Morgado et al. Stress affects pavlovian-to-instrumental transfer presented randomly over the next 40 min and lever presses were registered. During each stimulus presentation, lever presses where considered correct if encoded the same reward as the audible sound. When different, actions were considered incorrect. STATISTICAL ANALYSIS Results are expressed as group means SE. Pavlovian, instrumen- tal behavior and results of transfer test were compared between and within groups using two-way ANOVA. Differences were considered to be signiﬁcant if p < 0.05. RESULTS Pavlovian training resulted in conditioning of animals both in control and stress groups. Comparison of head entries on CS pre- sentation and on ITI during Pavlovian training (Figure 1) shows that all animals associate the stimuli to the outcome[head entries: F (1,28) D 88.762, p < 0.001] without differences between experi- mental groups [stress exposure: F (1,28) D 0.163, p D 0.689], thus implying that CUS does not affect Pavlovian conditioning. In what regards to instrumental training, the number of lever presses per minute increased during the task indicating that ani- mals in both groups can learn it equally well (Figure 2). This is con- ﬁrmed by the results of the outcome devaluation test performed at the end of instrumental conditioning, in which animals of both groups [stress exposure: F (1,28) D 1.019, p D 0.321]could correctly associate each reward to a speciﬁc lever[lever: F (1,28) D 25.787, p < 0.001; Figure 2. These results are in accordance with our pre- vious data (Dias-Ferreira et al., 2009) showing that CUS does not impair outcome devaluation when performed early during the period of training. Subsequently, we assessed the Pavlovian-instrumental trans- fer. Figure 3 displays the number of lever presses per minute when the conditioned sound predicted the same outcome as the response (same) and the number of lever presses per minute when the conditioned sound predicted a different outcome (diff). Our results (Figure 3A) show that stress signiﬁcantly impairs the transfer [stress exposure: F (1,28) D 5.397, p D 0.028], preventing exposed animals, contrary to controls, [interaction: F (1,28) D 7.558, p D 0.010] from associating levers to the corre- sponding sound cues [lever: F (1,28) D 7.630, p D 0.010]. Importantly, we also assessed whether these stress-induced effects were sustainable in time after the end of the exposure to stress and found that these effects of stress were reversible. In fact, a similar assessment of stressed-recovered animals and con- trols (Figure 3B) failed to show any difference between groups [stress-recovery exposure: F (1,28) D 0.976, p D 0.332], with all ani- mals from both groups [interaction: F (1,28) D 0.178, p D 0.676] being able to associate conditioned sound and appropriate responses[matching vs. non-matching lever: F (1,28) D 18.217, p < 0.001]. DISCUSSION The present results show for the ﬁrst time that chronic stress dis- rupts the modulation of instrumental responses by conditioned cues and that these stress-induced impairments are transient, being absent after a 6-weeks recovery period. This is of rele- vance for decision-making, as it is well established that environ- mental cues can have a strong modulatory effect upon instru- mental responses (Estes, 1948), which are the basis of most decision-making processes. Chronic stress has a strong modulatory inﬂuence (either nega- tive or positive) on learning processes, including spatial memory (Sousa et al., 2000), working memory (Mizoguchi et al., 2000; Cerqueira et al., 2007), and behavioral ﬂexibility (Cerqueira et al., 2007), but also in decision-making processes by biasing instru- mental actions to habits (Dias-Ferreira et al., 2009). In the present study we show that chronic stress does not affect Pavlovian condi- tioning nor instrumental learning. Although the effects of chronic stress upon Pavlovian conditioning have never been reported, the latter ﬁnding is in accordance with a previous report showing that chronic stress promotes the transfer from goal-directed actions to habit based behaviors without affecting instrumental learning per se (Dias-Ferreira et al., 2009). Indeed, in that study, when tested on a devaluation paradigm after 8 days of training (similar to the FIGURE 1 | Pavlovian conditioning. There were no differences between groups, with all animals increasing the number of head entries during conditioned stimulus exposure. ITI – intertrial interval between presentations of conditioned stimuli CS – conditioned stimulus.*p < 0.05. www.frontiersin.org June 2012 | Volume 6 | Article 93 | 3