Morgado et al. Stress affects pavlovian-to-instrumental transfer predictions on instrumental performance (Corbit and Balleine, 2011). In addition, these regions are known to regulate the activity of cortical sites integrating affective stimuli with executive com- mands, such as the mPFC and the OFC (Christakou et al., 2004; Kelley, 2004; Pasupathy and Miller, 2005; Saddoris et al., 2005; Stalnaker et al., 2007). Surprisingly, given the fact that stress inﬂuences many of the above mentioned areas involved in PIT, the impact of stress in PIT is largely unknown. Indeed, while acute stress was shown to enhance Pavlovian learning (Shors et al., 2000) and chronic stress failed to inﬂuence instrumental learning (Dias-Ferreira et al., 2009), no study addressed the effect of either acute or chronic stress on the interaction between these two key processes and thus on the impact of conditioned clues in goal-directed behavior. Thus, in the present study, we tested the impact of chronic stress in the modulation of instrumental behavior according to cues previously associated with rewards by studying the behavior of control and stressed rats in a PIT paradigm using a two-lever operant chamber. Moreover, to assess whether the impairments are reversible after chronic exposure to stress, PIT was also assessed after a period free of exposure to stressful stimuli. MATERIALS AND METHODS ANIMALS All experiments were conducted in accordance with local reg- ulations (European Union Directive 86/609/EEC) and National Institutes of Health guidelines on animal care and experimentation and approved by Direção Geral Veterinária (DGV; the Portuguese National Institute of Veterinary). Thirty-two adult male Wistar rats (Charles River Laborato- ries, Barcelona, Spain; 250–300 g at the start of the experiment), aged 3 months and weighing 400–500 g, were housed in groups of two under standard laboratory conditions with an artiﬁcial light- dark cycle of 12:12 h (lights on from 8:00 a.m. to 8.00 p.m.) in a temperature- and humidity-controlled room. Animals were given 2 weeks to acclimate to the housing conditions with ad libitum access to food and water. A food deprivation regimen was initiated 24 h before the initiation of training and testing to maintain the subjects at approximately 90% of their free-feeding body weight. Rats had free access to water while in the home cage. CHRONIC UNPREDICTABLE STRESS PARADIGM Animals assigned to the chronic unpredictable stress (CUS) group were exposed during 60 min once a day to one of ﬁve different stressors: cold water (18˚C), vibration, restraint, overcrowding, and exposure to a hot air stream. Stressors were randomly dis- tributed throughout a 28-day period. This type of chronic stress paradigm, mixing different stressors (including physical and psy- chological components) presented in an unpredictable schedule, was shown previously to result in persistently elevated plasma lev- els of corticosterone (for details, see Sousa et al., 1998) and is thought to better mimic the variability of stressors encountered in daily life (Sousa et al., 1998). Controls were handled daily during the same period. To assess the impact of chronic stress exposure in Pavlovian- instrumental transfer but also ascertain its reversibility, a ﬁrst group of animals (eight stressed and eight controls) were behav- iorally characterized immediately after stress while a similar group (eight stressed and eight controls) was left to recover for 6 weeks before being tested. This recovery period was set-up in light of previous studies showing that, at least, 4 weeks are necessary to complete reversion of behavioral and structural changes induced by CUS treatment (Sousa et al., 2000). Importantly, animals were randomly allocated to each of the four groups before the beginning of stress exposure. PAVLOVIAN-INSTRUMENTAL TRANSFER Behavior was assessed using the Pavlovian-instrumental transfer protocol as described by Ostlund and Balleine (2007). This task took place in operant chambers (30.5 cm L 24.1 cm W 21.0 cm H, MedAssociates, CA, USA) housed within sound attenuating cubicles. Each chamber was equipped with two retractable levers on either side of the food magazine and a house light (3W, 24V) mounted on the opposite side of the chamber. Reinforcers were delivered into the magazine through a pellet dispenser that deliv- ered 45 mg regular “chow” pellets or a liquid dipper that delivered 0.1 ml of 20% sucrose solution. A computer equipped with MED- PC IV software controlled the equipment and recorded lever presses and head entries. As described previously, animals were placed in a food deprivation schedule, having access to food dur- ing 1 h per day after the training or testing session, allowing them to maintain a body weight above 90% of their baseline weight. Water was removed for 2 h before each daily session. Training began with eight daily sessions of Pavlovian condi- tioning in which each of two auditory conditioned stimuli (tone and white noise) were paired with a different outcome (pellets and sucrose). Each CS was presented four times per session using a pseudo-randomized order and a variable ITI (mean 5 min). In the ninth day, animals were submitted to an outcome devaluation to ensure they were able to associate each outcome to the condi- tioned stimulus; this was assessed by comparing the number of head entries into the food dispenser during stimuli presentation and during ITI. Animals were then trained to obtain two different outcomes (pellets and sucrose) by pressing left and right levers. Training was performed in two separate daily sessions and the order of training was alternated during days (average interval between the two daily sessions was 3 h). Each session ﬁnished after 15 outcome deliver- ies or 30 min. In the ﬁrst 2 days, lever pressing was continuously reinforced (CRF) which means that each action resulted in one outcome delivered (p D 1.0). The probability of getting a reward decreased according the following sequence: days 3–4, p D 0.2; days 5–6, p D 0.1; and days 7–9, p D 0.05. Two sessions of outcome devaluation (by free access to the reward until satiety) were then performed, 48 h apart. In order to do this, one of the two outcomes (pellets or sucrose) was given ad libitum during 1 h before each session. Then the rats were placed during 5 min into the testing operant chamber where both levers were inserted but no outcome was delivered. Forty-eight hours later, subjects were placed in the operant chamber to test Pavlovian-instrumental transfer with both levers inserted. After an initial period of response extinction that lasts for 8 min, four blocks of each auditory conditioned stimulus were Frontiers in Neuroscience | Decision Neuroscience June 2012 | Volume 6 | Article 93 | 2