INTRODUCTION

In humans, experimental and anecdotal evidence points to astrong correlation between exposure to chronic stress andincreased alcohol consumption (Sinha, 2001); an effect attri-buted to alcohol’s ability to alleviate tension.

Unlike human studies, the experimental evidence linkingstress to increased alcohol consumption in laboratory animals,particularly rats, is equivocal. In animal models, chronic stresshas been reported to increase (Anisman and Waller, 1974;Volpicelli et al., 1990), decrease (van Erp and Miczek, 2001)or not change alcohol intake (Fidler and LoLordo, 1996;Myers and Holman, 1967). Interestingly, and in contrast to thedivergent findings on stress-induced alcohol consumption inlaboratory animals, it has been repeatedly shown that exposureto acute stress evokes reinstatement of alcohol-seeking (Leand Shaham, 2002; Martin-Fardon et al., 2000). The reasonsfor this discrepancy are not readily apparent; however, severalfactors may contribute to the lack of correlation betweenchronic stress and increased alcohol consumption in rats. Forexample, stress may have different effects on alcohol consump-tion in non-dependent rats compared to dependent rats, or inanimals that have had a prior history of dependence. Inaddition, stressors of a different nature may have a differentimpact upon alcohol consumption. Moreover, to our knowl-edge, the effects of different types of stress on the alcoholdeprivation effect (ADE), the temporary increase in alcoholconsumption seen after periods of abstinence (Spanagel andHolter, 2000), has not been previously examined.

Footshock has been the most commonly used stressor inthe animal literature to model stress-induced changes inalcohol consumption in humans (Anisman and Waller, 1974;Fidler and LoLordo, 1996; Le and Shaham, 2002; Le et al.,1998; Martin-Fardon et al., 2000; Myers and Holman, 1967;

Volpicelli et al., 1990). Interestingly, footshock has giveninconsistent results with respect to alcohol consumption, but ithas been repeatedly shown to reinstate alcohol-seekingbehaviour in rats (Le and Shaham, 2002; Martin-Fardon et al.,2000). To contrast the effects of chronic footshock on alcoholconsumption, animals were exposed to repeated injection ofthe endotoxin lipopolysaccharide (LPS), a model of chronicstress thought to use different stress-sensitive pathways totrigger activation of the hypothalamic-pituitary-adrenal (HPA)axis (Dayas et al., 2001; Li et al., 1996). Like footshock, LPSrobustly activates the HPA axis culminating in peripheral glucocorticoid release and is also accompanied by changes incentral monoamine levels (Besedovsky and del Rey, 1996;Grinevich et al., 2001). Glucocorticoid release in responseto LPS plays an important role as an immunosuppressant,subsequently acting to limit the degree of inflammation(Grinevich et al., 2001).

In the present study, the effects of different types of chronicstress on the alcohol deprivation effect (ADE), the temporaryincrease in alcohol consumption seen after periods ofabstinence, was assessed in rats made dependent on alcohol bya liquid diet. To address these issues, animals were subjectedto a week of forced abstinence from alcohol self-administration,during which they were subjected to either no stress, sevendays of chronic mild footshock or gradually increasing dosesof the endotoxin LPS. Alcohol-reinforced responding was thenmeasured daily for 3 weeks.

MATERIALS AND METHODS

Twenty-one, male, group housed (12 h light : 12 h dark cycle,lights off 18:00 hours) Wistar rats (Charles River, Kingston,NY) weighing 300 g on arrival were trained to self-administeralcohol in operant conditioning chambers (CoulbornInstruments, Allentown, PA) enclosed in sound-attenuatingcubicles, equipped with two retractable levers. Food and waterwere available ad libitum except during the initiation of operantself-administration training. After 1 week of acclimatization inthe vivarium, during which the rats were handled for 5 min

Alcohol & Alcoholism Vol. 39, No. 3, pp. 190–196, 2004doi:10.1093/alcalc/agh046, available online at www.alcalc.oupjournals.org

CHRONIC FOOTSHOCK, BUT NOT A PHYSIOLOGICAL STRESSOR, SUPPRESSESTHE ALCOHOL DEPRIVATION EFFECT IN DEPENDENT RATS

C. V. DAYAS*, R. MARTIN-FARDON, A. THORSELL and F. WEISS

Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA, USA

(Received 25 September 2003; first review notified 1 December 2003; in revised form 6 December 2003; accepted 29 December 2003)

Abstract — Aims: Unlike in humans, the link between chronic stress and increased alcohol consumption in laboratory animals isequivocal. Two factors may contribute to this: a lack of studies examining the effects of stress on consumption in dependent rats anddifferences in the nature of the stressor. Moreover, to our knowledge, the effects of different types of stress on the alcohol deprivationeffect (ADE), the temporary increase in alcohol consumption seen after periods of abstinence, has not been previously examined.Methods: In the present study, dependent rats previously trained to self-administer alcohol, received either no stress, chronic dailyintermittent footshock (10 min/day for 7 days) or daily (for 7 days) injections of lipopolysaccharide, a physiological stressor. Alcohol-reinforced responding was then measured for 20 days. Results: Only control animals and those treated with LPS exhibited an alcoholdeprivation effect and increased consumption. Conclusions: Taken together, these data suggest that chronic footshock may not be anappropriate paradigm to study the impact of stress on alcohol consumption.

Alcohol & Alcoholism Vol. 39, No. 3 © Medical Council on Alcohol 2004; all rights reserved

*Author to whom correspondence should be addressed at: Department ofNeuropharmacology (CVN–15), The Scripps Research Institute, La Jolla,CA 92037, USA. Tel.: +858 784 7233; Fax: +858 784 7405; E-mail:cvdayas@scripps.edu

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each day, rats were trained to orally self-administer alcohol indaily 30-min sessions using a sweet solution fading procedure(Martin-Fardon et al., 2000). Briefly, water availability waslimited to 2 h/day for 3 days. At this time, self-administrationtraining sessions were initiated by extension of the right lever,and each lever press was reinforced by 0.1 ml of a 0.2% (w/v)saccharin solution. Water was then made available againad libitum in the home cage and, on day four, alcohol self-administration was initiated by adding 5% (v/v) alcohol to thesaccharin solution. Over the next 3 weeks, the concentrationof alcohol was gradually increased to 10% (v/v) while con-comitantly saccharin was eliminated. During this time, theresponse requirement was increased to a fixed ratio (FR) of 3,and each reinforced response paired with a white cue light(24 W) above the lever illuminated for 0.5 s. Self-administrationof 10% alcohol under these contingencies continued in dailysessions for 7 weeks.

Dependence induction

After 50 days of alcohol self-administration (10%), rats wereexposed to an alcohol liquid diet in order to induce depend-ence, as previously reported (Zorrilla et al., 2001). Briefly, ratchow and water were replaced with a nutritionally complete,10% (v/v) alcohol-containing liquid diet consisting of achocolate-flavoured, liquid nutritional supplement (BOOST;Mead Johnson, Evansville, IN) supplemented with a vitaminand mineral salt mixture (0.3 and 0.5 g/100 ml, respectively;ICN Nutritional Biochemicals, Aurora, OH), distilled waterand 95% alcohol diluted to a final concentration of 10% (v/v).Rats in the alcohol condition received ad libitum access to thealcohol liquid diet. The diet was prepared daily and admin-istered at the onset of the reverse dark cycle for 21 days.

After withdrawal, alcohol self-administration was re-established for an additional 4 weeks using the same FR-3schedule as described above.

Blood alcohol levels

To determine blood alcohol levels (BAL), tail blood (approx.500 µl) was collected weekly into heparinized tubes 4–5 h afterthe onset of the dark cycle. Samples were centrifuged (10 min,5000 r.p.m.) and plasma assayed for alcohol content byinjection into an oxygen-rate alcohol analyzer (AnaloxInstruments, Lunenburg, MA).

Alcohol deprivation and stressor exposure

Once stable baseline alcohol self-administration was re-established, animals were randomly assigned to three groupsand underwent 1 week of abstinence from alcohol self-administration. During this week of abstinence, animals fromgroup one (n = 7) received no stress but once daily sterile salineinjections (i.p. 1 ml/kg). Animals from group two (n = 7) wereexposed to chronic daily intermittent footshock (n = 7,10 min/day, 0.5 mA, train length 0.5 s, administered via thegrid floor under a variable-interval 40-s schedule, intervalrange: 10–70 s for 7 days). Animals from group three (n = 7)were injected daily (for 7 days) with gradually increasing dosesof lipopolysaccharide (LPS, Escherichia coli serotype 0111(Sigma, St Louis, MO) 25, 50, 75, 100, 150, 170, 250 µg/100 gdissolved in sterile saline, 1 ml/kg) to simulate an immunechallenge. Incremental doses of LPS were administered as perprevious studies (Grinevich et al., 2001), because it is knownthat repeated injection of LPS desensitizes cytokine production.

After 7 days, alcohol reinforced-responding was measuredfor 20 days.

Data analysis

BAL from the last week of the liquid diet were compared usingone-way ANOVA to ensure that all animals were equallyintoxicated. Alcohol-reinforced responding pre- and post liquiddiet, the effect of stress on body weight, alcohol-reinforcedresponding and intake (g/kg) following stress were analysedusing two-way ANOVA with repeated measures on one factor.After statistically significant effects were observed, post-hoccomparisons were conducted using Fisher’s LSD tests.Statistical significance was set at P < 0.05.

RESULTS

All rats acquired responding for alcohol (10%) and devel-oped stable rates of alcohol-reinforced responding duringthe initial self-administration procedure. Moreover, the threegroups showed statistically indistinguishable levels of alcohol-reinforced responding before and after the intoxication period[F(2,18) = 1.1; P = 0.36]. Baseline levels of alcohol-reinforcedresponding prior to the liquid diet (average over 7 weeks) in thethree groups (i.e. control, footshock and LPS) were 45.4 ± 3.5,36.7 ± 1.8 and 42.6 ± 3.7 (mean ± SEM) respectively. Afterdependence induction, animals were allowed to self-administeralcohol for an additional 4 weeks. The mean (± SEM) num-ber of alcohol-reinforced responses in the control, footshockand LPS groups were 37.3 ± 4.2; 33.9 ± 2.7 and 39.3 ± 3.8respectively (Fig. 1).

After the first week of the alcohol liquid diet, the averageBAL in the three groups were 5 ± 1, 6 ± 1 and 7 ± 1 mg% inthe control no stress, footshock and LPS groups, respectively.No differences in BAL were observed after the last week ofintoxication (control, 96 ± 11, footshock 84 ± 13 and LPS81 ± 15 mg% [F(2,18) = 0.35, P = 0.7]).

In the 3 groups (see Fig. 1 insets for comparison), 1 weekof forced abstinence and stress had a significantly differenteffect on body weight, as reflected by a significant maineffect on the repeated measure (i.e. weight) [F(8,144) = 26.6,P < 0.001] and a significant stress � weight interaction[F(16,144) = 26.4, P < 0.001)]. In the control group (i.e. nostress) the animals demonstrated a gradual increase in bodyweight over the treatment period that was significantlydifferent from baseline on days 5–8 (Fisher’s LSD tests: days5–7, P < 0.05; day 8, P < 0.001), day 8 being the day ofre-introduction to the self-administration chamber (Fig. 1A,inset). Chronic daily footshock produced a gradual decreasein body weight compared to pre-stress levels reachingsignificance on days 2 (P < 0.05), 4 (P < 0.05), 5 (P < 0.01)and 6 and 7 (P < 0.001) (Fig. 1B inset; maximum loss wasapproximately 3% on days 6 and 7). Injections of LPS induceda more dramatic decrease in body weight, significantly dif-ferent from pre-stress levels on days 2–8 of treatment (Fisher’sLSD tests: P < 0.001) (Fig. 1C, inset; maximum loss wasapproximately 11% on day 7).

Alcohol reinforced responding, after 1 week of alcoholdeprivation and either no stress, footshock or LPS wassignificantly different to baseline (Fig. 1A–C), as reflected bya significant main effect of the repeated factor: days of self-administration [F(38,342) = 1.9, P < 0.01] and a significant

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group � day of self-administration interaction [F(38,342) =1.95, P < 0.01]. In control rats, 1 week of forced abstinenceproduced a typical ADE (i.e. increased alcohol-reinforcedresponding compared to baseline) the day of return to the

alcohol self-administration chamber (Fisher’s LSD test,P < 0.01 vs. baseline; Fig. 1A). This increase in alcohol-reinforced responding was also noticeable on several otherdays (days 6–8, 12–15 and 18, P < 0.01 vs. baseline; Fig. 1A).

Fig. 1. Effects of stress on responding for alcohol and body weight (insets). (A) Animals exposed to no stress displayed the typical alcohol deprivationeffect on day 1 and increased responding on days 6–8, 12–15 and 18. (B) Animals that underwent chronic daily footshock during the week of forcedabstinence showed no significant increase of responding for alcohol. (C) On the first day after the last lipopolysaccharide (LPS) challenge, the animalsdid not show an alcohol deprivation effect (ADE) but on days 6, 7 and 9 the rats showed an increase of alcohol-reinforced responding compared totheir baseline level. Control rats gradually increased their body weight reaching statistically different levels compared to baseline over the last fourdays of treatment (A, inset). Rats exposed to footshock gradually decreased their body weight returning to baseline levels by the end of treatment(B, inset). LPS-treated rats exhibited a typical decrease in body weight that persisted throughout the treatment period (C, inset). Fishers LSDtests: *P < 0.05, **P < 0.01, ***P < 0.001, compared to pre-stress baseline. B, baseline; R-SA, day of return to self-administration; SA,

self-administration; T1–7, treatment days.

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Animals that underwent chronic daily footshock during theweek of forced abstinence showed no significant increase ofalcohol-reinforced responding (Fig. 1B). Animals exposed togradually increasing doses of LPS displayed the typical behavi-oural response to an immune challenge consisting of piloerectionand decreased locomotor activity 3–5 h after each injection. Onthe first day after the last LPS challenge, the animals did notshow an ADE but on days 6, 7 and 9 the rats showed anincrease of alcohol-reinforced responding compared to their base-line level (Fisher’s LSD test, P < 0.05 vs. baseline; Fig. 1C).

To confirm that alcohol-reinforced responding in response tostress was not a function of increased body weight in the con-trol group over the treatment period, alcohol consumption wasreanalysed in g/kg. As described above, after 1 week of alcoholdeprivation and either no stress, footshock or LPS alcohol con-sumption in g/kg was significantly different to baseline (Fig. 2),as reflected by a significant main effect of the repeated factor:days of self-administration [F(38,342) = 3.2, P < 0.001] and asignificant group � day of self-administration interaction[F(38,342) = 2.1, P < 0.001]. In control rats, an identical pat-tern to levels of alcohol-reinforced responding was observed,namely 1 week of forced abstinence produced a typical ADE(i.e. increased alcohol consumption compared to baseline), theday of return to the alcohol self-administration chamber(Fisher’s LSD test, P < 0.01 vs. baseline; Fig. 2A). This increasein alcohol intake was also noticeable on days 6–8, 12–15 and18 (Fisher’s LSD test, P < 0.01 vs. baseline, Fig. 2). In therepeated footshock group, no significant change in alcoholintake (g/kg) was observed (Fig. 2B). Animals chronicallytreated with LPS did not show an ADE on day 1 but on days 6,7 and 9 the rats showed an increase of alcohol intake comparedto their baseline level (Fisher’s LSD test, P < 0.05, P < 0.001,P < 0.01 respectively, vs. baseline; Fig. 2C). In contrast to ouranalysis using alcohol-reinforced responses, an increase ofalcohol intake was also observed on day 16 compared tobaseline when expressed in g/kg (Fisher’s LSD test, P < 0.05vs. baseline; Fig. 2C).

DISCUSSION

The present findings demonstrate that in dependent rats thatwere subjected to a week of forced abstinence from alcoholself-administration, but receiving no stress, a typical ADE andan increase in alcohol-reinforced responding was observed.Somewhat surprisingly, animals subjected to repeated footshockshowed no ADE and no significant change in alcohol intake.Finally, animals that received repeated LPS injections showeda delayed ADE.

Studies in humans support a direct link between chronicstress and increased alcohol consumption (for review see(Sinha, 2001)), while in rats, a link between chronic stress andincreased alcohol consumption has been harder to establish. Incontrast, stress-induced relapse to alcohol-seeking has beenreported in both humans and rats (Brown et al., 1995; Le andShaham, 2002; Martin-Fardon et al., 2000; Sinha, 2001). Thepresent study examined several important variables that mightcontribute to this disassociation; whether a history of depend-ence or different stressors has an effect on the alcohol depri-vation effect. We examined these issues in rats with a historyof dependence; BAL corresponding to levels previously

reported to produce signs of alcohol withdrawal (Ciccocioppoet al., 2003).

Previous studies have shown that similar levels of alcohol-reinforced responding, as observed in the present study, producepharmacologically relevant BAL (Ciccocioppo et al., 2002;Roberts et al., 1999). It is noteworthy that animals made depend-ent on the alcohol liquid diet did not appear to increase theirintake in self-administration sessions. Increased alcohol con-sumption in dependent animals is a highly complex and variablephenomenon, often requiring repeated cycles of withdrawal(Roberts et al., 1996). In the present study our aim was to inves-tigate the effects of stress on the ADE. Therefore we deliberatelyavoided repeated cycles of abstinence and withdrawal.Regardless, animals had a long history of ethanol exposurewhether self-administering or in the homecage, the latter pro-ducing significant elevations in blood alcohol levels.

Physically dependent non-stressed rats that underwent1 week of forced abstinence demonstrated an ADE that wasobserved on the first day following re-exposure to the self-administration chamber; a finding consistent with previousstudies in both rats and humans (Spanagel and Holter, 2000).Moreover, these animals displayed an elevated level ofalcohol-reinforced responding on days 6–8, 12–15 and 18.Potentially, the augmented level of alcohol intake reflects self-medication of adverse symptoms that commonly accompanyalcohol withdrawal (Markou et al., 1998). While there aresome reports using a progressive ratio schedule suggestingthat the ADE produces an increase in the reinforcing value ofalcohol (Spanagel and Holter, 2000), it cannot be ruled outthat a decrease in the reinforcing effects of alcohol account forthe increase in responding observed here. Further experimentsare required to pursue this possibility. The exact mechanismsunderlying the ADE are not well characterized; however,drugs successful in treating alcoholism in humans, such asnaltrexone, acamprosate and baclofen, are also capable of sup-pressing the ADE in rats (Addolorato et al., 2002; Colomboet al., 2003; Spanagel and Holter, 2000), suggesting that dys-regulation of opioid, glutamatergic and or gabaergic systemsmay underlie this phenomenon.

In contrast to animals that received no stress, animals exposedto chronic footshock during the week of forced abstinencedemonstrated no ADE and no increase of alcohol intake. Whilesomewhat unexpected, the literature concerning stress-inducedincreases in alcohol consumption in rats suggests that the presentdata fit within the spectrum of studies showing either an increase(Anisman and Waller, 1974; Volpicelli et al., 1990; Vengeliene,2003), decrease (van Erp and Miczek, 2001) or no changefollowing exposure to a stressor (Fidler and LoLordo, 1996;Myers and Holman, 1967); most commonly chronic footshock.van Erp and Miczek, attempting to understand these inconsistentfindings have highlighted the importance of controlling for themany variations inherent in the stress/alcohol consumption field,such as animal housing, ratio of alcohol to an additionalsweetener, intensity of the stressor and importantly, the timing ofstressor exposure with respect to re-acquaintance with thealcohol solution. They reported a decrease in alcoholconsumption when alcohol access occurred before a briefexposure to a social stressor or no change when alcohol accessoccurred after the stressor (van Erp and Miczek, 2001).

The present results confirm previous studies showing nochange in alcohol consumption (Fidler and LoLordo, 1996;

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Myers and Holman, 1967) in response to chronic footshockstress but more importantly, footshock suppressed the normalincrease in alcohol intake observed in response to alcoholdeprivation. The reason for the suppression of the ADE byfootshock in the present study is not readily apparent, but one

parsimonious explanation is that chronic footshock results in apassive rather than active (increased alcohol consumption)coping strategy (Bandler et al., 2000).

Animals that underwent repeated LPS injections displayedthe typical symptoms of sickness associated with an immune

Fig. 2. Effects of stress on alcohol intake expressed as g/kg. (A) Animals exposed to no stress displayed the typical alcohol deprivation effect on day 1and increased intake on days 6–8, 12–15 and 18. (B) Animals that underwent chronic daily footshock during the week of forced abstinence showed nosignificant increase of alcohol intake. (C) On the first day after the last lipopolysaccharide (LPS) challenge, the animals did not show an alcoholdeprivation effect (ADE) but on days 6, 7 and 9 the rats showed an increase of alcohol intake compared to their baseline level. When alcohol intake wasexpressed in g/kg a significant increase in alcohol consumption was also observed on day 16 (C). Fishers LSD tests: *P < 0.05,

**P < 0.01, ***P < 0.001, compared to pre-stress baseline.

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SUPPRESSION OF THE ADE BY FOOTSHOCK AND NOT LPS 195

challenge, as reflected by a decrease in body weight across thetreatment period (Kent et al., 1992). These animals showed aninitial suppression of alcohol-reinforced responding (Spanageland Holter, 2000) lasting for up to five days following thecessation of the LPS injections. Despite this initial sup-pression, a delayed ADE was observed on days 6, 7, 9 and 16.In fact, in absolute terms, on day 6 this group displayed a levelof alcohol-reinforced responding that was greater than on anyother day and across any of the groups. The reasons for whyanimals exposed to an immune challenge would demonstratesuch a robust rebound in responding 6 days after the cessationof repeated LPS is not self-evident, but arguably, this reboundreflects the negative reinforcement associated with the adversesymptoms that accompany withdrawal. Also, recent attentionhas been given to the similarity of the symptoms induced byan LPS-mediated immune challenge to the symptoms of depres-sion such as suppression of appetite, sleep pattern distur-bances, lack of energy and fatigue (Dantzer et al., 1999). Thus,an interesting possibility is that the increase in alcohol-reinforced responding seen in immune challenged animalsreflects an attempt to self-medicate, a suggestion consistentwith clinical data supporting a correlation between anxiety-related disorders and drug abuse (Markou et al., 1998).

CONCLUSIONS

Cautiously interpreted, the present data suggest that chronicfootshock is not an appropriate paradigm to model the pheno-menon of increased alcohol consumption in response to stressas reported in humans (Sinha, 2001). This is in distinctcontrast to the many reports demonstrating that exposure tofootshock facilitates self-administration of other commonlyabused drugs including amphetamines (Piazza et al., 1990),morphine (Alexander et al., 1978; Shaham and Stewart, 1994)and cocaine (Covington and Miczek, 2001; Miczek andMutschler, 1996), that acute footshock has been shown toevoke reinstatement of heroin-, cocaine-, nicotine- andalcohol-seeking (Le and Shaham, 2002) and that acutefootshock used in the reinstatement paradigms does seem tocorrelate with reports of stress-induced relapse in humans(Brown et al., 1995). It seems highly likely then, at least inrats, that the mechanism(s) that trigger stress-induced re-instatement of drug-seeking and the facilitative effects ofstress on stimulant and opioid self-administration, arefootshock sensitive, whereas that triggering increased alcoholconsumption is not (Sinha, 2001). Further work is required toidentify paradigms suitable to study the phenomenon of stress-induced alcohol consumption in humans (Sinha, 2001).

Acknowledgements — This is publication number 16035-NP from The ScrippsResearch Institute. This work was supported by a NIH/NIAAA grant AA 10531program grant (F.W.) and by a C. J. Martin Post-doctoral Fellowship (NationalHealth and Medical Research Council of Australia) awarded to C.V.D. The authorsthank Jeff Simms and Maury Cole for expert technical assistance and Mike Arendsfor assistance with the preparation of this manuscript.

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