Inbred lines of HAD1 (n = 6), HAD2 (n = 6), LAD1 (n = 6) and LAD2 (n = 6) male rats were generously supplied by the Indiana University Alcohol Research Center. These lines of animals were chosen because they were selected from the N/NIH heterogeneous stock (which provides the advantage of a diverse population of starting genes) and because there are two independent replicates available, which greatly decreases the probability that a gene unrelated to alcohol consumption becomes fixed in both replicates. Upon receipt, HAD1 animals weighed 307 ± 10 g, LAD1 animals weighed 347 ± 6 g, HAD2 animals weighed 292 ± 10 g and LAD2 animals weighed 263 ± 9 g. Rats were housed in the Department of Comparative Medicine at Oregon Health & Science University an AAALAC-approved facility. All procedures were approved by the appropriate Institutional Animal Care and Use Committee and adhered to NIH Guidelines. To facilitate training and maintain responding 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 maintained at 90% of their free-feeding age-adjusted body weights with supplemental chow given immediately following each day’s test session.
We used eight identical (Med Associates, St. Albans, VT, U.S.A.) modular rat test chambers housed individually within melamine sound-attenuating cabinets. Chambers had acrylic front and back panels and stainless steel side panels. A fan provided constant ventilation and low-level background noise. A house light was mounted in the center of one of the stainless steel panels, and a response clicker was also mounted on the outside of this panel. Three non-retractable levers were mounted on the opposing panel directly 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 nose-pokes.
Order of Experiments
HAD1 and LAD1 rats completed the delay discounting task, followed by the probability discounting task and sucrose drinking. HAD2 and LAD2 rats completed the probability discounting task first, then delay discounting, sucrose drinking and locomotor activity. The discounting tasks were counterbalanced to offset potential order effects.
Training began after 3 days of food restriction and was divided into 3 phases (for detailed description see Supplement
). Phase 1 of training exposed subjects to a progressively delayed noncontingent reward to acquaint the animals with the location of the reinforcer, and a response-contingent reinforcer, to train the animals to press levers and earn a sucrose reward. In Phase 2 of training, animals were trained to press the middle lever to activate the outer “choice” levers. The purpose of this was to eliminate position bias by forcing the animals to move near the middle of the chamber. Once activated, an outer lever press resulted in sucrose reward delivery and deactivation of both levers until a new trial began (5 s ITI) and the middle lever was pressed. Animals were also introduced to forced choice trials, whereby animals that chose either the right or left lever on two consecutive trials were forced to press the opposite lever on the subsequent trial. Phase 3 of training incorporated time contingencies, requiring animals to successfully complete a middle lever press and a left or right lever press within 24 s of the start of a trial. Each trial was 40 s long. Illumination of the middle light signaled the start of a new trial. A subsequent middle lever press extinguished the middle light and illuminated the right and left lights, indicating that both levers were active. One of the outer levers was assigned to be the immediate/certain lever, while the other was the delayed or probabilistic lever (during training, no delays greater than 0 s or probabilities less than 1.0 were experienced in training). Delayed/Probabilistic levers were counter-balanced between subjects and remained the same throughout both delay and probability discounting experiments. The size of the immediate/certain reward (initially 75 µl) adjusted throughout the experimental session; choice 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 reward). Each sucrose reinforcer was delivered to the right or left nose pokes in combination with an auditory stimulus (response clicker). 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 LAD2 rat was unable to complete the training requirements and was therefore not included in the results.
Delay Discounting Task
The adjusting amount procedure was adapted from a procedure described in Richards et al. (1997)
. Experimental sessions were as described in Phase 3 of training, except that a response on the delayed lever resulted in delivery of a 150 µl sucrose reinforcer, delayed by 0, 2, 4, 8, or 16 s. The delay remained constant within a session but varied between sessions according to a Latin square design. Animals experienced each delay a minimum of 6 times, but only data from occasions 2–6 was included in the analyses. The purpose of the experiment was to assess the subjective value of the delayed sucrose by titrating the volume of the immediate reward until animals became indifferent between the two choices.
Probability Discounting Task
Experimental sessions were carried out exactly as described in Phase 3 of training, except that a response on the probabilistic lever resulted in the delivery of a 150 µl sucrose reinforcer with probabilities of 1.00, 0.75, 0.50, 0.25 or 0.125. Probability remained constant within a session but varied between sessions according to a Latin square design. Animals experienced each probability a minimum of 6 times, but only data from occasions 2–6 was included in the analyses. The purpose of the experiment was to assess the subjective value of the probabilistic sucrose by titrating the size of the immediate reward until animals became indifferent between the two choices.
Two-bottle sucrose preference
The purpose of this experiment was to determine if HAD and LAD rats differed in consumption or preference when offered a choice between a sucrose solution and water. Differences in sucrose consumption or preference might be critical to the interpretation of the discounting data. Replicates 1 and 2 were tested 4 days per week with 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 rat was placed in a rat drinking cage for 50 minutes (the approximate length of a discounting session) and given access to two bottles; one containing a sucrose solution and the other containing water. To eliminate the potential for side bias, placement of sucrose and water bottles was alternated daily. The amount of solution consumed was determined by weighing the bottles before and after each test session. The average amount of sucrose consumed per 50 min test session was calculated in grams of sucrose/kg body weight. In addition, sucrose preference was calculated as the ratio of the amount of sucrose consumed to the total amount of fluid (water plus sucrose) consumed. Again, preference was averaged over each of the test days. To maintain relevance to discounting procedures, animals were food deprived as described for the discounting procedure.
Locomotor activity was assessed in the HAD2 and LAD2 animals. Timing of the decision to collect activity data meant that the first replicate of animals was unavailable to complete the task. Food was available ad-libitum during this phase of the experiment. Four Accuscan (Accuscan Instruments Inc., Columbus, Ohio, U.S.A.) automated activity monitors were used for this study. Chambers consisted of a 40 × 40 × 30 cm clear acrylic test cage placed inside a monitoring unit that recorded photocell beam breaks, which were translated into distance traveled (in cm). Eight or sixteen evenly spaced photocells and receptors were located 2 cm above the chamber floor. Monitors and test cages were housed in black acrylic chambers that were lined with foam to attenuate external noise. A fluorescent light and fan located within the test chamber were on during testing to provide illumination, ventilation and low level background noise. Each rat was assigned to a locomotor activity chamber. Locomotor activity was recorded for 30 minutes on 5 consecutive days and converted to horizontal distance traveled.
Analyses of delay and probability discounting data were similar to those described in Mitchell et al (2006)
and Wilhelm et al. (2007)
. The main dependent variable was the amount of sucrose solution delivered from the immediate/certain, adjusting alternative at the “indifference point”, i.e., the amount at which the immediate/certain and delayed/probabilistic alternatives were selected equally often. Based on previous findings (Richards et al. 1997
), animals reach the indifference point after the first 30 trials of a session, thus the median volume of sucrose associated with the adjusting lever over trials 31–60 is an index of the subjective value of the alternate choice. demonstrates that animals chose each lever with roughly equal frequency over this period. The median, rather than the mean, was used as a measure of central tendency because changes in the adjusting amount on successive trials were proportions of the amount on the prior trial, resulting in a skewed distribution. Data from sessions where fewer than 40 trials were completed was excluded from analysis (percent of delay discounting sessions with fewer than 40 responses: HAD1 = 2.0%, LAD1 = 0%, HAD2 = 0%; LAD2 = 3% of delay discounting sessions; percent of probability discounting sessions with fewer than 40 responses: HAD1 = 0%, LAD1 = 1%, HAD2 = 3%, and LAD2 = 14% of probability discounting sessions). Hyperbolic equations were fitted to each animal’s average indifference points (modified from Mazur 1987
) using Graphpad Prism 3.0 (Graphpad Software Incorporated, San Diego, CA):
Percent choice during the final 30 choice trials
where V represents the value of the adjusting reward at indifference in µl; A represents the amount of sucrose solution from the delayed or probabilistic alternative (150 µl); X is either the delay to receiving the reinforcer (0, 2, 4, 8, or 16 s), or the odds against receiving the reinforcer ((1-probability)/probability: 0, 0.33, 1, 3, 7), and the bias parameter, b, is a fitted parameter indicative of bias, or side preference in the absence of delay (0 s delay condition) or uncertainty (odds against = 0)‥ The discount parameter (k) is a fitted parameter, and indexes the rate of discounting or overall sensitivity to delayed or probabilistic rewards. Larger values of k indicate steeper discount functions, and stronger aversion to delayed or probabilistic rewards. Mixed model ANOVAs were used to examine indifference points, with LINE and REPLICATE as between-subjects factors and DELAY or ODDS AGAINST as within-subjects factors. Similar ANOVAs with the additional inclusion of CHOICE as a within-subjects factor was used to examine reaction times and choice reaction times. Two-way ANOVAs were used to examine k values and b values, with LINE and REPLICATE as between-subjects factors.
The main dependent variables in the two-bottle choice study were the grams of sucrose consumed /kg of body weight and the preference ratio (ml of sucrose consumed/total ml consumed). A mixed model ANOVA with concentration of sucrose (CONCENTRATION) as a within subjects factor and LINE and REPLICATE as between subjects factors was used for analysis. For locomotor activity, the main dependent variables were the horizontal distance traveled on Days 1 through 5 and the difference in horizontal distance traveled from Day 1 to Day 5 (habituation). A mixed model ANOVA with DAY as a within subjects factor and LINE as a between subjects factor was used for analysis.
ANOVA and other statistical tests were carried out using SPSS version 15.0 (SPSS Inc., Chicago, IL). Huynh-Feldt corrections were applied as necessary, and adjusted degrees of freedom are provided.