Ratio and Interval Training Figure 1. Interval training leads to habitual responding. A) Mice were trained to lever press for food pellets across 14 days. 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. B) Mice trained on RR schedules showed a selective reduction in responding on the lever leading to the devalued outcome indicating that behavior was goal-directed. Mice trained on RI schedule did not exhibit a selective reduction in responding indicating that behavior was habitual. Error bars represent 6 SEM. *p,0.05. doi:10.1371/journal.pone.0048227.g001 procedure prior to a choice extinction test [14]. Following the last day of lever press training, we devalued one of the food pellets by giving mice ad libitum access to it for 60 minutes. After this period, a 10-minute extinction test was conducted where both levers were present but no reward was delivered. As shown in Figure 1B, mice trained on ratio schedules selectively reduced responding on the lever that was associated with the devalued outcome (main effect of lever, F1, 10 = 8.3, p,0.05). This indicates that animals learned the relationship between their actions and the outcomes they produce. In contrast, mice trained on interval schedules did not show a selective reduction in responding, which indicates that their behavior was habitual (no effect of lever, F1, 10 = 2.2, p.0.05). To examine the motivational impact of Pavlovian cues on instrumental responding, mice were trained to associate two conditional stimuli (CSs) with reward. Each CS was paired with one of the pellets used during instrumental training. Both ratio and interval groups showed an increase in conditional responding (magazine entries) across 8 training days (Figure 2A) (no effect of group, F1,21 = 1.12, p.0.05; main effect of day, F7,147 = 12.163, p,0.05; no group by day interaction, F ,1). The data are expressed as an elevation ratio = (CS entries/ITI entries). After Pavlovian training, the mice received a transfer test. During this test, both levers were extended but no reward was delivered. Each CS was presented 4 times and lever presses observed during the stimulus periods were compared to respond- ing during the ITI (Figure 2B). Following 8 days of Pavlovian training, no transfer effects were observed in the ratio group. The number of lever presses was the same during the CS presentations and the ITI (no effect of stimulus period, F2,22 = 1.26, p.0.05). In contrast, the interval group showed significant transfer as lever pressing was elevated during the CS periods (main effect of stimulus period, F2,20 = 6.97, p,0.05). Post-hoc tests (Fisher’s PLSD) revealed that responding was elevated during the pre- sentation of both CSs relative to the ITI (p values ,.05). However, selective transfer was not observed as responding increased on the lever leading to the same outcome as the CS and the lever leading to a different outcome (Fisher’s PLSD, p.0.05). A direct comparison of responding during the CS periods revealed that the interval group pressed more than the ratio group when reward cues were presented (main effect of group, F1,21 = 4.12, p = 0.05). These results suggest that habits are more sensitive to the motivational impact of reward cues than goal-directed actions [13]. Using similar procedures, we previously showed that reward cues can motivate goal-directed actions in mice [21]. To determine if our current animals required more training, we conducted an additional 8 days of Pavlovian conditioning (Figure 2C). Across days, interval and ratio groups showed similar levels of magazine entries in response to the CSs (no effect of group, F1,21 = 1.07, p.0.05; main effect of day, F7,147 = 3.14, p,0.05). There was a significant group x day interaction (F7,147 = 2.3, p,0.05) but post-hoc tests (Fisher’s PLSD) found no differences between ratio and interval groups on any of the training days (all p values ..05). After additional Pavlovian training, we observed positive transfer in both groups (Figure 2D). Mice trained on ratio schedules showed a significant increase in responding during the CS presentations (effect of stimulus period, F2,22 = 4.6, p,0.05), Post-hoc tests (Fisher’s PLSD) revealed that responding was elevated during the presentation of both CSs relative to the ITI (p values ,.05). However, selective transfer was not observed as responding increased on the lever leading to the same outcome as the CS and the lever leading to a different outcome (Fisher’s PLSD, p.0.05). The interval group, once again, increased responding during both CS periods relative to the ITI (effect of stimulus period, F2,20 = 13.8, p,0.05) (Fisher’s PLSD p values ,0.05). Similar to the ratio group, selective transfer was not observed as responding was equivalent during the presentation of both CSs (Fisher’s PLSD, p.0.05). Finally, a direct comparison of responding during the CS periods revealed that the interval group pressed more than the ratio group when reward cues were presented (main effect of group, F1,21 = 15.17, p,0.05). Therefore, even though positive transfer was observed in both groups the effect was larger for habits than goal-directed actions. Discussion Consistent with previous studies, we found that interval training led to habitual responding while ratio training produced goal- directed behaviors [17–19]. This fact was demonstrated by PLOS ONE | www.plosone.org 3 October 2012 | Volume 7 | Issue 10 | e48227