of the procedure used to assess swim stress effects on acute sensitivity to ethanol is depicted in Fig. 1a, and on ethanol consumption in Fig. 7a. Unless otherwise indicated, mice were stressed daily for 0, 1, 3, or 14 days and then tested for ethanol-related behaviors 24 h later. 2.4. Behavioral and neuroendocrine responses to forced swim stress To test for changes in depression-related behavior with repeated swim stress exposures, mice were observed for the presence/absence of immobility (cessation of limb movements except minor involuntary movements of the hind limbs) every
Fig. 1. Procedure for assessing effects of swim stress on ethanol-related behaviors, and behavioral and neuroendocrine responses to repeated swim stress. (a) Effects of swim stress on ethanol-related behaviors were measured 24 h after 0, 1, 3 or 14 daily exposures. (b) Repeated daily exposure to swim stress produced a progressive increase in percent time immobile, a measure of ‘depression-related’ behavior (n=16). (c) Plasma corticosterone levels were elevated following either 1 or 14 days of stress exposure and significantly greater following 1 than 14 exposures (n =4–7/stress condition). ⁎⁎P b 0.01 vs. 0; ##P b 0.01 vs. 1. Data in this figure as well as in Figs. 2 –7 are Means ±SEM.
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5 s during the last 8 min of each 10 min test session, as previously described [59]. To measure hypothalamic–pituitary–adrenal (HPA)-axis activation following acute or repeated swim stress, mice were subjected to 1 or 14 days of stress and corticosterone levels were determined by radioimmunoassay (RIA). After the final stressor, mice were returned to the home cage for 30 min and then sacrificed via rapid cervical dislocation and decapitation to collect trunk blood. Non-stressed controls were sacrificed within 30 s of removal from the home cage. To minimize disturbance of non-stressed controls, all mice were individually housed 24 h prior sacrifice. Blood samples were centrifuged at 13,000 rpm for 30 s. Serum was extracted and assayed for total corticosterone (bound and free) using the Coat-a-Count RIA TKRC1 kit (limit of detection: 5.7 ng/ml; Diagnostic Products Corp, Los Angeles). 2.5. Effects of stress on ethanol- and pentobarbital-induced sedation/hypnosis Ethanol-induced sedation/hypnosis was assessed as previously described [57]. Immediately after injection with ethanol, mice were placed in the supine position in Plexiglas ‘V’-shaped chambers. The time from ethanol injection to recovery of the righting reflex (turning onto all 4 paws twice in 30 s after initial self-righting) was defined as sleep time. One cohort of mice was exposed to 0, 1 or 3 days of stress and 24 h later tested for sleep time response following intraperitoneal (i.p.) injection with 3 or 4 g/kg ethanol. A separate cohort was tested in the same manner following 0 or 14 days of stress. To test for possible changes in rates of ethanol clearance following stress, mice were tested for sleep time responses to 3 or 4 g/kg ethanol following 0 or 14 days of stress as above and, on recovery of the righting reflex, were sacrificed to collect trunk blood. Blood ethanol concentrations were measured using the Analox AM1 alcohol analyzer (Analox Instruments USA Inc, Lunenburg, MA). To test whether stress-induced changes in ethanol-induced sleep time occurred in response to another sedative/hypnotic, mice were exposed to 0 or 14 days of stress, injected i.p. with 40 or 50 mg/kg pentobarbital (Sigma, St. Louis, MO) and tested for sleep time as above. Doses of pentobarbital were based on those previously shown to induce sleep time in C57BL/6J mice [60,61]. 2.6. Effects of stress on ethanol-induced hypothermia Ethanol-induced hypothermia was assessed as previously described [57]. Core body temperature was measured by inserting a Thermalert TH-5 thermometer (Physitemp, Clifton, NJ) 2 cm into the rectum to obtain a stable reading for 10 s. Temperature was measured immediately prior to and 30, 60, 90, and 120 min after i.p. injection with 3 or 4 g/kg ethanol. Mice were placed into individual holding cages following the ethanol injection for the remainder of the experiment. Temperature change from pre-ethanol baseline was taken as the dependent measure. Ambient room temperature was 23 °C. One cohort of mice was exposed to 0, 1 or 3 days of stress and tested for
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hypothermic responses 24 h later following i.p. injection with 3 or 4 g/kg. A separate cohort was tested in the same manner following 0 or 14 days of stress. 2.7. Effects of stress on ethanol-induced ataxia on the accelerating rotarod Ethanol-induced ataxia on the accelerating rotarod was assessed as previously described [57]. The apparatus was a Med Associates rotarod (St. Albans, VT) typically used for testing rats (model # ENV-577). The 7 cm-diameter dowel was covered with 320 Grit sandpaper to provide a uniform surface that prevented mice gripping the rubberized dowel. Mice were placed onto the dowel with the rotarod rotating at 4 rpm, and the rotarod was then accelerated at a constant rate of 8 rpm/min from 4 rpm up to 40 rpm. The latency to fall to the floor 10.5 cm below was automatically recorded by photocell beams, with a maximum cutoff latency of 300 s. Mice were first given 10 consecutive training trials each separated by a 30 s inter-trial-interval. Stress groups were matched for average performance on the ninth and tenth training trials and exposed to 0, 1 or 3 days or, in a separate cohort, 0 or 14 days of stress. Twenty-four h after the final stressor, mice were given 3 baseline trials, injected i.p. with 0, 1.75, or 2.25 g/kg ethanol and returned to the home cage. Thirty min later, mice were given 3 test trials. Average performance on the second and third test trials was taken as post-ethanol performance. 2.8. Ethanol-induced anxiolytic-like behavior in the elevated plus-maze Ethanol-induced anxiolytic-like behavior was tested using the elevated plus-maze test as previously described [57]. The apparatus consisted of 2 open arms (30 × 5 cm; 90 lx) and 2 closed arms (30 × 5 × 15 cm; 20 lx) extending from a 5 × 5 cm central area and elevated 20 cm from the ground (San Diego Instruments, San Diego, CA). The walls were made from black ABS plastic and the floor from white ABS plastic. A 0.5 cm raised lip around the perimeter of the open arms prevented mice from falling off the maze. Testing was conducted under 65 dB white noise to minimize noise disturbance. This experiment focused upon the effects of 14 days of stress on the basis of data showing stress effects on other ethanol-related behaviors were most robust with this chronic regimen. Twenty-four h after 0 or 14 days of stress, mice were injected i.p. with 0, 1.0, or 1.5 g/kg ethanol and immediately placed in the center facing an open arm and allowed to explore the apparatus for 5 min. Time spent in the open arms, and entries into the open and closed arms were measured by the Ethovision videotracking system (Noldus Information Technology Inc., Leesburg, VA). 2.9. Effects of stress on voluntary ethanol consumption Voluntary ethanol consumption was measured using a 2-bottle choice paradigm as previously described [57]. Mice were individually-housed in ‘Space Saver’ cages (Model 1145T, Tecniplast, Buguggiate, Italy) with lids fitted for 2 fluid bottles
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(Model 1145T482SUDB-Polysulfone cage-top to accommodate 2 water bottles, top flow design; Tecniplast, Buguggiate, Italy). Two bottles, 1 containing ethanol and the other containing tap water, were available. Every 2 days the ethanol and water were changed and the left/right position of the bottles switched to control for any side bias. Food was available ad libitum. After 1 week of acclimation to the housing conditions, mice received increasing concentrations of ethanol: 3% (4 days), 5% (4 days), 7% (4 days), 9% (6 days), and 11% (10 days). Mice were then matched for average pre-stress 11% ethanol consumption and exposed to 0, 1 or 14 days of stress. Eleven percent ethanol was offered during the stress period and for 10 days after the 14-day-stress group received the final stressor. Ethanol and water consumption was measured every 24 h correcting for evaporation and spillage (measured in an empty chamber) and body weight was measured every 48 h throughout the study. These data were used to calculate ethanol consumption and preference for the ethanol-containing solution over water (ethanol consumption/total fluid consumption).
tests where appropriate. The effect of trial number on rotarod latency to fall, and the effect of ethanol concentration on voluntary ethanol consumption and preference, was analyzed using repeated measures ANOVA. Statistical significance was set at P b.05. 3. Results 3.1. Repeated forced swim stress activates the HPA-axis and increases depression-related behavior As shown in Fig. 1b, there was a significant, progressive increase in immobility with repeated exposure to forced swim stress [F(15,195) = 13.15, P b.01]. As shown in Fig. 1c, swim stress produced a significant increase in plasma corticosterone as compared to non-stressed controls [F(2,13) = 162.70, P b.01], with significantly higher corticosterone levels following either 1 or 14 exposures to swim stress than no-stress baseline, and a significantly greater level following 1 than 14 exposures.
2.10. Drugs For ethanol injections, ethanol (200 prf) was prepared in 0.9% w/v saline to produce 20% v/v solutions (0.9% w/v saline served as the vehicle control). Pentobarbital (Sigma, St. Louis, MO) was prepared in 0.9% w/v saline and injected at a volume of 10 ml/kg. 2.11. Statistical analysis The effects of stress and ethanol dose were analyzed using analysis of variance (ANOVA) and Newman–Keuls post-hoc
3.2. Stress increases sensitivity to ethanol-induced sedation/ hypnosis As shown in Fig. 2a, mice exposed to 3, but not 1, days of swim stress exhibited significantly longer sleep time responses to 4, but not 3, g/kg ethanol as compared to non-stressed controls [stress × dose interaction, F(2,68)= 4.56, P b.05]. In a separate cohort of mice, exposure to 14 days of swim stress led to significantly longer sleep time responses to both 3 and 4 g/kg ethanol, as compared to non-stressed controls [stress × dose
Fig. 2. Swim stress increased sensitivity to the sedative/hypnotic effects of ethanol. (a) Exposure to 3 but not 1 day of stress significantly increased sleep time responses to 4 but not 3 g/kg ethanol as compared to non-stressed controls (n = 11–14/stress condition/dose). (b) Exposure to 14 days of stress significantly increased sleep time responses to 3 and 4 g/kg ethanol relative to non-stressed controls (n = 9–11/stress/dose). (c) In a replicate cohort, exposure to 14 days of stress increased sleep time responses to 4 but not 3 g/kg ethanol relative to non-stressed controls, and (d) blood ethanol concentrations were significantly lower at sleep time recovery in stressed than non-stressed mice (n = 6–9/stress condition/dose). ⁎P b 0.05 vs. 0 d stress/same dose.
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Fig. 3. Chronic swim stress decreased sensitivity to the sedative/hypnotic effects of pentobarbital. Exposure to 14 days of stress (black bars) significantly decreased sleep time responses to pentobarbital as compared to non-stressed controls (white bars), regardless of dose (n = 11–14/stress condition/dose).
interaction, F(1,36) = 6.13, P b.05] (Fig. 2b). In a third cohort of mice, exposure to 14 days of stress showed significantly longer sleep time responses to 4, but not 3, g/kg ethanol as compared to non-stressed controls [stress × dose interaction, F(1,25) = 7.86, P b.05] (Fig. 2c). In this third cohort, blood ethanol concentrations were significantly lower at recovery in the stressed mice in comparison to the non-stressed controls and lower in mice treated with 3 g/kg than 4 g/kg ethanol regardless of stress [main effect of stress, F(1,24) = 6.29, P b.05; main effect of dose, F(1,24)= 9.68, P b.01] (Fig. 2d).
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dependently decreased latency to fall, with no difference between stressed and non-stressed groups [main effect of dose, F(2,89) = 17.70, P b.01]. In a separate cohort, there was again significant motor learning over 10 training trials prior to stress [main effect of trial number, F(9,504) = 23.90, P b.01] (Fig. 5c). Fourteen days of stress did not alter pre-ethanol performance (0 d stress = 95.3 ±5.4, 14 d stress = 100.7 ± 7.0). As shown in Fig. 5d, ethanol dose-dependently decreased latency to fall, with no difference between stressed and non-stressed mice [main effect of dose, F(2,52) = 18.50, P b.01]. 3.6. Stress did not affect ethanol-induced anxiolytic-like behavior As shown in Fig. 6a, mice exposed to 14 days of stress showed no differences in baseline open arm time, as compared
3.3. Stress decreases sensitivity to pentobarbital-induced sedation/hypnosis As shown in Fig. 3, mice exposed to 14 days of swim stress showed significantly shorter sleep time responses to pentobarbital as compared to non-stressed controls and sleep time response was significantly altered by pentobarbital dose [main effect of stress, F(1,40) = 5.60, P b.05; main effect of dose, F(1,40) = 55.40, P b.01]. 3.4. Stress increases sensitivity to ethanol-induced hypothermia Mice exposed to 1 or 3 days of stress did not show altered ethanol-induced hypothermia as compared to non-stressed controls; 4 g/kg produced a greater hypothermic effect than 3 g/kg regardless of stress [main effect of dose, F(2,86) = 38.43, P b.01] (Fig. 4a). In a separate cohort, mice exposed to 14 days of stress showed significantly greater hypothermic responses to 4, but not to 3, g/kg ethanol than non-stressed controls, as evidenced by lower core body temperature at 90 and 120 min post-ethanol [stress × dose × time interaction, F(3,102) = 4.96, P b.01] (Fig. 4b). 3.5. Stress did not affect ethanol-induced ataxia Mice showed motor learning over 10 rotarod training trials [main effect of trial: F(9,855) = 93.62, P b.01] (Fig. 5a). Neither 1 nor 3 days of stress altered pre-ethanol rotarod performance (0 d stress = 77.6 ± 4.3, 1 d stress = 79.7 ± 6.5, 3 d stress = 75.8 ± 7.0 s latency to fall). As shown in Fig. 5b, ethanol dose-
Fig. 4. Chronic swim stress increased sensitivity to the hypothermic effects of ethanol. (a) Exposure to neither 1 nor 3 days of stress altered hypothermic responses to 3 or 4 g/kg ethanol (n = 13–18/stress condition/dose). (b) Exposure to 14 days of stress significantly increased hypothermic responses to 4 but not 3 g/kg ethanol relative to non-stressed controls (n = 9–11/stress condition/dose). ⁎P b 0.05 vs. 0 d stress/same time point.
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Fig. 5. Stress did not alter the ataxic effects of ethanol on the accelerating rotarod. Motor learning prior to (a) 0, 1 or 3 days of stress, or (c) 0 or 14 days of stress. (b) Exposure to 1 nor 3 days of stress did not alter ataxic responses 1.75 or 2.25 g/kg ethanol (n = 10–13/stress condition/dose). (d) Exposure to 14 days of stress did not alter ataxic responses to 1.75 or 2.25 g/kg ethanol (n = 9–10/stress condition/dose).
Fig. 6. Stress did not alter the anxiolytic-like effects of ethanol in the elevated plus-maze. (a) Exposure to 14 days of stress did not alter percent open time at baseline or in response to treatment with 1.0 or 1.5 g/kg ethanol, as compared to non-stressed controls. (b) Neither stress nor ethanol treatment altered closed entries (n = 9–11/stress condition/dose).
Fig. 7. Stress does not affect ethanol self-administration in the 2-bottle free choice test (a) Schematic representation of the experimental procedure. Stress did not alter either (b) ethanol consumption or (c) ethanol preference, either during or in the 10 days following the end of stress, as compared to non-stressed controls.
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to non-stressed controls, and ethanol increased open arm time [main effect of dose, F(2,53) = 3.93, P b.05]. Neither stress nor ethanol dose significantly altered closed arm entries. 3.7. Stress did not affect voluntary ethanol consumption Prior to stress, increasing ethanol concentrations influenced rates of ethanol consumption (3% ethanol = 0.59 ± 0.08, 5% = 2.98 ± 0.18, 7% = 8.45 ± 0.38, 9% = 5.86 ± 0.32, 11% = 6.39 ± 0.36 g/kg ethanol consumed/day) [F(4,100) = 200.28, P b.01] and preference (3% ethanol = 44.3 ± 0.4, 5% = 71.8 ± 0.4, 7% = 70.1 ± 0.2, 9% = 71.4 ± 0.3, 11% = 67.0 ± 0.3 preference for the ethanol containing fluid over water) [F(4,100) = 23.82, P b.01]. There was no difference between stressed and non-stressed in ethanol consumption (Fig. 7b) or ethanol preference (Fig. 7c) either during the 14-day stress period or in the 10 days following the cessation of stress. Non-stressed and 3day stressed mice showed an increase in body weight over the 14day stress period, whereas the 14-day stress mice did not [stress × day interaction, F(2,23) = 15.17, P b.01] (data not shown). 4. Discussion Repeated exposure to forced swim stress over fourteen days produced a modest, but significant increase in passive immobility; a profile suggestive of an increase in depressionlike behavior [53]. In addition, mice exhibited stress-induced elevation of plasma corticosterone following exposure to fourteen days of swim stress indicating that the stressor maintained its potency with this chronic stress regimen. These data are consistent with previous data showing that C57BL/6 mice do not fully habituate to repeated exposure to another form of swim stress [62]. The main novel finding of the present study was that C57BL/6J mice exposed to chronic swim stress exhibited prolonged sleep time responses to ethanol; an effect replicated in three independent cohorts of mice. Stress effects on sensitivity to ethanol's sedative/hypnotic effects were largely limited to a relatively prolonged stress regimen. Thus, in contrast to the effects of fourteen days of swim stress, changes in sleep time responses to ethanol were not consistently seen with more acute (i.e., one or three days) stress regimens. A similar pattern of stress-related effects was observed for changes in hypothermic responses to ethanol; i.e. hypothermic responses were potentiated by exposure to chronic but not acute swim stress. In addition, increases in both the sedative/hypnotic and hypothermic effects of ethanol following chronic stress were both specific to a relatively high dose of ethanol (4 but not 3 g/kg ethanol). By contrast, neither the ataxia-inducing effects of (1.75–2.25 g/kg) ethanol in the accelerating rotarod, nor the anxiolytic-like effects of (1.0–1.5 g/kg) ethanol in the elevated plus-maze were altered by chronic swim stress. Taken together these demonstrate that stress altered sensitivity to ethanol's effects in manner that was specific to certain behavioral responses and to a high dose of ethanol. Stress-induced changes in sedation/hypnosis and hyperthermia were measured twenty-four hours after the cessation of
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stress, indicating that the effect was produced by relatively longlasting alteration in ethanol's mechanistic effects rather than by an acute alteration in ethanol absorption caused by stressinduced increases in corticosterone [63]. It should be noted, however, that plasma corticosterone levels following 14 days of swim stress exposure were relatively lower than following 1 day of stress, raising the possibility that stress-induced alterations in the HPA-axis may have contributed to altered ethanol sensitivity following the more chronic stress regimen. Present data also showed that longer sleeping stressed mice had modestly, but significantly, lower blood ethanol concentrations at awakening than non-stressed controls. This suggests that longer sleep time responses were not an artifact of slower ethanol clearance following stress. The fact that stress effects were specific to a higher dose of ethanol would also argue against a general change in ethanol pharmacokinetics. Nonetheless, the fact that positive effects of stress were seen only for behaviors measured relatively long after ethanol injection raises the possibility that subtle shifts in ethanol clearance may have contributed to these effects. Detailed studies of ethanol absorption and clearance following exposure to swim stress would help to clarify this issue. As noted in the Introduction, previous studies have found varying effects of stress on measures of acute sensitivity to ethanol in rodents. Stressors including exposure to bright light, tail-shock and rectal-probing increased sensitivity to ethanolinduced hypothermia and, in some but not all studies, ataxia in rats, while repeated restraint stress increased sensitization to the locomotor stimulant effects of ethanol in mice [45–50]. Mice exposed to footshock stress or social-isolation stress displayed decreased sedative/hypnotic and hypothermic responses to ethanol and rats exposed to swim stress showed decreased ataxic responses to ethanol [46,47,51,52]. The reason for this variability is not clear but might relate to differences in stressor type, duration of stress exposure or the strain of rat or mouse tested, as well as the methods used to measure ethanol's effects. Because none of these previous studies have used the same swim stress procedure as employed in the current study it is difficult to draw clear comparisons between this and previous studies. Nonetheless, our data show that our stress procedure produces a replicable effect on certain measures of acute sensitivity to ethanol's acute intoxicating effects, and provide a starting point for further investigations of the neurocircuitry involved and correlating changes in other ethanol-related behaviors. In this context, neither acute nor chronic swim stress altered voluntary ethanol consumption or preference in the two-bottle choice paradigm. This finding is not without precedent in a literature indicating that stress does not have robust and reliable effects on ethanol drinking in rats or mice. However, of particular relevance to current data previous studies have demonstrated that two or three days of forced swim stress can produce significant increases in ethanol (and water) consumption in mice [54–56]. The reason for the discrepancy with current data is not clear but may stem from methodological differences including swim duration (e.g., 5 min previously vs. 10 min in our study), tank-size (notspecified vs. 20 cm-diameter) and water temperature (21 vs. 24 °C). Another potentially critical factor is genetic background. Those
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studies observing swim stress-induced increases in ethanol consumption typically employed mice on a hybrid genetic background [54–56], while we used C57BL/6J. A comparison of swim stress effects on ethanol drinking across different mouse strains could help to determine whether C57BL/6J is atypically insensitive to the effects this stressor on ethanol self-administration. Another avenue for future studies is delineating the neural and molecular basis of stress-induced changes in the sedative/ hypnotic and hypothermic effects of ethanol. There are multiple candidate mechanisms that could be implicated. However, given evidence of an important role for the GABAA receptor in mediating certain behavioral actions of ethanol [65], an interesting observation was that the same chronic swim stress procedure that increased sleep time responses to ethanol, and decreased sleep time responses to pentobarbital, a prototypical barbiturate sedative/hypnotic that acts at the GABAA receptor. The opposing effects of the stress on the actions of the two drugs may relate to differential effects on specific GABAA receptor subunits. For example, mice lacking the γ2 or β3 subunits of the GABAA receptor exhibit normal sedative/ hypnotic responses to ethanol and pentobarbital, while deletion of either the α1 or β2 subunits appears to decrease sensitivity to the sedative/hypnotic effects of ethanol but not pentobarbital in some, although not all, studies [61,66–68]. In addition, mice exposed to a similar procedure as employed herein (i.e., fourteen days of 10 min swim stress) exhibit significant decreases in α1 mRNA in the hippocampus [69]. However, other forms of chronic stress have been found to produce either increases or decreases in α1 and β2 subunit mRNA in the rat hippocampus [70–72]. Earlier work has also found both decreased [63–65] and increased [49] sleep time responses to pentobarbital after various forms of stress. Thus, while these data suggest that our chronic swim stress paradigm may have produced specific alterations in the expression and/or function of α1 or β2 subunits that contributed to the observed alterations in sleep time responses to ethanol, it is difficult to posit a clearer hypothesis in the absence of further studies. 5. Conclusions In summary, the present study demonstrated that chronic swim stress potentiated acute intoxicating effects of ethanol in mice. These effects were specific to the sedative/hypnotic and hypothermic effects of ethanol, and did not generalize to the ataxic or anxiolytic-like effects of ethanol or to ethanol selfadministration. These data extend evidence that various forms of stress can alter sensitivity to ethanol's behavioral effects in rodents and humans and may ultimately provide insight into the relationship between stress and alcohol. Acknowledgements We are grateful to Lisa Wiedholz, Michael Feyder, Rebecca Yang, and Keri Boyce for their technical assistance. Research supported by the Intramural Research Programs of the National Institute of Alcohol Abuse and Alcoholism (Z01-AA000411) and the National Institute of Mental Health (Z01-MH002784).
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Chronic swim stress alters sensitivity to acute behavioral effects of ethanol in mice Janel M. Boyce-Rustay a,⁎, Heather A. Cameron b , Andrew Holmes a a
Section on Behavioral Science and Genetics, Laboratory for Integrative Neuroscience, National Institute on Alcohol Abuse and Alcoholism, Rockville, MD, United States b Unit on Neuroplasticity, Mood and Anxiety Disorders Program, National Institute of Mental Health, Bethesda, MD, United States Received 18 August 2006; received in revised form 9 January 2007; accepted 31 January 2007
Abstract Epidemiological data support a strong link between stress, stress-related disorders and risk for alcoholism. However, precisely how stress might impact sensitivity to the intoxicating effects of ethanol or the willingness to voluntary consume ethanol remains unclear. The present study assessed the effects of daily exposure to forced swim stress on subsequent sensitivity to the sedative/hypnotic, hypothermic, ataxic (measured using accelerating rotarod), and anxiolytic-like (measured using elevated plus-maze) effects of ethanol, and ethanol consumption and preference in a two-bottle choice paradigm, in male C57BL/6J mice. Stress effects on the sedative/hypnotic effects of the barbiturate pentobarbital were also tested. Results showed that chronic (fourteen days) but not acute (one or three days) swim stress significantly potentiated the sedative/hypnotic and hypothermic effects of 4 g/kg, but not 3 g/kg, ethanol. The sedative/hypnotic effects of pentobarbital were attenuated by chronic swim stress. Irrespective of chronicity, swim stress did not alter the ataxic or anxiolytic-like effects of ethanol, or alter ethanol self-administration either during or after stress. These data provide further evidence that stress alters the intoxicating effects of high doses of ethanol in a behaviorally selective manner. Published by Elsevier Inc. Keywords: Ethanol; Stress; Drinking; Anxiety; Sedation; Mouse; Hypothermia; Ataxia; Forced swim; Depression; Alcohol; Pentobarbital
1. Introduction There is considerable comorbidity between mood disorders and alcoholism [1,2]. Individuals with a history of stress and mood disorders such as anxiety and depression have approximately three times the risk for developing an alcohol-related disorder, and alcoholics with a co-morbid mood disorder tend to drink more heavily and have a poorer prognosis [3–6]. These epidemiological data have lent support to the notion that mood abnormalities represent a major risk factor for alcoholism, possibly because individuals abuse alcohol (ethanol) for its ‘anti-stress’ or ‘anti-tension’ properties [2,7,8]. The association between stress, mood disturbances and alcoholism appears to be complex. For example, while a history of adverse life events positively correlates with increased rates of alcoholism, a ⁎ Corresponding author. R4N5, AP9A/L018, Abbott Laboratories, 100 Abbott Park Rd, Abbott Park, IL 60064-6126. Tel.: +1 847 937 2559; fax: +1 847 938 1656. E-mail address: [email protected] (J.M. Boyce-Rustay). 0031-9384/$ - see front matter. Published by Elsevier Inc. doi:10.1016/j.physbeh.2007.01.024
proportion of individuals actually exhibit higher than normal rates of abstinence following stress [9–16]. These data suggest that individual differences, possibly at the levels of modifying psychosocial, biological and genetic factors, determine the relationship between stress and alcoholism. In this context, a number of studies have shown that individuals with a family history of alcoholism (family history positive, FHP) are less sensitive to certain behavioral effects of ethanol than family history negatives (FHN), apparently due to increased acute functional tolerance to the drug's effects [17,18]. Moreover, some studies show that FHP's also exhibit greater hypothalamic–pituitary–adrenal (HPA)-axis activation than FHN's following stress or ethanol challenge [19–25]. In addition, FHP's show more pronounced neuroendocrine and autonomic responses to ethanol itself than FHN's [26–28]. While these findings suggest that decreased sensitivity to ethanol and increased risk for alcoholism is associated with abnormal stress responsivity, how stress might impact sensitivity to ethanol intoxication has not been fully elucidated. Previous studies have shown that non-alcoholics exposed to laboratory
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stressors report increased feelings of sedation in response to ethanol challenge [29,30]. In rodents, while stress reinstates ethanol-seeking behavior after periods of abstinence [31,32], the effects of various types of stress on voluntary self-administration have been inconsistent [33–44]. Moreover, to-date there have been relatively few studies examining the effects of stress on sensitivity to the acute intoxicating effects of ethanol in rodents, with existing studies again report conflicting results [45–52]. The aim of the present study was to further examine the effects of stress on sensitivity to ethanol-related behaviors including self-administration in mice. Forced swim stress was employed as a stressor. Swim stress was used because it is a simple and readily replicable procedure and variants of this test are commonly used to assay depression-related behaviors in rats and mice [53]. Previous studies have also shown that swim stress can increase ethanol self-administration in mice [54–56]. In the present study, C57BL/6J mice were subjected to either acute or chronic swim stress and subsequently tested for ethanol-induced sedation/hypnosis, hypothermia, ataxia in the accelerating rotarod, and anxiety-like behavior in the elevated plus-maze. The effects of stress on ethanol consumption were measured using a standard two-bottle free choice paradigm. 2. Animals, materials and methods 2.1. Ethics All experimental procedures were approved by the National Institute on Alcohol Abuse and Alcoholism Animal Care and Use Committee, and followed the National Institute of Health guidelines outlined in ‘Using Animals in Intramural Research.’ 2.2. Animals Subjects were male C57BL/6J mice obtained from The Jackson Laboratory (Bar Harbor, ME) and aged 10–15 weeks
at the time of testing. Mice were housed in groups of 3–5 (except for the voluntary ethanol consumption experiment) in a temperature- and humidity-controlled vivarium under a 12 h light/dark cycle (lights on 0600 h). Separate cohorts of experimentally-naïve mice were used for each behavioral (and neuroendocrine) assay. The number of animals used in each experiment is shown in the corresponding table and figures legends. Ethanol doses were chosen to elicit an appropriate behavioral response based on previous studies [57]. With the exception of the voluntary ethanol consumption experiment, mice were given a 1 h acclimation period in the test room prior to testing, and apparatuses were cleaned with 70% v/v ethanol and dried between subjects. 2.3. Forced swim stress Mice were stressed by being placed into a transparent Plexiglas cylinder (20 cm diameter) filled halfway with water for 10 min, as previously described [58]. Water temperature was 24 ± 1 °C; in line with that typically used for studies employing the forced swim test as an assay for depressionrelated behavior and which likely produces a mild hypothermia [53]. A schematic representation of the procedure used to assess swim stress effects on acute sensitivity to ethanol is depicted in Fig. 1a, and on ethanol consumption in Fig. 7a. Unless otherwise indicated, mice were stressed daily for 0, 1, 3, or 14 days and then tested for ethanol-related behaviors 24 h later. 2.4. Behavioral and neuroendocrine responses to forced swim stress To test for changes in depression-related behavior with repeated swim stress exposures, mice were observed for the presence/absence of immobility (cessation of limb movements except minor involuntary movements of the hind limbs) every
Fig. 1. Procedure for assessing effects of swim stress on ethanol-related behaviors, and behavioral and neuroendocrine responses to repeated swim stress. (a) Effects of swim stress on ethanol-related behaviors were measured 24 h after 0, 1, 3 or 14 daily exposures. (b) Repeated daily exposure to swim stress produced a progressive increase in percent time immobile, a measure of ‘depression-related’ behavior (n=16). (c) Plasma corticosterone levels were elevated following either 1 or 14 days of stress exposure and significantly greater following 1 than 14 exposures (n =4–7/stress condition). ⁎⁎P b 0.01 vs. 0; ##P b 0.01 vs. 1. Data in this figure as well as in Figs. 2 –7 are Means ±SEM.
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5 s during the last 8 min of each 10 min test session, as previously described [59]. To measure hypothalamic–pituitary–adrenal (HPA)-axis activation following acute or repeated swim stress, mice were subjected to 1 or 14 days of stress and corticosterone levels were determined by radioimmunoassay (RIA). After the final stressor, mice were returned to the home cage for 30 min and then sacrificed via rapid cervical dislocation and decapitation to collect trunk blood. Non-stressed controls were sacrificed within 30 s of removal from the home cage. To minimize disturbance of non-stressed controls, all mice were individually housed 24 h prior sacrifice. Blood samples were centrifuged at 13,000 rpm for 30 s. Serum was extracted and assayed for total corticosterone (bound and free) using the Coat-a-Count RIA TKRC1 kit (limit of detection: 5.7 ng/ml; Diagnostic Products Corp, Los Angeles). 2.5. Effects of stress on ethanol- and pentobarbital-induced sedation/hypnosis Ethanol-induced sedation/hypnosis was assessed as previously described [57]. Immediately after injection with ethanol, mice were placed in the supine position in Plexiglas ‘V’-shaped chambers. The time from ethanol injection to recovery of the righting reflex (turning onto all 4 paws twice in 30 s after initial self-righting) was defined as sleep time. One cohort of mice was exposed to 0, 1 or 3 days of stress and 24 h later tested for sleep time response following intraperitoneal (i.p.) injection with 3 or 4 g/kg ethanol. A separate cohort was tested in the same manner following 0 or 14 days of stress. To test for possible changes in rates of ethanol clearance following stress, mice were tested for sleep time responses to 3 or 4 g/kg ethanol following 0 or 14 days of stress as above and, on recovery of the righting reflex, were sacrificed to collect trunk blood. Blood ethanol concentrations were measured using the Analox AM1 alcohol analyzer (Analox Instruments USA Inc, Lunenburg, MA). To test whether stress-induced changes in ethanol-induced sleep time occurred in response to another sedative/hypnotic, mice were exposed to 0 or 14 days of stress, injected i.p. with 40 or 50 mg/kg pentobarbital (Sigma, St. Louis, MO) and tested for sleep time as above. Doses of pentobarbital were based on those previously shown to induce sleep time in C57BL/6J mice [60,61]. 2.6. Effects of stress on ethanol-induced hypothermia Ethanol-induced hypothermia was assessed as previously described [57]. Core body temperature was measured by inserting a Thermalert TH-5 thermometer (Physitemp, Clifton, NJ) 2 cm into the rectum to obtain a stable reading for 10 s. Temperature was measured immediately prior to and 30, 60, 90, and 120 min after i.p. injection with 3 or 4 g/kg ethanol. Mice were placed into individual holding cages following the ethanol injection for the remainder of the experiment. Temperature change from pre-ethanol baseline was taken as the dependent measure. Ambient room temperature was 23 °C. One cohort of mice was exposed to 0, 1 or 3 days of stress and tested for
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hypothermic responses 24 h later following i.p. injection with 3 or 4 g/kg. A separate cohort was tested in the same manner following 0 or 14 days of stress. 2.7. Effects of stress on ethanol-induced ataxia on the accelerating rotarod Ethanol-induced ataxia on the accelerating rotarod was assessed as previously described [57]. The apparatus was a Med Associates rotarod (St. Albans, VT) typically used for testing rats (model # ENV-577). The 7 cm-diameter dowel was covered with 320 Grit sandpaper to provide a uniform surface that prevented mice gripping the rubberized dowel. Mice were placed onto the dowel with the rotarod rotating at 4 rpm, and the rotarod was then accelerated at a constant rate of 8 rpm/min from 4 rpm up to 40 rpm. The latency to fall to the floor 10.5 cm below was automatically recorded by photocell beams, with a maximum cutoff latency of 300 s. Mice were first given 10 consecutive training trials each separated by a 30 s inter-trial-interval. Stress groups were matched for average performance on the ninth and tenth training trials and exposed to 0, 1 or 3 days or, in a separate cohort, 0 or 14 days of stress. Twenty-four h after the final stressor, mice were given 3 baseline trials, injected i.p. with 0, 1.75, or 2.25 g/kg ethanol and returned to the home cage. Thirty min later, mice were given 3 test trials. Average performance on the second and third test trials was taken as post-ethanol performance. 2.8. Ethanol-induced anxiolytic-like behavior in the elevated plus-maze Ethanol-induced anxiolytic-like behavior was tested using the elevated plus-maze test as previously described [57]. The apparatus consisted of 2 open arms (30 × 5 cm; 90 lx) and 2 closed arms (30 × 5 × 15 cm; 20 lx) extending from a 5 × 5 cm central area and elevated 20 cm from the ground (San Diego Instruments, San Diego, CA). The walls were made from black ABS plastic and the floor from white ABS plastic. A 0.5 cm raised lip around the perimeter of the open arms prevented mice from falling off the maze. Testing was conducted under 65 dB white noise to minimize noise disturbance. This experiment focused upon the effects of 14 days of stress on the basis of data showing stress effects on other ethanol-related behaviors were most robust with this chronic regimen. Twenty-four h after 0 or 14 days of stress, mice were injected i.p. with 0, 1.0, or 1.5 g/kg ethanol and immediately placed in the center facing an open arm and allowed to explore the apparatus for 5 min. Time spent in the open arms, and entries into the open and closed arms were measured by the Ethovision videotracking system (Noldus Information Technology Inc., Leesburg, VA). 2.9. Effects of stress on voluntary ethanol consumption Voluntary ethanol consumption was measured using a 2-bottle choice paradigm as previously described [57]. Mice were individually-housed in ‘Space Saver’ cages (Model 1145T, Tecniplast, Buguggiate, Italy) with lids fitted for 2 fluid bottles
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(Model 1145T482SUDB-Polysulfone cage-top to accommodate 2 water bottles, top flow design; Tecniplast, Buguggiate, Italy). Two bottles, 1 containing ethanol and the other containing tap water, were available. Every 2 days the ethanol and water were changed and the left/right position of the bottles switched to control for any side bias. Food was available ad libitum. After 1 week of acclimation to the housing conditions, mice received increasing concentrations of ethanol: 3% (4 days), 5% (4 days), 7% (4 days), 9% (6 days), and 11% (10 days). Mice were then matched for average pre-stress 11% ethanol consumption and exposed to 0, 1 or 14 days of stress. Eleven percent ethanol was offered during the stress period and for 10 days after the 14-day-stress group received the final stressor. Ethanol and water consumption was measured every 24 h correcting for evaporation and spillage (measured in an empty chamber) and body weight was measured every 48 h throughout the study. These data were used to calculate ethanol consumption and preference for the ethanol-containing solution over water (ethanol consumption/total fluid consumption).
tests where appropriate. The effect of trial number on rotarod latency to fall, and the effect of ethanol concentration on voluntary ethanol consumption and preference, was analyzed using repeated measures ANOVA. Statistical significance was set at P b.05. 3. Results 3.1. Repeated forced swim stress activates the HPA-axis and increases depression-related behavior As shown in Fig. 1b, there was a significant, progressive increase in immobility with repeated exposure to forced swim stress [F(15,195) = 13.15, P b.01]. As shown in Fig. 1c, swim stress produced a significant increase in plasma corticosterone as compared to non-stressed controls [F(2,13) = 162.70, P b.01], with significantly higher corticosterone levels following either 1 or 14 exposures to swim stress than no-stress baseline, and a significantly greater level following 1 than 14 exposures.
2.10. Drugs For ethanol injections, ethanol (200 prf) was prepared in 0.9% w/v saline to produce 20% v/v solutions (0.9% w/v saline served as the vehicle control). Pentobarbital (Sigma, St. Louis, MO) was prepared in 0.9% w/v saline and injected at a volume of 10 ml/kg. 2.11. Statistical analysis The effects of stress and ethanol dose were analyzed using analysis of variance (ANOVA) and Newman–Keuls post-hoc
3.2. Stress increases sensitivity to ethanol-induced sedation/ hypnosis As shown in Fig. 2a, mice exposed to 3, but not 1, days of swim stress exhibited significantly longer sleep time responses to 4, but not 3, g/kg ethanol as compared to non-stressed controls [stress × dose interaction, F(2,68)= 4.56, P b.05]. In a separate cohort of mice, exposure to 14 days of swim stress led to significantly longer sleep time responses to both 3 and 4 g/kg ethanol, as compared to non-stressed controls [stress × dose
Fig. 2. Swim stress increased sensitivity to the sedative/hypnotic effects of ethanol. (a) Exposure to 3 but not 1 day of stress significantly increased sleep time responses to 4 but not 3 g/kg ethanol as compared to non-stressed controls (n = 11–14/stress condition/dose). (b) Exposure to 14 days of stress significantly increased sleep time responses to 3 and 4 g/kg ethanol relative to non-stressed controls (n = 9–11/stress/dose). (c) In a replicate cohort, exposure to 14 days of stress increased sleep time responses to 4 but not 3 g/kg ethanol relative to non-stressed controls, and (d) blood ethanol concentrations were significantly lower at sleep time recovery in stressed than non-stressed mice (n = 6–9/stress condition/dose). ⁎P b 0.05 vs. 0 d stress/same dose.
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Fig. 3. Chronic swim stress decreased sensitivity to the sedative/hypnotic effects of pentobarbital. Exposure to 14 days of stress (black bars) significantly decreased sleep time responses to pentobarbital as compared to non-stressed controls (white bars), regardless of dose (n = 11–14/stress condition/dose).
interaction, F(1,36) = 6.13, P b.05] (Fig. 2b). In a third cohort of mice, exposure to 14 days of stress showed significantly longer sleep time responses to 4, but not 3, g/kg ethanol as compared to non-stressed controls [stress × dose interaction, F(1,25) = 7.86, P b.05] (Fig. 2c). In this third cohort, blood ethanol concentrations were significantly lower at recovery in the stressed mice in comparison to the non-stressed controls and lower in mice treated with 3 g/kg than 4 g/kg ethanol regardless of stress [main effect of stress, F(1,24) = 6.29, P b.05; main effect of dose, F(1,24)= 9.68, P b.01] (Fig. 2d).
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dependently decreased latency to fall, with no difference between stressed and non-stressed groups [main effect of dose, F(2,89) = 17.70, P b.01]. In a separate cohort, there was again significant motor learning over 10 training trials prior to stress [main effect of trial number, F(9,504) = 23.90, P b.01] (Fig. 5c). Fourteen days of stress did not alter pre-ethanol performance (0 d stress = 95.3 ±5.4, 14 d stress = 100.7 ± 7.0). As shown in Fig. 5d, ethanol dose-dependently decreased latency to fall, with no difference between stressed and non-stressed mice [main effect of dose, F(2,52) = 18.50, P b.01]. 3.6. Stress did not affect ethanol-induced anxiolytic-like behavior As shown in Fig. 6a, mice exposed to 14 days of stress showed no differences in baseline open arm time, as compared
3.3. Stress decreases sensitivity to pentobarbital-induced sedation/hypnosis As shown in Fig. 3, mice exposed to 14 days of swim stress showed significantly shorter sleep time responses to pentobarbital as compared to non-stressed controls and sleep time response was significantly altered by pentobarbital dose [main effect of stress, F(1,40) = 5.60, P b.05; main effect of dose, F(1,40) = 55.40, P b.01]. 3.4. Stress increases sensitivity to ethanol-induced hypothermia Mice exposed to 1 or 3 days of stress did not show altered ethanol-induced hypothermia as compared to non-stressed controls; 4 g/kg produced a greater hypothermic effect than 3 g/kg regardless of stress [main effect of dose, F(2,86) = 38.43, P b.01] (Fig. 4a). In a separate cohort, mice exposed to 14 days of stress showed significantly greater hypothermic responses to 4, but not to 3, g/kg ethanol than non-stressed controls, as evidenced by lower core body temperature at 90 and 120 min post-ethanol [stress × dose × time interaction, F(3,102) = 4.96, P b.01] (Fig. 4b). 3.5. Stress did not affect ethanol-induced ataxia Mice showed motor learning over 10 rotarod training trials [main effect of trial: F(9,855) = 93.62, P b.01] (Fig. 5a). Neither 1 nor 3 days of stress altered pre-ethanol rotarod performance (0 d stress = 77.6 ± 4.3, 1 d stress = 79.7 ± 6.5, 3 d stress = 75.8 ± 7.0 s latency to fall). As shown in Fig. 5b, ethanol dose-
Fig. 4. Chronic swim stress increased sensitivity to the hypothermic effects of ethanol. (a) Exposure to neither 1 nor 3 days of stress altered hypothermic responses to 3 or 4 g/kg ethanol (n = 13–18/stress condition/dose). (b) Exposure to 14 days of stress significantly increased hypothermic responses to 4 but not 3 g/kg ethanol relative to non-stressed controls (n = 9–11/stress condition/dose). ⁎P b 0.05 vs. 0 d stress/same time point.
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Fig. 5. Stress did not alter the ataxic effects of ethanol on the accelerating rotarod. Motor learning prior to (a) 0, 1 or 3 days of stress, or (c) 0 or 14 days of stress. (b) Exposure to 1 nor 3 days of stress did not alter ataxic responses 1.75 or 2.25 g/kg ethanol (n = 10–13/stress condition/dose). (d) Exposure to 14 days of stress did not alter ataxic responses to 1.75 or 2.25 g/kg ethanol (n = 9–10/stress condition/dose).
Fig. 6. Stress did not alter the anxiolytic-like effects of ethanol in the elevated plus-maze. (a) Exposure to 14 days of stress did not alter percent open time at baseline or in response to treatment with 1.0 or 1.5 g/kg ethanol, as compared to non-stressed controls. (b) Neither stress nor ethanol treatment altered closed entries (n = 9–11/stress condition/dose).
Fig. 7. Stress does not affect ethanol self-administration in the 2-bottle free choice test (a) Schematic representation of the experimental procedure. Stress did not alter either (b) ethanol consumption or (c) ethanol preference, either during or in the 10 days following the end of stress, as compared to non-stressed controls.
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to non-stressed controls, and ethanol increased open arm time [main effect of dose, F(2,53) = 3.93, P b.05]. Neither stress nor ethanol dose significantly altered closed arm entries. 3.7. Stress did not affect voluntary ethanol consumption Prior to stress, increasing ethanol concentrations influenced rates of ethanol consumption (3% ethanol = 0.59 ± 0.08, 5% = 2.98 ± 0.18, 7% = 8.45 ± 0.38, 9% = 5.86 ± 0.32, 11% = 6.39 ± 0.36 g/kg ethanol consumed/day) [F(4,100) = 200.28, P b.01] and preference (3% ethanol = 44.3 ± 0.4, 5% = 71.8 ± 0.4, 7% = 70.1 ± 0.2, 9% = 71.4 ± 0.3, 11% = 67.0 ± 0.3 preference for the ethanol containing fluid over water) [F(4,100) = 23.82, P b.01]. There was no difference between stressed and non-stressed in ethanol consumption (Fig. 7b) or ethanol preference (Fig. 7c) either during the 14-day stress period or in the 10 days following the cessation of stress. Non-stressed and 3day stressed mice showed an increase in body weight over the 14day stress period, whereas the 14-day stress mice did not [stress × day interaction, F(2,23) = 15.17, P b.01] (data not shown). 4. Discussion Repeated exposure to forced swim stress over fourteen days produced a modest, but significant increase in passive immobility; a profile suggestive of an increase in depressionlike behavior [53]. In addition, mice exhibited stress-induced elevation of plasma corticosterone following exposure to fourteen days of swim stress indicating that the stressor maintained its potency with this chronic stress regimen. These data are consistent with previous data showing that C57BL/6 mice do not fully habituate to repeated exposure to another form of swim stress [62]. The main novel finding of the present study was that C57BL/6J mice exposed to chronic swim stress exhibited prolonged sleep time responses to ethanol; an effect replicated in three independent cohorts of mice. Stress effects on sensitivity to ethanol's sedative/hypnotic effects were largely limited to a relatively prolonged stress regimen. Thus, in contrast to the effects of fourteen days of swim stress, changes in sleep time responses to ethanol were not consistently seen with more acute (i.e., one or three days) stress regimens. A similar pattern of stress-related effects was observed for changes in hypothermic responses to ethanol; i.e. hypothermic responses were potentiated by exposure to chronic but not acute swim stress. In addition, increases in both the sedative/hypnotic and hypothermic effects of ethanol following chronic stress were both specific to a relatively high dose of ethanol (4 but not 3 g/kg ethanol). By contrast, neither the ataxia-inducing effects of (1.75–2.25 g/kg) ethanol in the accelerating rotarod, nor the anxiolytic-like effects of (1.0–1.5 g/kg) ethanol in the elevated plus-maze were altered by chronic swim stress. Taken together these demonstrate that stress altered sensitivity to ethanol's effects in manner that was specific to certain behavioral responses and to a high dose of ethanol. Stress-induced changes in sedation/hypnosis and hyperthermia were measured twenty-four hours after the cessation of
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stress, indicating that the effect was produced by relatively longlasting alteration in ethanol's mechanistic effects rather than by an acute alteration in ethanol absorption caused by stressinduced increases in corticosterone [63]. It should be noted, however, that plasma corticosterone levels following 14 days of swim stress exposure were relatively lower than following 1 day of stress, raising the possibility that stress-induced alterations in the HPA-axis may have contributed to altered ethanol sensitivity following the more chronic stress regimen. Present data also showed that longer sleeping stressed mice had modestly, but significantly, lower blood ethanol concentrations at awakening than non-stressed controls. This suggests that longer sleep time responses were not an artifact of slower ethanol clearance following stress. The fact that stress effects were specific to a higher dose of ethanol would also argue against a general change in ethanol pharmacokinetics. Nonetheless, the fact that positive effects of stress were seen only for behaviors measured relatively long after ethanol injection raises the possibility that subtle shifts in ethanol clearance may have contributed to these effects. Detailed studies of ethanol absorption and clearance following exposure to swim stress would help to clarify this issue. As noted in the Introduction, previous studies have found varying effects of stress on measures of acute sensitivity to ethanol in rodents. Stressors including exposure to bright light, tail-shock and rectal-probing increased sensitivity to ethanolinduced hypothermia and, in some but not all studies, ataxia in rats, while repeated restraint stress increased sensitization to the locomotor stimulant effects of ethanol in mice [45–50]. Mice exposed to footshock stress or social-isolation stress displayed decreased sedative/hypnotic and hypothermic responses to ethanol and rats exposed to swim stress showed decreased ataxic responses to ethanol [46,47,51,52]. The reason for this variability is not clear but might relate to differences in stressor type, duration of stress exposure or the strain of rat or mouse tested, as well as the methods used to measure ethanol's effects. Because none of these previous studies have used the same swim stress procedure as employed in the current study it is difficult to draw clear comparisons between this and previous studies. Nonetheless, our data show that our stress procedure produces a replicable effect on certain measures of acute sensitivity to ethanol's acute intoxicating effects, and provide a starting point for further investigations of the neurocircuitry involved and correlating changes in other ethanol-related behaviors. In this context, neither acute nor chronic swim stress altered voluntary ethanol consumption or preference in the two-bottle choice paradigm. This finding is not without precedent in a literature indicating that stress does not have robust and reliable effects on ethanol drinking in rats or mice. However, of particular relevance to current data previous studies have demonstrated that two or three days of forced swim stress can produce significant increases in ethanol (and water) consumption in mice [54–56]. The reason for the discrepancy with current data is not clear but may stem from methodological differences including swim duration (e.g., 5 min previously vs. 10 min in our study), tank-size (notspecified vs. 20 cm-diameter) and water temperature (21 vs. 24 °C). Another potentially critical factor is genetic background. Those
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studies observing swim stress-induced increases in ethanol consumption typically employed mice on a hybrid genetic background [54–56], while we used C57BL/6J. A comparison of swim stress effects on ethanol drinking across different mouse strains could help to determine whether C57BL/6J is atypically insensitive to the effects this stressor on ethanol self-administration. Another avenue for future studies is delineating the neural and molecular basis of stress-induced changes in the sedative/ hypnotic and hypothermic effects of ethanol. There are multiple candidate mechanisms that could be implicated. However, given evidence of an important role for the GABAA receptor in mediating certain behavioral actions of ethanol [65], an interesting observation was that the same chronic swim stress procedure that increased sleep time responses to ethanol, and decreased sleep time responses to pentobarbital, a prototypical barbiturate sedative/hypnotic that acts at the GABAA receptor. The opposing effects of the stress on the actions of the two drugs may relate to differential effects on specific GABAA receptor subunits. For example, mice lacking the γ2 or β3 subunits of the GABAA receptor exhibit normal sedative/ hypnotic responses to ethanol and pentobarbital, while deletion of either the α1 or β2 subunits appears to decrease sensitivity to the sedative/hypnotic effects of ethanol but not pentobarbital in some, although not all, studies [61,66–68]. In addition, mice exposed to a similar procedure as employed herein (i.e., fourteen days of 10 min swim stress) exhibit significant decreases in α1 mRNA in the hippocampus [69]. However, other forms of chronic stress have been found to produce either increases or decreases in α1 and β2 subunit mRNA in the rat hippocampus [70–72]. Earlier work has also found both decreased [63–65] and increased [49] sleep time responses to pentobarbital after various forms of stress. Thus, while these data suggest that our chronic swim stress paradigm may have produced specific alterations in the expression and/or function of α1 or β2 subunits that contributed to the observed alterations in sleep time responses to ethanol, it is difficult to posit a clearer hypothesis in the absence of further studies. 5. Conclusions In summary, the present study demonstrated that chronic swim stress potentiated acute intoxicating effects of ethanol in mice. These effects were specific to the sedative/hypnotic and hypothermic effects of ethanol, and did not generalize to the ataxic or anxiolytic-like effects of ethanol or to ethanol selfadministration. These data extend evidence that various forms of stress can alter sensitivity to ethanol's behavioral effects in rodents and humans and may ultimately provide insight into the relationship between stress and alcohol. Acknowledgements We are grateful to Lisa Wiedholz, Michael Feyder, Rebecca Yang, and Keri Boyce for their technical assistance. Research supported by the Intramural Research Programs of the National Institute of Alcohol Abuse and Alcoholism (Z01-AA000411) and the National Institute of Mental Health (Z01-MH002784).
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