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ARTICLE IN PRESSG ModelBR 9132 1–9

Behavioural Brain Research xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Behavioural Brain Research

jou rn al hom epage: www.elsev ier .com/ locate /bbr

esearch report

ubgroup-dependent effects of voluntary alcohol intake on behavioralrofiles in outbred Wistar rats

hima Momeni ∗, Erika Romaneuropharmacology, Addiction & Behaviour, Department of Pharmaceutical Biosciences, Uppsala University, P.O. Box 591, SE-751 24</postal>, Uppsala,weden

i g h l i g h t s

Large individual differences in alcohol intake were demonstrated.High-drinking rats displayed an alcohol deprivation effect.High-drinking animals showed a lower anxiety-like behavior after alcohol intake.High-drinking animals may consume alcohol for its anxiolytic properties.The present study highlights the importance of studying subgroups.

r t i c l e i n f o

rticle history:eceived 26 May 2014eceived in revised form 26 August 2014ccepted 30 August 2014vailable online xxx

eywords:nxietylcoholultivariate concentric square field (MCSF)

ntermittent accessndividual differencelcohol deprivation effect

a b s t r a c t

Experimental animal models are critical for understanding the genetic, environmental and neurobio-logical underpinnings of alcohol use disorders. Limited studies investigate alcohol-induced effects onbehavior using free-choice paradigms. The aims of the present experiment were to study voluntary alco-hol intake using a modified intermittent access paradigm, investigate the effects of voluntary alcoholintake on behavioral profiles in water- and alcohol-drinking rats, and select extreme low- and high-drinking animals for a more detailed behavioral characterization. Sixty outbred male Wistar rats wererandomized into water and alcohol groups. Behavioral profiles in the multivariate concentric squarefieldTM (MCSF) test were assessed prior to and after voluntary alcohol intake. The animals had intermit-tent access to 20% alcohol and water for three consecutive days per week for seven weeks. The resultsrevealed increased alcohol intake over time. No major alcohol-induced differences on behavior profileswere found when comparing water- and alcohol-drinking animals. The high-drinking animals displayedan alcohol deprivation effect, which was not found in the low-drinking animals. High-drinking rats had

lower risk-taking behavior prior to alcohol access and lower anxiety-like behavior after voluntary alcoholintake compared to low-drinking rats. In conclusion, the modified intermittent access paradigm may beuseful for pharmacological manipulation of alcohol intake. With regard to behavior, the present findingshighlights the importance of studying subgroup-dependent differences and add to the complexity ofindividual differences in behavioral traits of relevance to the vulnerability for excessive alcohol intake.

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. Introduction

Alcohol use disorders (AUD) are heterogeneous with regardo etiology, vulnerability for addiction and response to treatmentnvolving complex interactions between multiple genes [1–3] and

Please cite this article in press as: Momeni S, Roman E. Subgroup-depin outbred Wistar rats. Behav Brain Res (2014), http://dx.doi.org/10.10

nvironmental factors [4]. Many different risk factors for AUD haveeen identified including comorbid psychiatric disorders [5] andersonality traits such as impulsivity [6,7]. In addition, clusters of

∗ Corresponding author. Tel.: +46 18 4714620; fax: +46 18 4714253.E-mail address: Shima.Momeni@farmbio.uu.se (S. Momeni).

ttp://dx.doi.org/10.1016/j.bbr.2014.08.058166-4328/© 2014 Published by Elsevier B.V.

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© 2014 Published by Elsevier B.V.

personality traits brought together into typologies have been linkedto vulnerability for AUD [8]. Due to numerous involving factors, westill do not know why some individuals are more vulnerable forAUD than others and therefore knowledge of individual differencesin vulnerability is needed.

Experimental animal models are critical for understanding thegenetic, environmental and neurobiological underpinnings of AUD.One of the most consistent findings is that animals showing a

endent effects of voluntary alcohol intake on behavioral profiles16/j.bbr.2014.08.058

high anxiety-like behavior also show higher alcohol intake [9–12],but contrasting results have also been reported indicating on thecomplexity [13–15]. In most studies on this topic forced admin-istration paradigms are used, where the animal is administrated

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Fig. 1. The experimental outline. The animals were first tested in the MCSF, then randomly assigned into water- and alcohol-drinking groups and introduced to a two-bottlef days pa lcoho( ed ana

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ree-choice paradigm with intermittent access to 20% alcohol for three consecutive

gain tested in the MCSF, after which the experiment ended. Based on the average aLD), intermediate-drinking (ID) and high-drinking (HD) subgroups for more detail

specific dose of alcohol. The limitation with this procedure ishat individual predisposition to excessive alcohol intake cannote investigated. We have recently shown that risk-taking behav-

or displayed minor associations with voluntary alcohol intake inutbred rats while animals showing a high risk-assessing behaviorad a higher alcohol intake and preference, and increased alcohol

ntake over time compared to low risk-assessing rats. These find-ngs indicate that certain aspects of risk-related behaviors imply onigher vulnerability for alcohol intake [16]. Moreover, the literaturen alcohol-induced effects on behavior after voluntary drinkings limited and contrasting results have been reported likely dueo different intake paradigms, duration of intake and tests used.or instance, rats having access to 6% alcohol solution for 51 con-ecutive days spent more time on the open arms of the elevatedlus maze compared to the water controls [17]. Using a 1 h-limitedccess paradigm with 10% sweetened alcohol for 35 days, alcoholrinking animals had decreased activity on the open arms in thelevated plus maze [18]. Furthermore, when profiling adolescentutbred rats prior to and after a period of alcohol access in order totudy alcohol-induced behavior modulation over time no evidentlcohol-induced effects were found and the predictive value of theehavioral profiling in relation to later alcohol intake was weak19].

The aim of this study was to analyze the effects of voluntary alco-ol intake on behavior profiles in adult male rats. We here introduce

free-choice intermittent access paradigm modified from the mod-ls used by Wise [20] and [21], summarized by Carnicella et al. [22].he animals had free access to 20% alcohol and water during threeonsecutive days followed by a four-day intermission with access toater only. The behavioral profiling of the animals was made using

he multivariate concentric square fieldTM (MCSF) [23,24] wherehe first trial was conducted before the seven-week period of accesso alcohol and the second trial was conducted 3–7 days after theast alcohol session. Alcohol-induced effects on behavioral profiles

ere investigated by comparing water- and alcohol-drinking rats.n addition, subgroups of extreme low- and high-drinking animals

ere selected based on the alcohol intake during the last week ofccess for a more detailed characterization of behavioral profilesrior to and after voluntary alcohol intake.

. Material and methods

.1. Animals and housing

Sixty outbred male Wistar rats (RccHanTM:WI) from Harlanaboratories B.V., Horst, The Netherlands were delivered to the

Please cite this article in press as: Momeni S, Roman E. Subgroup-depin outbred Wistar rats. Behav Brain Res (2014), http://dx.doi.org/10.1

nimal facility at seven weeks of age. The rats were group-oused (three rats per cage) in transparent polysulfone cages59 cm × 38 cm × 20 cm) containing wood-chip bedding materialnd two paper sheets (40 cm × 60 cm; Cellstoff, Papyrus) for

er week for seven weeks. After the period of alcohol access, the animals were oncel intake during the last week of access, the animals were divided into low-drinkinglysis of behavioral profiles in LD and HD rats prior to and after access to alcohol.

enrichment purpose. The cages were placed in temperature-(21 ± 1 ◦C) and humidity-controlled (50 ± 10%) housing cabinetswith a reversed 12 h light/dark cycle (lights off at 07.00 h), and witha masking background noise. The rats were maintained on standardrat chow (R36; Lantmännen, Kimstad, Sweden) and water ad libi-tum. All animal experiments were approved by the Uppsala AnimalEthical Committee and followed the guidelines of the Swedish Leg-islation on Animal Experimentation (Animal Welfare Act SFS1998:56) and the European Communities Council Directive (86/609/EEC).

2.2. Experimental procedures

The outline of the experimental procedure is illustrated in Fig. 1.Upon arrival, the rats were undisturbed for two weeks to allowacclimatization to the animal facility and the reversed light/darkcycle. The week following the acclimatization period the rats werehandled daily using a procedure similar to that of previous exper-iments [24,25], consisting of individual handling, weighing, andadaptation to the transportation bucket that was used to trans-port the animals from the home cage to the testing room. The ratswere then tested in the MCSF test. Subsequently, after the MCSFtest, the animals were given access to alcohol solution and waterin the home cage for seven weeks. Finally, 3–7 days after the lastalcohol session, the animals were once again tested in the MCSFtest with the same conditions as in the first MCSF trial.

2.2.1. The multivariate concentric square fieldTM (MCSF)The MCSF test is an ethologically founded test and unlike many

other tests, it is unprejudiced in the sense that the animal is exposedto stimuli with different quality and can freely choose betweendifferent environments designed to include opportunity for explo-ration risk assessment, risk taking and shelter seeking. In this waya behavioral profile is generated. The details of the MCSF arenahave been described elsewhere [23,24] and in brief in Fig. S1. Thetest was performed in a room separate from the housing room,with the same conditions with regard to temperature, humidityand masking background noise to those in the housing room. Theanimals were tested using a running schedule to avoid disturb-ance in the home cages and to avoid order biases. All observationswere carried out during the dark period of the light/dark cycle. Thearena was divided into ten different zones, which forms the basisof the description and the variables of the animals’ performancein this test. The animals were started in the center zone facingthe wall with no openings, between the center and bridge (Fig.S1). Each animal was tested for 20 min. After each test the floorwas wiped with a cloth containing 10% alcohol solution and was

endent effects of voluntary alcohol intake on behavioral profiles016/j.bbr.2014.08.058

allowed to dry before the next animal was placed in the arena. Theapproximate light conditions (lux) were as following: dark cornerroom < 1; center approximately 20; corridors and hurdle 10–20;slope approximately 30; and bridge: 600–650.

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The behavior was recorded by a video camera placed above therena and observed from an adjacent room. The number of rearings,roomings and stretched attend postures (SAPs) from the corri-ors to the center was manually recorded by direct observation.he number of fecal boli, urinations and number of head dips intohe hole board on the hurdle was noted after each trial. A blindedbserver scored the behavior manually using the program Score 3.3Copyright Soldis, Uppsala, Sweden). Latency (LAT, s), frequencyFRQ) and duration (DUR, s) of visits to each different zone wereegistered. Ethovision version 2.3 (Noldus Information Technologync., Wageningen, The Netherlands) was used for automatic track-ng in order to obtain mean velocity (cm/s) and total distance (cm)ravelled. An operational categorization of the various parametersenerated from the MCSF with regard to function (i.e., general activ-ty, exploration, risk assessment, risk taking and shelter seeking)s used in the interpretation of results. In addition a rank-orderrocedure referred to as the trend analysis is used [26].

.2.2. Voluntary alcohol intake and preferenceThe animals were randomly assigned into water (n = 30)

nd alcohol (n = 30) groups and placed individually in cages42 cm × 26 cm × 18 cm) with wooden houses for enrichment pur-ose. The alcohol-drinking animals were introduced to alcoholsing an intermittent two-bottle free-choice paradigm, modifiedrom Wise [20] and [21], and summarized by Carnicella et al. [22].n Tuesdays, Wednesdays, and Thursdays every week, the ratsere given 24 h access to one bottle of water and one bottle of

0% alcohol (v/v). On the other days, the animals had access towo bottles of water. Bottle positions were changed for each alco-ol session to avoid position preference. Alcohol solutions wereade from 96% ethanol (Solveco Etanol A 96%; Solveco AB, Rosers-

erg, Sweden) and tap water. Water and alcohol solution at roomemperature was available in 150 ml plastic bottles with ball-valveipples (Scanbur AB, Sollentuna, Sweden) with minimal spillage.ater and alcohol intake was measured at the end of each 24 h

ccess period by weighing the bottles. Alcohol preference was cal-ulated as alcohol intake (g) divided by the total fluid intake (g). Inrder to minimize disturbance during intake measures, cages werehanged and animals were weighed on Friday afternoons.

Based on the average alcohol intake during the last week ofccess, the alcohol-drinking animals were divided into three differ-nt groups; low-, intermediate- and high-drinking animals (LD, IDnd HD, respectively; n = 10 rats/group). This classification enabled

more detailed description of drinking patterns in the three groupss well as detailed analyses of behavioral profiles prior to and afterccess to alcohol in rats with a high and low voluntary alcoholntake, respectively.

.2.3. Statistical analysesStatistica 10.0 (StatSoft Inc., Tulsa, OK) was used for all statis-

ical analyses. Differences were considered statistically significantt p ≤ 0.05. Most data were not normally distributed according tohe Shapiro–Wilk’s W test. The non-parametric Mann–Whitney U-est was used to compare descriptive MCSF parameters and intake

easures in water- and alcohol-drinking groups, and LD and HDroups. To compare behavior parameters in the first and the sec-nd MCSF trial and to compare fluid intake during week seven tohat during week one, the Wilcoxon matched pair test was used.he Spearman Rank Order Correlation was used for analysis of cor-elations. Further analysis of MCSF data was done by using the trend

Please cite this article in press as: Momeni S, Roman E. Subgroup-depin outbred Wistar rats. Behav Brain Res (2014), http://dx.doi.org/10.10

nalysis [26]. Due to the ranking in the trend analysis, the data isormally distributed and comparisons between the groups wereade using one-way ANOVA and over time comparing the first and

he second MCSF trial using repeated measurements ANOVA.

PRESSin Research xxx (2014) xxx–xxx 3

3. Results

3.1. Alcohol intake and preference using the modifiedintermittent two-bottle free-choice paradigm

The animals had free-choice intermittent access to 20% alcoholevery Tuesday, Wednesday and Thursday. The pattern of alcoholintake (g/kg) per session and per week is shown in Fig. 2A and B,respectively. The median alcohol intake increased over time witha significant difference [Z = 3.5, p < 0.001] between the first and thelast week of alcohol access (Fig. 2B). Weekly alcohol preference(%) is shown in Fig. 2C. The median alcohol preference reflectedthe alcohol intake and increased over time with a significant differ-ence [Z = 2.6, p < 0.01] between the first and the last week of alcoholaccess (Fig. 2C). The alcohol intake during session 12 displayed amarked difference compared to the other sessions, affecting intakeand preference during week four. During this time the experimentalperson was changed for practical reasons but both experimenterswere female [27].

The median (quartile range, QR) total fluid intake (g/kg) alsoincreased over time with a significant difference [Z = 2.6, p < 0.01]between the first (55.1 (9.6)) and the last (57.7 (15.3)) week of alco-hol access. The total fluid intake did not differ between the alcohol-and water-drinking animals (data not shown). No body weight dif-ference between the alcohol and water group was found (data notshown).

3.2. Behavioral profiles in water- and alcohol-drinking rats

3.2.1. Trial IThe animals were tested in the MCSF and randomly divided

into water- and alcohol-drinking groups prior to alcohol access.The descriptive parameters from the 20-min trial in the MCSF areshown in Table S1. Only minor differences between the groups werenoted. The animals assigned to alcohol access had higher durationper visit in the hurdle and made more nose pokes into the hurdlehole board compared to animals assigned to drink water. No otherdifferences were found. The results from the MCSF trend analysis ofthe functional categories general activity, exploratory activity, riskassessment, risk taking and shelter seeking revealed no significantdifferences between animals assigned to drink water and alcohol(data not shown).

3.2.2. Trial IIThe animals were repeatedly tested after the seven-week period

of alcohol access and the descriptive parameters are shown in TableS1. Besides a higher number of nose pokes into the hurdle holeboard in alcohol-drinking rats compared to water-drinking rats,no differences were found when comparing the water- and thealcohol-drinking animals. The results from the MCSF trend analysisrevealed no significant group differences (data not shown).

3.2.3. Trial I and trial II over timeComparing trial I with trial II over time, differences within the

water- and alcohol-drinking groups were found (Table S1). Differ-ences over time were similar in both groups with the exception ofrisk-taking behavior in the central circle, which was significantlylower in the alcohol-drinking group only.

3.3. Alcohol intake and preference in low-drinking,intermediate-drinking and high-drinking rats

endent effects of voluntary alcohol intake on behavioral profiles16/j.bbr.2014.08.058

The animals were split by a tertiary split into low-drinking (LD),intermediate-drinking (ID) and high-drinking (HD) groups basedon the average intake during the last week of access to alcohol.The ID animals are shown to illustrate how this group relates to

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Please cite this article in press as: Momeni S, Roman E. Subgroup-dependent effects of voluntary alcohol intake on behavioral profilesin outbred Wistar rats. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.08.058

ARTICLE IN PRESSG ModelBBR 9132 1–9

4 S. Momeni, E. Roman / Behavioural Brain Research xxx (2014) xxx–xxx

A.

B.

C.

Fig. 2. Alcohol intake (g/kg) and preference (%) patterns during the period of intermittent access to 20% alcohol and water for three consecutive days per week in all animalswith access to alcohol. (A) Alcohol intake per session during the 21 sessions. (B) Alcohol intake per week during the seven weeks of access. (C) Alcohol preference per weekduring the seven weeks of access. Data are shown as median and quartile range. **p < 0.01, ***p < 0.001 comparing week 1 and week 7 (Wilcoxon matched pair test).

Please cite this article in press as: Momeni S, Roman E. Subgroup-dependent effects of voluntary alcohol intake on behavioral profilesin outbred Wistar rats. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.08.058

ARTICLE IN PRESSG ModelBBR 9132 1–9

S. Momeni, E. Roman / Behavioural Brain Research xxx (2014) xxx–xxx 5

Fig. 3. Alcohol intake (g/kg) and preference (%) during the period of intermittent access to 20% alcohol and water for three consecutive days per week in low-drinking (LD),intermediate-drinking (ID) and high-drinking (HD) animals. The ID group is included in order to show that this is a stable intermediate group, but the ID rats were notincluded in the statistical analysis. (A) Weekly alcohol intake during the seven weeks of access. (B) The increase in alcohol intake from the first to the last week of access. (C)Alcohol preference per week during the seven weeks of access. (D) The increase in alcohol preference from the first to the last week of access. (E) Average alcohol intake onthe three consecutive days of access, i.e. Tuesdays, Wednesdays and Thursdays. Data are shown as median and quartile range. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001comparing LD and HD groups (Mann–Whitney U-test); #p < 0.05, ##p < 0.01 compared to the intake on Tuesdays within the respective LD and HD group; @p < 0.05 comparedto the intake on Wednesdays within the respective LD and HD group (Wilcoxon matched pair test).

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he LD and HD groups, respectively. However, in the statisticalnalysis focus is on the extreme groups, i.e. LD and HD animals.s shown in Fig. 3A, the weekly alcohol intake was significantlyigher in the HD animals compared to the LD animals during alleeks of alcohol access except for week four. When comparing the

lcohol intake week one to week seven, a significant increase overime was seen in the HD group only [Z = 2.7, p < 0.01] (Fig. 3B). The

edian (min-max) weekly cumulative alcohol intake (g/kg) aftereven weeks of alcohol access was 18.6 (16.1–20.4) for the LD ani-als and 28.4 (24.6–33.1) for the HD animals with a significant

ifference between the two groups [U = 00.0, Z = 3.7, p < 0.0001]. Theedian (min–max) alcohol load (g) after seven weeks of alcohol

ccess was 53.8 (41.5–76.1) and 76.9 (63.3–88.7) for the LD andD animals, respectively, with a significant difference between the

wo groups [U = 7.0, Z = 3.2, p < 0.001].The alcohol preference (Fig. 3C) followed a pattern similar to that

f the alcohol intake and the weekly preference was significantlyigher in the HD group during all weeks of alcohol access excepteek one and week four. When comparing the alcohol preferenceeek one to week seven, a significant increase over time was seen

n the HD group only [Z = 2.7, p < 0.01] (Fig. 3D).The average alcohol intake on Tuesdays, Wednesdays and Thurs-

ays is shown in Fig. 3E. The HD animals had a significantly higherlcohol intake on Tuesdays [U = 0.0, Z = 3.7, p < 0.0001], WednesdaysU = 4, Z = 3.4, p = 0.0001], and Thursdays [U = 10.0, Z = 3.0, p = 0.001]ompared to the LD animals. In the HD group, the alcohol intaken Tuesdays was significantly higher than that on Wednesdaysnd Thursdays, respectively; 30% higher on Tuesdays comparedo Wednesdays and 20% higher compared to Thursdays. The pat-ern was somewhat different in the LD group with a significantlyower intake on Wednesdays relative to Tuesdays and Thursdays,espectively.

The weekly water intake (g/kg) and total fluid intake (g/kg) ishown in Table S2. During week five the water intake was sig-ificantly lower in the HD animals compared to the LD animals.o other significant differences were found. No weight differenceetween the LD and the HD rats was found (data not shown).

.4. Behavioral profiles in low-drinking and high-drinking rats

.4.1. Trial IThe descriptive parameters from the 20-min trial in the MCSF

n LD and HD animals prior to alcohol access are shown in Table S3.nly a few differences between the groups were found. Notably,

hese differences were related to the functional category risk tak-ng. The HD animals spent significantly longer time per visit inhe center compared to the LD animals. Moreover, the HD ratsad a shorter duration, percentage duration and a trend towards ahorter duration per visit (p = 0.06) in the central circle, indicatingigher thigmotactic behavior in the HD rats relative to the LD rats.o other significant differences were found. The results from theCSF trend analysis of the functional categories general activity,

xploratory activity, risk assessment, risk taking and shelter seek-ng (Fig. 4A–E) revealed no significant differences between the LDnd the HD groups. However, when the risk-taking category wasplit into bridge- and central circle-related parameters (Fig. 4F–G)ignificantly lower risk-taking behavior in the central circle wasound in HD relative to LD rats [F = 5.6, p = 0.03] (Fig. 4G).

.4.2. Trial IIThe descriptive parameters from the second MCSF trial, after the

even-week long period of access to alcohol, are shown in Table S3.

Please cite this article in press as: Momeni S, Roman E. Subgroup-depin outbred Wistar rats. Behav Brain Res (2014), http://dx.doi.org/10.1

he HD animals had a shorter duration and percentage durationn the dark corner room (DCR, i.e. the sheltered area) compared tohe LD animals. No other significant differences were found. Inter-retations about aspects of anxiety-like behavior revealed a lower

PRESSin Research xxx (2014) xxx–xxx

anxiety-like behavior in the HD group compared to the LD group[U = 22.0, Z = 2.07, p = 0.04]. The results from the MCSF trend analy-sis of the functional categories general activity, exploratory activity,risk assessment, risk taking and shelter seeking (Fig. 4) revealedlower shelter-seeking behavior in HD relative to LD rats [F = 4.5,p = 0.05] (Fig. 4E).

3.4.3. Trial I and trial II over timeComparing trial I and trial II over time revealed differences

within the LD and HD group, respectively (Table S3). HD rats spentmore time in the corridors in the second trial and LD animals madefewer visits to and spent less time in the hurdle in the second rela-tive to the first trial. Activity in the central circle was significantlylower in LD animals in trial II than in trial I. With regard to shelterseeking, LD animals spent more time and longer time per visit inthe DCR in the second trial. Anxiety-like behavior increased in theLD animals only in trial II relative to trial I. The results from theMCSF trend analysis of the functional categories general activity,exploratory activity, risk assessment, risk taking and shelter seek-ing (Fig. 4A–G) revealed no significant differences over time withineither the LD or the HD group.

4. Discussion

When subgrouping animals based on the alcohol intake dur-ing the last week of alcohol access using the modified intermittentaccess paradigm, the HD group not only had a higher alcohol intakeand preference compared to the LD group, but also had an increasedalcohol intake over time, which was not seen in the LD group. More-over, the HD group displayed a higher intake on Tuesdays relativeto Wednesdays and Thursdays, indicating on an alcohol depriva-tion effect, which was not found in the LD group. With regard tobehavior, the MCSF is used as a complementary methodologicalpossibility to understand mechanisms underlying a broader behav-ioral repertoire rather than focusing on any particular behavior.Minor overall differences in alcohol-induced effects on behavioralprofiles were found when comparing water- and alcohol-drinkinganimals. However, when focusing on the most extreme subgroupsamong the alcohol-drinking animals, the HD animals were char-acterized by lower risk-taking behavior prior to alcohol access andlower anxiety-like behavior after the period of alcohol consumptionrelative to LD rats.

4.1. Alcohol intake using three consecutive days of intermittentaccess

One aspect of human drinking patterns is high alcohol intake onconsecutive days, especially on weekends [28]. The modified modelused in this study, with alcohol access during three consecutivedays followed by a deprivation period for four days was introducedto mimic human weekend consumption. Using the modified inter-mittent access paradigm with three consecutive days of accessto 20% alcohol for seven weeks, the median alcohol intake dur-ing the last week of access in all alcohol-drinking animals was3.2 g/kg (1.8–5.8 g/kg) and the preference was 36.4% (16.0–46.2%).This is in agreement with previous studies using intermittent accessparadigms in which alcohol consumption up to 6 g/kg has beendescribed [22,29–32] and higher compared to studies in whichWistar rats were given continuous alcohol access [20,31,33]. More-over, compared to our recent study [16] in which the animals hadintermittent access to 20% alcohol on Mondays, Wednesdays andFridays the modified model used herein resulted in a higher alcohol

endent effects of voluntary alcohol intake on behavioral profiles016/j.bbr.2014.08.058

intake and a higher preference as well as an increased intake andpreference over time. One likely explanation for the higher intakeand preference found is the choice of Wistar sub-strain used in thisstudy [16,30].

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Please cite this article in press as: Momeni S, Roman E. Subgroup-dependent effects of voluntary alcohol intake on behavioral profilesin outbred Wistar rats. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.08.058

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Fig. 4. The MCSF trend analysis in low-drinking (LD) and high-drinking (HD) animals prior to (trial I) and after (trial II) access to alcohol. Sum rank values for parametersincluded in the functional categories general activity (A), exploratory activity (B), risk-assessment (C), risk-taking (D) and shelter-seeking (E) behavior, and risk-takingbehavior related to the bridge (F) and risk-taking behavior related to the central circle (CTRCI) (G). Values represent mean ± S.E.M. *p < 0.05 comparing LD and HD animals(one-way ANOVA).

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A large individual variation in alcohol intake was found amongll alcohol-drinking animals, which enabled subgrouping of ani-als with high, intermediate and low alcohol intake. This finding

s in line with previous studies [22,32]. Since one part of this studys devoted to the introduction of and the characterization of alco-ol intake using the modified intermittent access paradigm the IDroup is shown in the figures despite being excluded in the sta-istical analysis, since the behavioral analysis was focused on the

ost extreme subgroups. However, this selection shows that theubgroups have a stable alcohol intake throughout the period ofccess to alcohol with preference stabilizing after the forth weekf access. The HD animals had significantly higher intake and pref-rence compared to the LD animals, and also significantly higherntake over time compared to the LD group in which the alco-ol intake was similar throughout the period of alcohol access. Ashown herein, not only the alcohol intake differed between theseroups, but also the drinking pattern. The HD animals had a sig-ificantly higher intake on Tuesdays, after the four day alcoholeprivation, compared to Wednesdays and Thursdays, which wasot seen in the LD group. This enhanced intake indicates that theD animals were more sensitive to the four days of interruption

o alcohol access, i.e. in the HD animals, this modified intermit-ent paradigm gave rise to an alcohol deprivation effect [34,35],hich is typically not seen when using intermittent access models

22] and to our knowledge only has been reported in one previoustudy [36]. The alcohol deprivation effect found herein was not asronounced as that described for some Wistar rats when deprivedrom alcohol after about two months of continuous access under

4-bottle free-choice paradigm [35]. However, this alcohol accessaradigm, with pronounced subgroup-dependent effects, includinglcohol deprivation effects, has clinical validity and may be usefuln future investigations of molecular, neuronal, neurochemical, orehavioral adaptations related to excessive alcohol intake as both

ndividuals that escalate their intake over time (HD animals) andow-drinking individuals (LD animals) without escalation are to beistinguished [22].

.2. Behavior in water- and alcohol-drinking rats

Overall, the predictive value of the behavior prior to alcoholccess on later alcohol intake was weak when comparing water-nd alcohol-drinking animals, which is in line with previous stud-es using the same behavior test to predict voluntary alcohol intaken adolescent animals [19]. We have previously shown that whennimals were split into groups based on risk-assessment behaviorn the MCSF prior to alcohol access, the high risk-assessing ratsad a higher alcohol intake when later given access to alcoholompared to the low-risk-assessing rats [16]. Herein, no asso-iation between risk-assessment behavior and voluntary alcoholntake was found, which may relate to the different study designsed.

The lack of differences in MCSF performance between water-nd alcohol-drinking rats following a period of voluntary alcoholntake is in line with previous studies in which other behav-oral tests were used [37,38] and also following after a periodf abstinence[38]. However, previous studies have revealed con-rasting results with both increased activity [17] and decreasedctivity [18] on the open arms of the elevated plus maze followingoluntary alcohol intake relative to water-drinking controls. Sinceo group-housed water-drinking control group was included the

Please cite this article in press as: Momeni S, Roman E. Subgroup-depin outbred Wistar rats. Behav Brain Res (2014), http://dx.doi.org/10.1

ffects of single housing masking potential alcohol-induced effectsannot be ruled out. It should also be emphasized that this studyesign represents non-dependent rats, which to a large extent mayxplain differences as compared to other findings in Ref. [22].

PRESSin Research xxx (2014) xxx–xxx

4.3. Behavior in low-drinking and high-drinking animals

Despite a lack of differences in MCSF performance betweenwater- and alcohol-drinking rats after the period with access toalcohol, differences within the alcohol-drinking animals emergedwhen studying the subgroups of LD and HD animals in more detail.The HD animals had a shorter duration in the central circle prior toalcohol access compared to the LD animals. As previous results haverevealed differences in risk-taking behavior in the central circleversus on the bridge [26,39], the functional category risk taking wasalso divided into performance on the bridge and in the central circle,respectively. The HD animals showed a lower risk-taking behaviorin central circle-related parameters compared to the LD animals.When repeatedly tested after the period with intermittent accessto alcohol, the HD animals spent shorter time in the DCR, had loweranxiety-like behavior as indicated by the higher risk/shelter indexand lower shelter-seeking behavior in the trend analysis, which alltogether is interpreted as lower anxiety-like behavior compared tothe LD rats. In addition, anxiety-like behavior increased over timein the LD animals only.

Considering the differences between LD and HD rats prior toalcohol access, an evident profile predisposing HD rats cannotbe found in the MCSF trend analysis. However, when taking thefindings together, the fact that the HD rats were characterizedby lower risk-taking behavior prior to alcohol access and loweranxiety-like behavior after alcohol consumption, compared to theLD rats, may indicate that the HD animals consumed alcoholfor its anxiolytic properties likely due to alcohol-induced neu-roadaptive changes. This finding is in line with previous studiesshowing the association of anxiety-like behavior and high alco-hol consumption (e.g. [10,12]) but contrasts our recent findingof risk-assessment behavior being associated with high volun-tary alcohol intake [16] indicating on the complexity in assessingbehavioral predispositions to alcohol intake as well as alcohol-induced effects on behavior. However, these results demonstratethat even if differences cannot be seen between the alcohol- andwater-drinking groups, there are individual differences among thealcohol-drinking animals that become evident when studying sub-groups of animals in more detail.

5. Conclusions

In this study, the effects of voluntary alcohol intake on behav-ior in non-dependent outbred Wistar rats were investigated. Largeindividual differences in alcohol intake were demonstrated whenusing the modified intermittent access paradigm. This enableddetailed analyses of subgroups of low and high alcohol-drinkingrats, respectively. An alcohol deprivation effect was observed inHD animals only, which may be of importance in further phar-macological studies. Moreover, the HD animals were interpretedas having lower anxiety-like behavior after alcohol intake. Thus,this subgroup of animals may consume alcohol for its anxiolyticproperties. Taken together, the present study adds to the complexliterature on interactions between voluntary alcohol intake andbehavioral traits but also highlights the importance of studyingsubgroup-dependent differences in alcohol intake and behav-ior.

Author contributions

endent effects of voluntary alcohol intake on behavioral profiles016/j.bbr.2014.08.058

Conceived and designed the experiment: ER; Performed theexperiment: SM; Analyzed the data: SM; Interpreted the resultsand wrote the manuscript: SM, ER.

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cknowledgements

The authors would like to thank Dr. Lena Bergström for valu-ble input on the experimental design and for comments on theanuscript, and Ms. Marita Berg for technical assistance. Financial

upport from the Alcohol Research Council of the Swedish Alcoholetailing Monopoly, and the Magnus Bergvall, Åke Wiberg, Fredrik

Ingrid Thuring (ER) and Facias (ER, SM) Foundations is gratefullycknowledged.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at http://dx.doi.org/10.1016/j.bbr.2014.08.058.

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