Materials and Methods Subjects Twenty-three adult male B6129 F1 hybrid mice from Taconic were used in these studies. Throughout the experiment, mice were kept in a temperature-controlled vivarium on a 12-hour light:dark cycle and housed two animals per cage. Behavioral procedures were conducted during the light phase of the cycle. Subject weights were maintained at , 85% of their original free-fed weight throughout the experiments. All experimental procedures were approved by the University of Virginia Animal Research Committee. Equipment Mice were trained in eight operant conditioning chambers (Med Associates; East Fairfield, VT, USA) housed in sound and light attenuating cubicles. Each cubicle contained an exhaust fan that was on during all sessions to increase air circulation and reduce background noise. Each operant chamber contained two retract- able levers mounted on the same wall, with the food magazine located in-between them. Each lever controlled a pellet dispenser that released 20 mg dustless precision pellets (Bio-Serv; French- town, NJ) into the magazine. The left lever produced grain pellets while the right lever produced chocolate pellets. All chambers were connected to a central computer running MED PC software (Med Associates; East Fairfield, VT, USA), which controlled the boxes during experimental sessions and automatically recorded the data. Magazine Training All mice received 2 days of magazine training for familiarization with reward delivery. Each day consisted of a 30-minute training session, during which the levers remained withdrawn and pellets were dispensed into the magazine on independent random interval schedules (60 s). Lever Press Training Following magazine training, mice were split into ratio and interval groups and trained to lever press for the delivery of the two food rewards. For all animals, left lever presses led to a grain pellet reward and right lever presses led to a chocolate pellet reward. Mice underwent 2 lever-press training sessions per day, one session for each lever, spaced at least 1 hour apart. The order of these sessions was reversed each day. Each session lasted either until animals received 20 reinforcers or a maximum time of 30 minutes was reached. A continuous schedule of reinforcement, where each lever press produced a reward delivery, was used for the first 6 days of training in all animals (data not shown). Following initial lever press acquisition, the two groups of animals continued instrumental training on two different reward delivery schedules. One group was trained to lever press on a random ratio (RR) schedule of reinforcement while the other group was trained on a random interval schedule (RI) of reinforcement. The RR group underwent 2 days of ratio training on a RR5 schedule, where the probability of reinforcement for each lever press was 0.2. During the same 2 days, the RI group underwent interval training on a RI15 schedule, where the average time between reinforcer availability was 15 seconds. This was followed by 2 days of a RR10 schedule for the RR group (probability of re- inforcement, P = 0.1) and a RI30 schedule for the RI group (reinforcers available every 30 s on average) and then 10 days of a RR20 schedule for the RR group (probability of reinforcement, P = 0.05) and a RI60 schedule for the RI group (reinforcers available every 60 s on average). Ratio and Interval Training Devaluation Test A devaluation test was performed on all animals 24-hours after their last day of instrumental training. Half of the animals in each group were given ad libitum access to the grain pellet for one hour while the other half of the animals were given ad libitum access to the chocolate pellet for the same amount of time. One mouse from the ratio group did not consume pellets during this period and was excluded from analysis. Immediately following this hour of outcome devaluation, animals were placed in the operant chambers for a 2-lever choice extinction test that lasted 10- minutes. Activity on both levers was recorded. Pavlovian Conditioning Following devaluation, mice received 8 days of Pavlovian conditioning. Each day consisted of a single hour-long session in which two conditional stimuli [(tone (85 dB, 2000 Hz) or white noise (80 dB)] were paired with reinforcement. The levers were retracted during these sessions. Each stimulus was presented four times during each session. The duration of each CS was 2 min, during which time reinforcement was delivered on a random interval schedule (30 s). The time between stimulus presentations was ,5 minutes. For all animals, white noise presentations were paired with grain pellet delivery and tone presentations were paired with chocolate pellet delivery. During these sessions, head- entry detectors recorded the number of magazine entries (the conditional response) for each animal during the 2 minutes prior to stimulus presentation as well as during CS presentation. The data are expressed across training days as an elevation ratio (entries per minute during cue presentations/entries per minute during the ITI). Pavlovian Instrumental Transfer (PIT) Test One day after the last Pavlovian conditioning session animals were placed in the operant chambers for a 45-minute 2-lever choice extinction test. Following an initial 8-minute baseline period, the 2 conditioned stimuli from Pavlovian training were presented intermittently to the animals. Each stimulus was presented a total of 4 times separated by ,5-minute ITI period. Lever presses were recorded during the CS presentations and during the ITI. Rates of responding were analyzed for the lever leading to the same outcome as the CS and the lever leading to a different outcome than the CS. After this test, the animals received an additional 8 days of Pavlovian training and a second PIT test was performed, identical to the first. Results Mice were first trained to lever press on a continuous re- inforcement schedule for 6 days. One lever produced grain pellets while the other led to chocolate pellets. After this period, mice were trained on random ratio (RR) or random interval (RI) reinforcement schedules for 14 days (Figure 1A). On days 1–2 the ratio group was trained on a RR5 schedule while the interval group was trained on a RI15 schedule. On days 3–4 the ratio group was trained on a RR10 schedule while the interval group was trained on a RI30 schedule. On days 5–14 the ratio group was trained on a RR20 schedule while the interval group was trained on a RI60 schedule. Both groups showed an increase in responding across training days (No effect of group F ,1; main effect of day F13, 273 = 38.7, p,0.05; no group by day interaction F ,1). Previous work showed that interval training promotes habitual responding while ratio training produces goal-directed actions [17,18,20]. This fact can be demonstrated by using a devaluation PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e48227