Genes, Brain and Behavior (2008) 7: 705–713 # 2008 The AuthorsJournal compilation # 2008 Blackwell Publishing Ltd/International Behavioural and Neural Genetics Society

Rats bred for high alcohol drinking are more sensitiveto delayed and probabilistic outcomes

C. J. Wilhelm*,† and S. H. Mitchell†,‡,§

†Department of Behavioral Neuroscience, and ‡Department of

Psychiatry, Oregon Health & Science University, Portland, OR,

and §Portland Alcohol Research Center, Portland, OR, USA

*Corresponding author: C. J. Wilhelm, PhD, Department of

Behavioral Neuroscience, L470 Oregon Health & Science Univer-

sity, 3181 SWSam Jackson Park Road, Portland, OR 97239, USA.

E-mail: wilhelmc@ohsu.edu

Alcoholics and heavy drinkers score higher on measures

of impulsivity than nonalcoholics and light drinkers. This

may be because of factors that predate drug exposure

(e.g. genetics). This study examined the role of genetics by

comparing impulsivity measures in ethanol-naive rats

selectively bred based on their high [high alcohol drinking

(HAD)] or low [low alcohol drinking (LAD)] consumption of

ethanol. Replicates 1 and 2 of the HAD and LAD rats,

developed by the University of Indiana Alcohol Research

Center, completed two different discounting tasks. Delay

discounting examines sensitivity to rewards that are

delayed in time and is commonly used to assess ‘choice’

impulsivity. Probability discounting examines sensitivity

to the uncertain delivery of rewards and has been used to

assess risk taking and risk assessment. High alcohol

drinking rats discounted delayed and probabilistic re-

wards more steeply than LAD rats. Discount rates associ-

ated with probabilistic and delayed rewards were weakly

correlated, while bias was strongly correlated with dis-

count rate in both delay and probability discounting. The

results suggest that selective breeding for high alcohol

consumption selects for animals that aremore sensitive to

delayed and probabilistic outcomes. Sensitivity to delayed

or probabilistic outcomes may be predictive of future

drinking in genetically predisposed individuals.

Keywords: Alcohol, delay discounting, genetics, impulsivity,probability discounting, rats, selected lines

Received 15 January 2008, revised 1 April 2008, accepted forpublication 8 April 2008

Drug abusers (including alcohol) exhibit heightened impulsiv-ity. Delay discounting procedures assess impulsivity by

offering subjects a choice between an immediate rewardand a larger reward available after a delay (e.g. Logue 1988;

Rachlin & Green 1972). Alcoholics and heavy drinkers prefer

small, immediate monetary rewards over larger but delayed

rewards comparedwith light drinkers (Petry 2001; Vuchinich &

Simpson 1998), suggesting that alcoholics and heavy drinkers

discount the hypothetical ‘value’ of the delayed reward more

than light drinkers. Heightened impulsivity may predict sub-

sequent alcohol abuse or may be the result of short-term and/

or long-term neurobiological adaptations to drinking. Or,

heightened impulsivity could predate drinking and become

further exacerbated by long-term alcohol use. Alcoholism

(e.g. Goodwin et al. 1974) and impulsivity have genetic

components (Anderson & Woolverton 2005; Isles et al.

2004), so the association of heightened impulsivity with

alcohol abuse suggests that the genes involved in alcohol

abuse and impulse control may overlap.To more closely model aspects of human drinking behav-

iors, numerous animal models have been selectively bred to

consume relatively high or low amounts of alcohol (for review,

see McBride & Li 1998). We chose to use the high-alcohol-

drinking and low-alcohol-drinking (HAD and LAD, respec-

tively) rat lines developed by the Indiana University Alcohol

Research Center because of their diverse genetic background

and the availability of two independent replicates. Steinmetz

et al. (2000) reported no differences between these lines on

a motor impulsivity (behavioral inhibition) task, but delay

discounting has yet to be examined.

Various factors can affect reinforcer value, including delay,effort and uncertainty costs associated with reinforcer deliv-

ery. Delay and probability discounting are positively correlated

(Richards et al. 1999) and can both be modeled with a similar

hyperbolic equation (Rachlin et al. 1991; Richards et al. 1999).

Probability discounting offers subjects a choice between

a certain reward and an uncertain reward and assesses each

subject’s sensitivity to probabilistic reinforcement. Little data

are available on probability discounting, and the available

studies are mixed (Mitchell 1999; Ohmura et al. 2005;

Reynolds et al. 2004; Yi et al. 2007). Delay and probability

discounting appear to share some underlying neural mecha-

nisms but are also dissociable (Acheson et al. 2006; Estle

et al. 2006). Therefore, we examined both delay and proba-

bility discounting.Isles et al. (2004) found that mice with higher levels of

spontaneous locomotor activity also preferred small imme-

diate rewards. Perry et al. (2005) found no such correlation in

rats. No studies have examined the relationship between

locomotor activity and probability discounting. We com-

pared locomotor activity between strains and correlated this

with performance on the delay and probability discounting

tasks to determine if there are species differences in task

performance. In addition, we compared sucrose drinking

between these rat lines to determine if there were

Portions of the abstract of this manuscript have been pre-viously published in the 2007 and 2008 annual meetings of

the Research Society on Alcoholism Supplement.

doi: 10.1111/j.1601-183X.2008.00406.x 705

differences and if sucrose consumption correlated withaspects of task performance.

The goal of this study was to determine if heighteneddiscounting of delayed or probabilistic rewards is selected

in concert with level of alcohol drinking in a rat model.Understanding the relationship among genetics, impulsivity

and alcohol consumption will enhance our knowledge ofthe behavioral pathways by which genes impact alcohol

use.

Materials and methods

Subjects

Inbred lines of HAD1 (n ¼ 6), HAD2 (n ¼ 6), LAD1 (n ¼ 6) and LAD2(n ¼ 6) male rats were generously supplied by the Indiana UniversityAlcohol Research Center. These lines of animals were chosenbecause they were selected from the N/National Institutes of Health(NIH) heterogeneous stock (which provides the advantage of a diversepopulation of starting genes) and because there are two independentreplicates available, which greatly decreases the probability thata gene unrelated to alcohol consumption becomes fixed in bothreplicates. Upon receipt, HAD1 animals weighed 307 � 10 g, LAD1animals weighed 347 � 6 g, HAD2 animals weighed 292 � 10 g andLAD2 animals weighed 263 � 9 g. Rats were housed in the Depart-ment of Comparative Medicine at Oregon Health & Science Univer-sity, an AAALAC-approved facility. All procedures were approved bythe appropriate Institutional Animal Care and Use Committee andadhered to NIH Guidelines. To facilitate training and maintainresponding in behavioral tasks, 3 days prior to the start of training,animals were food deprived to approximately 90% of their free-feeding body weights. Unless otherwise noted, animals were main-tained at 90% of their free-feeding age-adjusted body weights withsupplemental chow given immediately following each day’s testsession.

Apparatus

We used eight identical (Med Associates Inc., St Albans, VT, USA)modular rat test chambers housed individually within melaminesound-attenuating cabinets. Chambers had acrylic front and backpanels and stainless steel side panels. A fan provided constantventilation and low-level background noise. A house light wasmounted in the center of one of the stainless steel panels, anda response clicker was also mounted on the outside of this panel.Three nonretractable levers were mounted on the opposing paneldirectly below circular lights and above recessed nose pokes. Thus,there were left, right and center lights, levers and nose pokes.Computer-controlled pumps were used to deliver sucrose reinforcers(10% w/v) to liquid cups located in the recesses of the outer nosepokes.

Order of experiments

High alcohol drinking 1 and LAD1 rats completed the delay discount-ing task, followed by the probability discounting task and sucrosedrinking. High alcohol drinking 2 and LAD2 rats completed theprobability discounting task first, and then delay discounting, sucrosedrinking and locomotor activity. The discounting tasks were counter-balanced to offset potential order effects.

Training

Training began after 3 days of food restriction and was divided intothree phases (for detailed description, see Appendix S1). Phase 1 of

training exposed subjects to a progressively delayed noncontingentreward to acquaint the animals with the location of the reinforcer anda response-contingent reinforcer to train the animals to press leversand earn a sucrose reward. In phase 2 of training, animals weretrained to press the middle lever to activate the outer ‘choice’ levers.The purpose of this was to eliminate position bias by forcing theanimals to move near the middle of the chamber. Once activated, anouter lever press resulted in sucrose reward delivery and deactivationof both levers until a new trial began (5 seconds inter-trial interval(ITI)), and themiddle lever was pressed. Animals were also introducedto forced-choice trials, whereby animals that chose either the right orthe left lever on two consecutive trials were forced to press theopposite lever on the subsequent trial. Phase 3 of training incorpo-rated time contingencies, requiring animals to successfully completea middle lever press and a left or right lever press within 24 secondsof the start of a trial. Each trial was 40 seconds long. Illumination ofthe middle light signaled the start of a new trial. A subsequent middlelever press extinguished the middle light and illuminated the right andleft lights, indicating that both levers were active. One of the outerlevers was assigned to be the immediate/certain lever, while theother was the delayed or probabilistic lever (during training, no delaysgreater than 0 seconds or probabilities less than 1.0 were experi-enced in training). Delayed/probabilistic levers were counterbalancedbetween subjects and remained the same throughout both delay andprobability discounting experiments. The size of the immediate/certain reward (initially 75 ml) adjusted throughout the experimentalsession; choice of the delayed/probabilistic lever increased the size ofthe immediate/certain reward by 10% and choice of the immediate/certain lever decreased the size of the immediate/certain reward by10% (forced-choice trials did not affect the size of the immediatereward). Each sucrose reinforcer was delivered to the right or the leftnose pokes in combination with an auditory stimulus (responseclicker). Sessions lasted until 60 free-choice trials occurred or60 min had elapsed. The majority of sessions were completed within50 min. To complete training, rats were required to respond on atleast 55 of the 60 possible free-choice trials on two consecutivesessions. One LAD2 rat was unable to complete the training require-ments and was therefore not included in the results.

Delay discounting task

The adjusting amount procedure was adapted from a proceduredescribed in Richards et al. (1997). Experimental sessions were asdescribed in phase 3 of training, except that a response on thedelayed lever resulted in delivery of a 150-ml sucrose reinforcer,delayed by 0, 2, 4, 8 or 16 seconds. The delay remained constantwithin a session but varied between sessions according to a Latinsquare design. Animals experienced each delay a minimum of sixtimes, but only data from occasions 2–6 were included in theanalyses. The purpose of the experiment was to assess the subjectivevalue of the delayed sucrose by titrating the volume of the immediatereward until animals became indifferent between the two choices.

Probability discounting task

Experimental sessionswere carried out exactly as described in phase 3of training, except that a response on the probabilistic lever resulted inthe delivery of a 150-ml sucrose reinforcer with probabilities of 1.00,0.75, 0.50, 0.25 or 0.125. Probability remained constant withina session but varied between sessions according to a Latin squaredesign. Animals experienced each probability a minimum of six times,but only data from occasions 2–6 were included in the analyses. Thepurpose of the experiment was to assess the subjective value of theprobabilistic sucrose by titrating the size of the certain reward untilanimals became indifferent between the two choices.

Two-bottle sucrose preference

The purpose of this experiment was to determine if HAD and LAD ratsdiffered in consumption or preference when offered a choice

706 Genes, Brain and Behavior (2008) 7: 705–713

Wilhelm and Mitchell

between a sucrose solution and water. Differences in sucroseconsumption or preference might be critical to the interpretation ofthe discounting data. Replicates 1 and 2 were tested 4 days per weekwith each concentration of sucrose in the following sequence: 0, 0.1,0.5, 1.0, 2.5, 5, 10, 20, 30 and 0%w/v sucrose. On test days, each ratwas placed in a rat-drinking cage for 50 min (the approximate length ofa discounting session) and given access to two bottles, one contain-ing a sucrose solution and the other containing water. To eliminate thepotential for side bias, placement of sucrose and water bottles wasalternated daily. The amount of solution consumed was determinedby weighing the bottles before and after each test session. Theaverage amount of sucrose consumed per 50-min test session wascalculated in grams of sucrose per kilogram body weight. In addition,sucrose preference was calculated as the ratio of the amount ofsucrose consumed to the total amount of fluid (water plus sucrose)consumed. Again, preference was averaged over each of the testdays. To maintain relevance to discounting procedures, animals werefood deprived as described for the discounting procedure.

Locomotor activity

Locomotor activity was assessed in the HAD2 and the LAD2 animals.Timing of the decision to collect activity data meant that the firstreplicate of animals was unavailable to complete the task. Food wasavailable ad libitum during this phase of the experiment. Four ACCUSCAN(Accuscan Instruments Inc., Columbus, OH, USA) automated activitymonitors were used for this study. Chambers consisted of a 40 � 40 �30 cm clear acrylic test cage placed inside a monitoring unit thatrecorded photocell beam breaks, which were translated into distancetraveled (in cm). Eight or 16 evenly spaced photocells and receptorswere located 2 cm above the chamber floor. Monitors and test cageswere housed in black acrylic chambers that were lined with foam toattenuate external noise. A fluorescent light and fan located within thetest chamber were on during testing to provide illumination, ventila-tion and low-level background noise. Each rat was assigned toa locomotor activity chamber. Locomotor activity was recorded for30 min on five consecutive days and converted to horizontal distancetraveled.

Data analysis

Analyses of delay and probability discounting data were similar tothose described in Mitchell et al. (2006) andWilhelm et al. (2007). Themain dependent variable was the amount of sucrose solution deliv-ered from the immediate/certain, adjusting alternative at the ‘indiffer-ence point’, i.e. the amount at which the immediate/certain anddelayed/probabilistic alternatives were selected equally often. Basedon previous findings (Richards et al. 1997), animals reach theindifference point after the first 30 trials of a session; thus, themedian volume of sucrose associated with the adjusting lever overtrials 31–60 is an index of the subjective value of the alternate choice.Table 1 shows that animals chose each lever with roughly equalfrequency over this period. The median, rather than the mean, wasused as a measure of central tendency because changes in theadjusting amount on successive trials were proportions of the amounton the prior trial, resulting in a skewed distribution. Data fromsessions where fewer than 40 trials were completed were excludedfrom analysis (percent of delay discounting sessions with fewer than40 responses: HAD1 ¼ 2%, LAD1 ¼ 0%, HAD2 ¼ 0% and LAD2 ¼3% of delay discounting sessions; percent of probability discountingsessions with fewer than 40 responses: HAD1 ¼ 0%, LAD1 ¼ 1%,HAD2 ¼ 3% and LAD2 ¼ 14% of probability discounting sessions).Hyperbolic equations were fitted to each animal’s average indifferencepoints (modified fromMazur 1987) using GRAPHPAD PRISM 3.0 (GraphpadSoftware Incorporated, San Diego, CA, USA):

V ¼ b�A=1 þ k�X ð1Þwhere V represents the value of the adjusting reward at indiffer-ence in ml; A represents the amount of sucrose solution from thedelayed or probabilistic alternative (150 ml); X is either the delay toreceiving the reinforcer (0, 2, 4, 8, or 16 seconds) or the odds

against receiving the reinforcer [(1 � probability)/probability: 0,0.33, 1, 3, 7] and the bias parameter, b, is a fitted parameterindicative of bias or side preference in the absence of delay(0 second delay condition) or uncertainty (odds against ¼ 0). Thediscount parameter (k) is a fitted parameter and indexes the rateof discounting or overall sensitivity to delayed or probabilisticrewards. Larger values of k indicate steeper discount functionsand stronger aversion to delayed or probabilistic rewards. Mixedmodel analysis of variances (ANOVAs) were used to examineindifference points, with LINE and REPLICATE as between-subjects factors and DELAY or ODDS AGAINST as within-subjects factors. Similar ANOVAs with the additional inclusion ofCHOICE as a within-subjects factor were used to examinereaction times and choice reaction times. Two-way ANOVAs wereused to examine k-values and b-values, with LINE and REPLI-CATE as between-subjects factors.The main dependent variables in the two-bottle choice study were thegrams of sucrose consumed per kilogram of body weight and thepreference ratio (ml of sucrose consumed/total ml consumed). Amixed model ANOVA with concentration of sucrose (CONCENTRA-TION) as a within-subjects factor and LINE and REPLICATE asbetween-subjects factors was used for analysis. For locomotoractivity, the main dependent variables were the horizontal distancetraveled on days 1 through 5 and the difference in horizontal distancetraveled from day 1 to day 5 (habituation). A mixed model ANOVA withDAY as a within-subjects factor and LINE as a between-subjectsfactor was used for analysis.

ANOVA and other statistical tests were carried out using SPSS version15.0 (SPSS Inc., Chicago, IL, USA). Huynh–Feldt corrections wereapplied as necessary, and adjusted degrees of freedom are provided.

Results

Delay discounting performance

The adjusting amount procedure determined the discountedvalue of a reinforcer given after a delay by adjusting the size of

an immediate alternative, until subjects became indifferentbetween the two options. Indifference points decreased

as the delay to reward increased (DELAY: F3.7,69.5 ¼ 140.9,

P < 0.001) (Fig. 1). High alcohol drinking rats were moresensitive to delayed rewards than LAD rats (DELAY � LINE:

F3.7,69.5 ¼ 12.4, P < 0.001), an effect that was consistent inboth replicates (REPLICATE: F1,19 < 1). Therewas also amain

effect of line (LINE: F1,19 ¼ 5.2, P ¼ 0.03). Follow-up ANOVAswith line and replicate as factors were carried out at each

Table 1: Percent choice during the final 30 choice trials

Delay (% Choice) Probability (% Choice)

Immediate

(%)

Delayed

(%)

Certain

(%)

Uncertain

(%)

HAD1 44.9 � 1.4 55.1 � 1.4 49.2 � 1.6 50.8 � 1.6

HAD2 48.0 � 1.4 52.0 � 1.4 47.1 � 1.1 52.9 � 1.1

LAD1 45.8 � 1.0 54.2 � 1.0 47.5 � 1.3 52.5 � 1.3

LAD2 47.6 � 1.0 52.4 � 1.0 46.4 � 1.4 53.6 � 1.4

Data shown are the mean � SEM per cent choice of the noted lever

during the final 30 choice trials of delay discounting and probability

discounting sessions. This shows that the animals chose each lever

with roughly the same frequency over this period, i.e. they had

reached an indifference point. HAD1 n ¼ 6, HAD2 n ¼ 6, LAD1

n ¼ 6 and LAD2 n ¼ 5.

Genes, Brain and Behavior (2008) 7: 705–713 707

Discounting by HAD and LAD rats

delay and suggest the main effect of line was driven by

differences at the 0 second delay (LINE: F1,19 ¼ 20.8,P < 0.001) but not at any of the other delays (LINE other

delays: all F 0s1,19 < 1).The hyperbolic discount function (Eqn 1) was fitted to the

data for each individual rat, resulting in the generation of a k-

value (discount rate) and b-value (bias) for each animal. Highalcohol drinking rats had higher k-values than LAD rats (LINE:

F1,19 ¼ 15.1, P ¼ 0.001) (Table 2), indicating that HAD ani-mals discount the value of delayed rewards more rapidly than

LAD animals. This effect was consistent in both replicates(REPLICATE: F1,19 < 1.0). For b-values, HAD1 and HAD2

animals were biased toward the delayed lever (b > 1.0),while LAD2 animals were biased toward the immediate lever

(b < 1.0) and LAD1 animals were relatively unbiased (b ¼ 1)(LINE: F1,19 ¼ 25.5, P < 0.001) (Table 2). Again, there was no

effect of replicate (REPLICATE: F1,19 < 1.0).There were no line differences in reaction time (the time

from trial start to middle lever press) (LINE: F1,19 ¼ 3.6,P ¼ 0.074) (Figure S1), choice reaction time (the time from

middle lever press to choice of either of the two outer levers)(LINE: F1,19 ¼ 1.8, P ¼ 0.311) (Figure S2) or on the number of

trials completed each session (LINE: F1,19 ¼ 1.0, P ¼ 0.708).High alcohol drinking 1 and LAD1 animals had faster reactions

times than HAD2 and LAD2 animals (REPLICATE:F1,19 ¼ 8.8, P ¼ 0.008). Reaction times were slower on trials

when the delayed alternative was chosen (CHOICE:F1,19 ¼ 7.9, P ¼ 0.011) and as the delay to reward increased

(DELAY: F4,76 ¼ 35.6, P < 0.001). Likewise, choice reactiontimes were slower on trials when the delayed alternative was

chosen (CHOICE: F1,19 ¼ 10.0, P ¼ 0.005) and as the delayto reward increased (DELAY: F3.0,56.7 ¼ 14.1, P < 0.001).

Choice reaction times were similar in both replicates (REPLI-CATE: F1,19 < 1). The number of trials completed also

decreased as the delay to reward increased (DELAY:F3.0,57.7 ¼ 22.9, P < 0.001), and HAD1 and LAD1 animals

completed more trials than the HAD2 and LAD2 animals(REPLICATE: F1,19 ¼ 12.0, P < 0.001). There were other

significant interactions; however, these were not systematicand are not discussed further.

Probability discounting performance

The adjusting amount procedure was used to assess the

discounted value of rewards given with varying probabilities(Fig. 2). Analysis of probability discounting is complicated

because choice of the uncertain lever leads to probabilistic

reward delivery. Therefore, experienced probability oftendiffers from the target probability. The average experienced

probabilities for the 0.125 probability sessions ranged from0.083 to 0.213. The averaged experienced probabilities by

line for the 0.125 condition were HAD1 ¼ 0.145 � 0.012,LAD1 ¼ 0.129 � 0.012, HAD2 ¼ 0.135 � 0.013 and LAD2 ¼0.147 � 0.020. One sample t-tests showed that experiencedprobabilities in all conditions were not different from pro-

grammed probabilities (all P 0s > 0.05). Therefore, to facilitatecomparisons, further analyses use the programmed probabili-

ties to calculate the odds against.Indifference points decreased as the odds against obtain-

ing a reward increased (ODDS AGAINST: F3.9,74.3 ¼ 46.1,P < 0.001). High alcohol drinking animals were more sensi-

tive to odds against than LAD animals (LINE � ODDSAGAINST: F3.9,74.3 ¼ 3.3, P ¼ 0.015). Post hoc analyses indi-

cate that line differences were most robust at the level of fullcertainty (odds against ¼ 0, LINE: F1,19 ¼ 4.25, P ¼ 0.053, all

other odds against, P 0s > 0.09). There was no effect of line(LINE: F1,19 ¼ 1.2, P ¼ 0.29) or replicate (REPLICATE:

F1,19 ¼ 1.2, P ¼ 0.29). As with the delay discounting task,the experimentally derived indifference points were fitted to

the hyperbolic discount function (Eqn 1). The resulting k-values were skewed (1.41); therefore, a natural logarithm

transformation was applied to reduce skewness anddecrease the influence of outliers (resulting skewness

¼ 0.12). High alcohol drinking rats had larger k-values thanLAD rats (LINE: F1,19 ¼ 5.54, P ¼ 0.03) (Table 2), with no

difference between replicates (REPLICATE: F1,19 < 1). Thelarge standard errors of the HAD2 and LAD2 animals were the

result of single outliers, which when excluded yield average k-values of HAD2 k ¼ 0.35 � 0.07 and LAD2 k ¼ 0.14 � 0.04.

Thus, HAD animals appear more sensitive to probabilistic

Figure 1: Mean 6 SEM indifference point for each line at

each delay tested (0, 2, 4, 8 and 16 seconds). Data for replicate

1 are shown in the top (a) graph and for replicate 2 in the bottom

(b) graph. A significant LINE � DELAY interaction suggests that

HAD animals were more sensitive to delayed rewards than LAD

animals. HAD1 n ¼ 6, HAD2 n ¼ 6, LAD1 n ¼ 6 and LAD2 n ¼ 5.

708 Genes, Brain and Behavior (2008) 7: 705–713

Wilhelm and Mitchell

outcomes. Similar to the results from the delay discounting

study, the HAD line was biased toward the uncertain lever,while LAD animals were unbiased or biased toward the

certain lever (LINE: F1,19 ¼ 5.17, P ¼ 0.04) (Table 2). Therewas no difference between replicates (REPLICATE:

F1,19 ¼ 1.38, P ¼ 0.26).There were no line differences in reaction time (the time

from trial start to middle lever press) (LINE: F1,19 ¼ 2.3,P ¼ 0.142) (Figure S3), choice reaction time (the time from

middle lever press to choice of either of the two outer levers)(LINE: F1,19 ¼ 3.7, P ¼ 0.071) (Figure S4) or on the average

number of trials completed per session (LINE: F1,19 ¼ 2.5,P ¼ 0.130). Reaction times were slower as the odds against

rece iv ing a reward increased (ODDS AGAINST:F4.0,75.1 ¼ 48.9, P < 0.001), and HAD1 and LAD1 rats had

faster reaction times than HAD2 and LAD2 rats (REPLICATE:F1,19 ¼ 85.8, P < 0.001). Choice reaction times were also

slower when the uncertain reward was chosen (CHOICE:F1,19 ¼ 16.5, P ¼ 0.001) and as the odds against receiving a

reward increased (ODDS AGAINST: F4,76 ¼ 3.8, P ¼ 0.007).The number of trials completed decreased as the odds

against receiving a reward increased (ODDS AGAINST:F3.6,69.0 ¼ 56.5, P < 0.001). High alcohol drinking 1and

LAD1 rats completed more trials than HAD2 and LAD2 rats

(REPLICATE: F1,19 ¼ 25.4, P < 0.001). There were othersignificant interactions; however, these were not systematic

and are not discussed further.

Two-bottle choice

Animals consumed more of the sucrose solution as sucrose

concentration increased (CONCENTRATION: F2.3,46.7 ¼340.2, P < 0.001) (Fig. 3a). There were also line differences

in consumption of sucrose (LINE: F1,20 ¼ 8.7, P ¼ 0.008). Tofurther examine this effect, post hoc ANOVAs were conducted

independently for each replicate. For HAD1 and LAD1 ani-mals, LAD1 animals drank more sucrose than HAD1 animals

(LINE: F1,10 ¼ 25.9, P < 0.001), and as the concentration ofsucrose increased, so did the amount of sucrose solution

consumed (CONCENTRATION: F2.7,26.6 ¼ 207.5, P < 0.001).The observed difference in sucrose consumption was not be-

cause of weight differences (average weights HAD1 ¼ 306� 14 g, LAD1 ¼ 321 � 9 g, t-test P ¼ 0.38). For HAD2 and

LAD2 animals, sucrose consumption increased with concen-tration (CONCENTRATION: F2.0,20.1 ¼ 149.2, P < 0.001);

however, consumption did not differ between lines (LINE:

F1,10 < 1, P ¼ 0.88). Thus, sucrose consumption differencesbetween HAD1 and LAD1 animals were responsible for the

observed line difference.

Table 2: HAD rats are more sensitive to delayed or probabilistic outcomes

Delay discounting Probability discounting

b k b k

HAD1 1.28 � 0.04*** 0.53 � 0.07** 1.14 � 0.10* 0.76 � 0.19*

HAD2 1.36 � 0.09*** 0.40 � 0.07** 1.12 � 0.09* 0.53 � 0.19*

LAD1 0.99 � 0.09 0.20 � 0.06 1.00 � 0.12 0.25 � 0.04

LAD2 0.80 � 0.11 0.25 � 0.04 0.76 � 0.14 0.44 � 0.30

Data shown are the mean � SEM for bias (b) and discount parameter (k) values as derived from the hyperbolic discount function (Eqn 1). Higher

values of k indicate greater sensitivity to delayed or probabilistic outcomes. Bias values above one indicate a preference for the cost (uncertain or

delayed) lever and bias values below 1.0 indicate a preference for the certain or immediate lever. HAD1 n ¼ 6, HAD2 n ¼ 6, LAD1 n ¼ 6 and

LAD2 n ¼ 5. Significant line differences are denoted as follows, *P < 0.05, **P ¼ 0.001, ***P < 0.001.

Figure 2: Mean 6 SEM indifference point for each line at

each odds against (0, 0.33, 1, 3 and 7). Data for replicate 1 are

shown in the top (a) graph and for replicate 2 in the bottom (b)

graph. A significant LINE � ODDS AGAINST interaction suggests

that HAD animals were more sensitive to probabilistic rewards

than LAD animals. HAD1 n ¼ 6, HAD2 n ¼ 6, LAD1 n ¼ 6 and

LAD2 n ¼ 5.

Genes, Brain and Behavior (2008) 7: 705–713 709

Discounting by HAD and LAD rats

Preference for sucrose increased with increasing concen-trations of sucrose (CONCENTRATION: F7.4,149.0 ¼ 66.7,

P < 0.001) and was similar in both lines (LINE: F1,20 ¼ 0.5,P ¼ 0.49) and replicates (REPLICATE: F1,20 ¼ 1.90, P ¼0.18). At higher concentrations of sucrose, all animalsshowed a strong preference for sucrose over water (Fig. 3b).

Furthermore, at the 10% sucrose concentration (the concen-tration used for the delay and probability discounting experi-

ments), all lines of animals showed a near exclusivepreference for the sucrose solution (one of the HAD1 animals

had a preference ratio of 0.5 at the 10% sucrose concentra-tion, while all other animals had a preference ratio of >0.92).

Locomotor activity

High alcohol drinking 2 animals were more active than LAD2

animals (LINE: F1,9 ¼ 31.6, P < 0.001) (Fig. 4). Activitytended to decrease as animals became habituated to the

chamber fo l l owing numerous exposures (DAY:F3.1,28.0 ¼ 3.2, P ¼ 0.035).

Correlational analysis

Table 3 summarizes correlations for the dependent measuresobtained in each component experiment of this study. Within

each discounting task, bias was correlated with k-values.Between discounting tasks, k-values were correlated but b-

values were not. Interestingly, consumption of 10% sucrosewas correlated with both bias and k-values derived from delay

discounting but not probability discounting tasks. This wasnot the result of using the programmed odds against descrip-

tor, as a correlation using probability discounting parametersincorporating experienced odds against yielded similar results

[correlations: probability discounting b (experienced probabil-ities) with sucrose, Spearman’s rho ¼ 0.078; probability dis-

counting k (experienced probabilities) with sucrose,Spearman’s rho ¼ �0.005].

Discussion

A genetic predisposition toward high alcohol

drinking is associated with greater sensitivity to

delayed and probabilistic outcomes.

Significant line differences were apparent in discounting of

both delayed and probabilistic rewards, with HAD animalsexhibiting greater sensitivity to both delayed and probabilistic

contingencies. The consistency of this finding in both HAD/LAD replicates adds additional support, suggesting that the

genes associated with the high or low alcohol consumptionphenotype are also associated with heightened sensitivity to

delayed or probabilistic outcomes. It is not clear if thesediscounting profiles will generalize to other genetically unre-

lated rat models of high alcohol drinking or low alcoholdrinking, but this could further strengthen these associations.

Differences in rearing may contribute to the observed pat-terns of discounting, although this is unlikely given that the

effect was consistent in both independently selected repli-cates. Bias or indifference points at 0 second delay and

0 odds against that are not equal to 150 ml accounted formuch of the differences between lines. It is not clear what

causes animals to undervalue or overvalue these conditions,but it is not unique to this study (Richards et al. 1997). Most

common in this study was for HAD rats to have bias valuesgreater than one, indicating a preference and subsequent

overvaluation of the delayed or probabilistic lever underconditions of 0 second delay or 0 odds-against. It is unlikely

that this is simply position bias, i.e. HAD rats being generallymore averse to the lever closest to the door because delayed

and probabilistic levers were counterbalanced. Instead, thismay be the result of differences in behavioral contrast

(Richards et al. 1997). High alcohol drinking rats may exhibita strong positive behavioral contrast under conditions of

0 second delay or 0 odds against, which would result inovervaluation of the delayed or probabilistic lever. By con-

trast, LAD2 rats undervalued the delayed or probabilisticlevers under these conditions, while LAD1 rats tended to

exhibit no bias (b � 1). Given the correlation between bias

Figure 3: Consumption and preference for reinforcer were

similar in both HAD and LAD rats. HAD1 n ¼ 6, HAD2 n ¼ 6,

LAD1 n ¼ 6 and LAD2 n ¼ 6. Panel a illustrates themean � SEM

amount of sucrose consumed by each strain in g of sucrose

consumed per kg of body weight. Sucrose concentrations ranged

from 0% to 30% sucrose (w/v). Panel b illustrates the mean �SEM preference ratio between the sucrose solution and water.

Preference ratio was calculated as volume of sucrose consumed/

total volume.

710 Genes, Brain and Behavior (2008) 7: 705–713

Wilhelm and Mitchell

and discount rate (k), the importance of bias cannot beunderestimated. Clearly, the cause of bias in the adjusting

amount procedure needs to be better understood. Neverthe-less, the slope of the discount curves for LAD rats were

consistently shallower than those for HAD rats, making thisthe first study to show that heightened sensitivity to delayed

or probabilistic outcomes exists in alcohol-naive rats predis-posed to HAD. This finding may have significant implications

for risk factors that contribute to human alcohol abusedisorders; however, it is not clear if such differences contrib-

ute to initiation of drug use and/or drug dependence over andabove the risk conferred by family history. Although it is

outside the scope of this study, gender differences may alsoplay a role in the genetics of alcohol abuse (Petry et al. 2002).

A single previous study in short term selectively bred micefound no difference in delay discounting between high alcohol

drinking and low alcohol drinking mice (Wilhelm et al. 2007).This may be explained by a number of factors. First, the

mouse background used to create the selected lines for the

previous study was a DBA/2J and C57BL/6J cross. A previousstudy found that the alcohol averse DBA/2J strain was more

sensitive to delayed reinforcement than the alcohol preferringC57BL/6J strain (Helms et al. 2006). Thus, the genes associ-

ated with heightened discounting and alcohol consumptionmay not be identical in rats and mice (see Discussion of

locomotor activity below). In addition, HAD and LAD rats havemost nonalcohol-related genes in common, while DBA/2J and

C57BL/6J mice have different alleles at many nonalcohol-related genes. Therefore, the differences in delay discounting

between these mouse lines may be because of factorsunrelated to their ethanol preference phenotypes. Further-

more, mice respond less reliably than rats in discountingprocedures (unpublished observation), reducing the sensitivity

of the assessment in mice.

Behavioral correlations

Some believe that delay and probability discounting areanalogous processes, i.e. choosing an uncertain reward with

a probability of 0.25, resulting in, on average, delivery ofa reinforcer every fourth trial is similar to having a long delay

between response and reinforcer delivery (Hayden & Platt2007; Mazur 1989). Other studies suggest that although delay

and probability discounting share some underlying processesand can be modeled with the same hyperbolic equation, they

are distinguishable from one another behaviorally and neuro-logically (Adriani & Laviola 2006; Green et al. 1999; Rachlin

et al. 1991). Studies in human subjects have found weakpositive relationships between probability and delay discount-

ing (Richards et al. 1999), indicating that some but not all

aspects of these tasks are related. If delay and probabilitydiscounting were essentially the same process, then bias (b)

and the discount parameter (k) should be correlated betweenthe two tasks. We found a weak positive correlation for k-

values, but no such correlation for bias between the two tasks.The relationship between delay discounting performance

and preference or consumption of reinforcer is not wellunderstood. Establishing that animals have similarly strong

preferences for the reinforcer is an important control. If,for example, we had used alcohol instead of sucrose as

a reinforcer, the results of the study would be difficult to

Figure 4: Mean (6SEM) of the total horizontal activity (in

cm) for each line during a 30-min session. HAD2 rats were

more active than LAD2 rats (* denotes main effect of LINE:

F1,9 ¼ 31.6, P < 0.001). Locomotor activity decreased as the

animals habituated to the chamber (main effect of DAY:

F3.1,28.0 ¼ 3.2, P ¼ 0.035). HAD2 n ¼ 6, and LAD2 n ¼ 6.

Table 3: Nonparametric correlations between experimental measures

DD b DD k PD b PD k 10% Sucrose D1 Loco† D1–D5 Loco†

DD b

DD k 0.687 (<0.001)**

PD b 0.326 (0.129) 0.177 (0.419)

PD k 0.241 (0.268) 0.460 (0.027)* 0.582 (0.004)**

10% Sucrose 20.498 (0.016)* 20.600 (0.002)** 0.093 (0.672) 0.043 (0.847)

D1 Loco† 0.564 (0.071) 0.255 (0.450) 0.364 (0.272) 0.355 (0.285) 0.236 (0.484)

D1–D5 Loco† 0.300 (0.370) 0.227 (0.502) �0.245 (0.467) �0.109 (0.750) �0.209 (0.537) 0.627 (0.039)*

PD, probability discounting; DD, delay discounting; 10% sucrose, the amount of sucrose consumed in g/kg body weight; D1 Loco, locomotor

activity on day 1; D1-D5 Loco, locomotor activity on day 5 minus locomotor activity on day 1 (habituation); †n ¼ 11, all other n ¼ 23.

Data shown are Spearman’s rho (P-value). *P < 0.05, **P < 0.01. Correlations between performance measures that were significant at the

P < 0.05 level are indicated by bold type.

Genes, Brain and Behavior (2008) 7: 705–713 711

Discounting by HAD and LAD rats

interpret simply because LAD rats are relatively averse toethanol, while HAD rats exhibit a much stronger preference

for alcohol. Thus, although differences in deprivation havelittle effect on choice behavior (Cardinal et al. 2000; Richards

et al. 1997), it is unclear how differences in preference orconsumption of reinforcers might affect responding on this

task. Consumption of 10% sucrose negatively correlated withboth b and k for the delay discounting task but did not

correlate with either in the probability discounting task. Thatis, the more the animals drink the 10% sucrose solution the

less sensitive they were to devaluation of the large 150 mlreinforcer as a result of delay. In contrast, the amount of

sucrose consumed was unrelated to devaluation caused byprobabilistically withholding the 150 ml reinforcer. Differencesin deprivation affect other aspects of the task such as reactiontime and choice reaction time (Richards et al. 1997), but there

were no line differences on these measures. Our resultssupport the hypothesis that delay and probability discounting

are dissociable experimental measures.Locomotor activity predicts delay discounting in mice (Isles

et al. 2004) but not rats (Perry et al. 2005). Our results supportthe finding by Perry and colleagues and suggest that locomo-

tor activity is not correlated with delay or probability discount-ing in rats. High alcohol drinking rats were significantly more

active than LAD rats, but this did not correlate with individualdifferences in discounting parameters. For the LAD2 rats, the

rats with the lowest levels of activity tended to have the

highest k-values within the line, thus nullifying the potentialpositive correlation. A significant correlation may have been

apparent if a larger number of rats were tested. Differences inlocomotor activity between HAD and LAD rats have not been

observed previously (Nowak et al. 2000; Overstreet et al.1997); however, the current study is the first to examine the

HAD2 and LAD2 replicate line. In contrast, the alcohol pre-ferring (P) and alcohol non-preferring (NP) rats do exhibit

a difference in locomotor activity, with P rats being moreactive than NP rats (Nowak et al. 2000). There were also line

differences in sucrose drinking, with LAD rats tending to drinkmore 10% sucrose than HAD rats. This effect was driven by

differences between HAD1 and LAD1 animals and was notobserved in the HAD2 and LAD2 lines. Inclusion of 10%

sucrose consumption as a covariate in the analysis of delaydiscounting did not alter the statistical outcome, suggesting

that differences in sucrose consumption were not solelyresponsible for the observed line differences. Differences

between HAD and LAD replicates are not uncommon; forinstance, HAD1 rats exhibited higher levels of operant ethanol

self-administration than HAD2 rats (Files et al. 1998). Nodifferences were observed between LAD1 and LAD2 rats in

this task. High alcohol drinking 2 rats consume more ethanolthan LAD2 rats by having larger and more frequent drinking

bouts per day, whereas HAD1 rats drink fewer bouts per daybut have larger bouts than HAD2 rats (Files et al. 1998).

Conclusions

This study found a significant and replicable relationshipbetween heightened sensitivity to delayed and probabilistic

rewards and a genetic predisposition to high alcohol con-

sumption. This indicates that heightened sensitivity to thesereward costs may exist prior to drug use and, therefore, may

serve as a risk factor in humans for developing an alcoholabuse disorder. This is the first study showing a strong

relationship between sensitivity to delayed or probabilisticoutcomes and a predisposition to high alcohol drinking in rats.

It is not clear if this finding will generalize to other selectivelybred lines for high alcohol drinking or to other models of

alcohol-associated behaviors such as sensitization or with-drawal. Future studies attempting to determine the specific

proteins/neurotransmitters involved in mediating this behav-ior may provide important and useful pharmacotherapeutic

targets that may aid in preventing the development of alcoholabuse disorders.

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Acknowledgments

We thank the National Institute on Alcohol Abuse and Alcoholism(NIAAA)-funded Indiana Alcohol Research Center (R24AA015512-01) for providing us with the HAD and LAD animalsused in this study and Kirigin Elstad for her assistance with datacollection. This article is supported by grant AA007468 (C.J.W.)and DA016727 (S.H.M.).

Supporting Information

Additional supporting information may be found in the onlineversion of this article.

Appendix S1: Detailed training description.Figure S1:Mean � SEM reaction time (time from the start

of each new free-choice trial until a middle lever press) foreach line and replicate combination at each delay. HAD and

LAD rats did not differ in reaction times during delaydiscounting.

Figure S2: Mean � SEM choice reaction time (time frommiddle lever press until one of the two outer levers was

pressed for free-choice trials) for each line and replicatecombination at each delay. HAD and LAD rats did not differ

in choice reaction time during delay discounting.Figure S3: Mean � SEM reaction time (time from the start

of each new free-choice trial until a middle lever press) foreach line and replicate combination at each probability (odds

against ¼ 0, 0.33, 1, 3, 7). HAD and LAD rats did not differ inreaction times during probability discounting.

Figure S4: Mean � SEM choice reaction time (time from

middle lever press until one of the two outer levers is pressedfor free-choice trials) for each line and replicate combination at

each probability (odds against ¼ 0, 0.33, 1, 3, 7). HAD andLAD rats did not differ in choice reaction time during

probability discounting.Please note: Wiley-Blackwell are not responsible for the

content or functionality of any supporting information sup-plied by the authors. Any queries (other thanmissing material)

should be directed to the corresponding author.

Genes, Brain and Behavior (2008) 7: 705–713 713

Discounting by HAD and LAD rats

Supplement: Detailed training description

Training began after 3 days of food restriction and was divided into 3 phases. In

Phase 1, the lights above the levers were illuminated to indicate that the levers were

active, and responses on these levers resulted in delivery of 150 µl of sucrose reinforcer

to the nose-poke immediately below the lever as well as activation of the response

clicker. In addition, a noncontingent reward (150 µl) was delivered after progressively

longer periods of inactivity. The initial period of inactivity was 30 s, but lengthened by

10 s after each delivery (i.e., the second noncontingent reward was delivered after no

presses occurred on the outer levers for 40 s, the third was delivered after 50 s and so

forth). Noncontingent rewards were delivered pseudorandomly to one of the two

recessed nose-poke holes immediately below the outer levers. The maximum number of

noncontingent rewards delivered in a session where no responses occurred was 24.

Training sessions lasted for 60 min or until 80 total (contingent + noncontingent)

reinforcers were delivered. Completion of this phase of training required subjects to earn

65 contingent reinforcers on two consecutive sessions.

In Phase 2, trials began when the light above the middle lever was illuminated.

When subjects pressed this lever, the center light extinguished, and the lights above the

outer levers were illuminated. The center lever press was included to minimize the

potential for position bias. The rat could then press either of the outer levers to earn a

150 µl sucrose reinforcer. When the rat earned a reinforcer, the house light was

extinguished and the response clicker sounded to signal food delivery. A 5-s intertrial

interval occurred before the beginning of the next trial. If the rat chose the same outer

lever on two consecutive trials, a forced choice trial followed, which required the rat to

pick the previously unchosen lever. Thus, rats were exposed to the contingencies on both

levers throughout each session. Rats were required to complete 80 free-choice trials in 60

min on two consecutive days to move to Phase 3.

Phase 3 incorporated all aspects of Phase 2 with the following additions. First, in

Phase 3 each trial was 40 s long. Second, one of the outer levers was assigned to be the

immediate, adjusting lever, while the other was the delayed or probabilistic lever

(although no delays greater than 0 s or probabilities less than 1.0 were experienced during

training). Lever assignments were counter-balanced between subjects but remained the

same throughout the experiment. Third, the size of the immediate/certain reward (initially

75 µl) is adjusted as it would be during experimental sessions; choices of the

delayed/probabilistic lever increased the size of the immediate/certain reward by 10%

and choice of the immediate/certain lever decreased the size of the immediate/certain

reward by 10% (forced-choice trials did not affect the size of the immediate/certain

reward). At the beginning of each trial, the rat was given 22 s to respond on the middle

lever, after which a response on the outer levers was allowed until 24 s had elapsed in the

trial. If no response was made during this time, all lights were extinguished until the next

trial began. If the rat responded within the time constraints, lights were extinguished, the

sucrose reinforcer was delivered and the response clicker sounded. A variable length

intertrial interval was used to make trials 40 s long, after which a new trial began.

Sessions lasted until 60 free-choice trials occurred, or 60 minutes had elapsed. The

majority of sessions were completed within 50 minutes. To complete training, rats were

required to respond on at least 55 of the 60 possible free-choice trials on two consecutive

sessions. One iLAD2 rat was unable complete the training requirements and is therefore

not included in the results.