Inactivation of the medial prefrontal cortex of the rat impairs strategy set-shifting, but not reversal learning, using a novel, automated procedure

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Behavioural Brain Research 190 (2008) 85–96

Research report

Inactivation of the medial prefrontal cortex of the rat impairs strategy set-shifting, but not reversal learning, using a novel, automated procedure Stan B. Floresco ∗ , Annie E. Block, Maric T.L. Tse Department of Psychology and Brain Research Centre, University of British Columbia, 2136 West Mall, Vancouver, B.C. V6T 1Z4, Canada Received 7 December 2007; received in revised form 30 January 2008; accepted 8 February 2008 Available online 15 February 2008

Abstract The medial prefrontal cortex (mPFC) of the rat plays an essential role in behavioral flexibility, as lesions or inactivations of this region impair shifting between strategies or attentional sets using a variety of different behavioral tests. In the present study, we assessed the effects of inactivation of the mPFC on strategy set-shifting and reversal learning, using a novel, automated procedure conducted in an operant chamber. In Experiment 1, inactivation of the mPFC with bupivacaine did not impair the initial learning of a visual-cue (i.e.; always press the lever with a cue light illuminated above it) or a response (i.e.; always press the left lever) discrimination. Control rats required greater number of trials to shift from using a visual-cue to a response strategy than the opposite shift. mPFC inactivation impaired performance of a visual-cue-response set-shift, but not the easier response-visual-cue shift. In Experiment 2, pre-exposure to the visual-cue stimulus lights increased the difficulty of the response-visual-cue shift, reflected by a greater number of trials required by control rats to achieve criterion relative to those in Experiment 1. Under these conditions, inactivation of the mPFC did impair performance of this set-shift. In contrast, mPFC inactivation did not affect reversal learning of a response discrimination. These findings highlight the utility of this automated procedure for assessing set-shifting mediated by the mPFC. Furthermore, they reveal that the relative difficulty of the type of shift rats are required to perform has a direct impact on whether or not the mPFC contributes to this form of behavioral flexibility. © 2008 Elsevier B.V. All rights reserved. Keywords: Prefrontal cortex; Set-shifting; Reversal learning; Perseveration; Drug discovery; Rat

A number of tasks have been developed to investigate the neural basis of different types of behavioral flexibility in rodents, using different sensory modalities and classes of stimuli. For example, Birrell and Brown [2] developed a procedure based on the intradimensional/extradimensional shifting task used with primate and human subjects [9]. In this task, rats discriminate between two bowls that can be distinguished based on a variety of features (digging media, odor, texture) to receive reinforcement. During the attentional (or extradimensional) set-shift, rats are presented with two novel bowls, and must attend to a previously irrelevant dimension (e.g., shift from texture to odor). Extradimensional shifts of attentional set are impaired by lesions of the medial prefrontal cortex (mPFC), but are unaffected by lesions of the orbitofrontal region of the PFC (OFC) [2,17]. Conversely, reversal learning assessed with this task is impaired by

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0166-4328/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2008.02.008

OFC, but not mPFC lesions [2,17]. One advantage of this task is that it tests different aspects of learning that may be required for behavioral flexibility (discrimination learning, reversals, intradimensional and extradimensional shifts). Furthermore, during the extradimensional shift, rats are presented with novel sets of stimuli. This procedure ensures that impairments in performance during this stage of the task are likely attributable to disruptions in the ability to shift attentional set to different aspects of compound stimuli, rather that an impaired ability to stop approaching a specific stimulus previously associated with reward. Another task used to assess set-shifting abilities is conducted on a cross-maze, where rats shift between visual-cue and egocentric spatial response-based discrimination strategies. Initially, rats learn to enter an arm with a visual-cue to receive reinforcement. During the set-shift, animals must turn in a specific direction (e.g; always turn left) regardless of the position of the visual-cue. Manipulations of the mPFC in rodents severely impair performance during the set-shift [11,22,26],

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whereas similar manipulations do not impair reversal learning using a similar protocol [22]. As opposed to other perceptual set-shifting tasks, where novel complex stimuli are used during the extradimensional shifts [2,17], the stimuli remain constant across different phases in this maze-based procedure, making it similar to the Wisconsin Card Sorting task used in humans. As a consequence, this task places a heavier emphasis on response conflict, because rats are presented with the same set of stimuli during the initial discrimination learning and during the set-shift, even though it requires a shift of attention from one stimulus dimension to another (e.g. visual-cue to turn direction) [25]. However, an advantage of this task is that it can be used to distinguish between different types of impairments in set-shifting, such as perseverative deficits or impairments in the ability to acquire and maintain a new strategy. We and others have used this task previously to delineate the distinct contributions that a number of brain regions connected with the PFC make to this form of set-shifting. For example, a neural circuit that includes mediodorsal thalamic and dopaminergic input to the mPFC plays a selective role in suppressing the use of a previously acquired strategy during the initial stages of the shift [3,11]. In contrast, the nucleus accumbens core and dorsomedial striatum mediate the maintenance of a novel strategy after perseveration has ceased [12,21], whereas a thalamo-accumbens circuit facilitates acquisition of a new strategy by eliminating inappropriate responses [3]. Furthermore, inactivation of the OFC does not affect set-shifting assessed in this manner, suggesting that successful performance of this task is not dependent on cognitive operations related to reversal learning [14]. A number of neuropsychiatric disorders are associated with impairments in set-shifting, including schizophrenia [18], depression [1] and attention deficit disorder [31]. Accordingly, there has been a growing interest in using animal models in conjunction with tests of behavioral flexibility to facilitate the discovery of novel pharmaceutical compounds that may ameliorate the cognitive impairments observed in these disorders. However, one drawback of the above-mentioned paradigms is that they are generally labor intensive. An experimenter can only test one animal at a time, and in some instances, testing may take 1–2 h per rat. Thus, the development of an automated set-shifting procedure would make these tests more amenable to high throughput screenings of novel compounds that may improve this domain of cognition. To this end, we developed a novel strategy set-shifting procedure conducted in an operant chamber, using the same basic rules and shifts as the maze-based procedure described above. In the present study, we tested the effects of reversible inactivations of the mPFC on strategy setshifting from a visual-cue to a response-based strategy, and vice versa. If this procedure assesses a similar form of behavioral flexibility (i.e.; set-shifting) as the above-mentioned paradigms, then it would be expected that inactivation of mPFC should impair performance. In addition, we also assessed the effects of mPFC inactivation on a simpler form of behavioral flexibility, namely, reversal learning of a response discrimination, which is sensitive to lesions of the OFC and its striatopallidal outputs, but not the mPFC [4,10,14,22].

1. Materials and methods 1.1. General method 1.1.1. Subjects and surgery Long Evans rat (300–400 g, Charles Rivers, Montreal QC) were anesthetized with 100 mg/kg of ketamine hydrochloride and 7 mg/kg xylazine, and bilaterally implanted with 23-gauge stainless-steel guide cannulae into the prelimbic region of the mPFC (flat skull: AP = +3.0 mm, ML = ±0.7 mm from bregma, and DV = −2.7 mm from dura). Thirty-gauge obdurators flush with the end of the guide cannulae remained in place until the injections were made. Each rat was given at least 7 days to recover from surgery prior to training. During this recovery period, animals were food restricted to 85% of their free feeding weight and were handled for at least 5 min per day. 1.1.2. Apparatus All testing was conducted in eight operant chambers (30.5 cm × 24 cm × 21 cm; Med-Associates, St. Albans, VT, USA) enclosed in sound-attenuating boxes. Boxes were equipped with a fan to provide ventilation and to mask extraneous noise. Each chamber was fitted with two retractable levers, one located on each side of a central food receptacle where food reinforcement (45 mg; BioServ, Frenchtown, NJ) was delivered by a pellet dispenser. A light emitting diode stimulus light was positioned centrally above each lever and served as a stimulus for visual-cue discrimination learning. Each chamber was illuminated by a single 100-mA house light located in the top-center of the wall opposite the levers. All experimental data were recorded by an IBM personal computer connected to the chambers via an interface. 1.1.3. Microinfusion procedure Infusions into the mPFC were made through 30-gauge injection cannulae extending 0.8 mm below the guide cannulae. The injection cannulae were attached by a polyethylene tube to a 10 ␮l syringe. Saline or the local anesthetic bupivacaine hydrochloride (0.75%; Abbott Laboratories Saint Laurent, Quebec) was infused at a rate of 0.5 ␮l/72 s by a microsyringe pump. Injection cannulae were left in place for an additional 1 min to allow for diffusion. Following the infusion, the rat was placed in the operant chamber. Behavioral testing commenced 10 min later. In Experiment 1, all rats received at least two and a maximum of three microinfusions. In Experiment 2, infusions were only delivered prior to the set-shift or reversal, thus rats received one or two infusions (see below). 1.1.4. Histology Upon completion of behavioral testing, the rats were sacrificed in a carbon dioxide chamber. Brains were removed and fixed in a 4% formalin solution. The brains were frozen and sliced in 50 ␮m sections prior to being mounted and stained with Cresyl Violet. Placements were verified with reference to the neuroanatomical atlas of Paxinos and Watson [19].

2. Experiment 1: effects of mPFC inactivation on strategy set-shifting from visual-cue to response strategies and vice versa 2.1. Pretraining On the day before initial exposure to the operant chamber, rats were given ∼ 20 reward pellets in their home cage. Before the animal was placed in the chamber on the first day of training, 2–3 crushed pellets were placed in the food cup and on the active lever. Rats were trained under a fixed-ratio 1 schedule to a criterion of 50 presses in 30 min, first for one lever, then the other (counterbalanced left/right between subjects). On subsequent days, rats were familiarized with the insertion of the levers into the chambers and were trained to press them within

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10 s of insertion. These sessions consisted of 90 training trials and began with the levers retracted and the chamber in darkness. Every 20 s, a trial began with illumination of the houselight and insertions of one of the two levers into the chamber. If the rat failed to respond on the lever within 10 s, the lever was retracted, the chamber darkened and the trial was scored as an omission. If the rat responded within 10 s, the lever retracted, a single pellet was delivered immediately and the houselight remained illuminated for another 4 s. In every pair of trials, the left or right lever was presented once, and the order within the pair of trials was random. Importantly, the stimulus lights above each of the levers were never illuminated during these training sessions. All rats received at least 4 daily training sessions. Rats had to achieve a criterion of less than 5 omissions over the 90 trials before proceeding to the next stage of testing. On the last day of pretraining, the side bias for the rat was determined. This procedure was modified from that used with maze-based set-shifting tasks that we have described previously [3,11,12]. These sessions were similar to pretraining session, except that both levers were inserted into the chamber. Again, the stimulus lights above the levers were not illuminated during these trials. On the first trial, a food pellet was delivered after responding on either lever. Upon subsequent insertion of the levers, food was delivered only if the rat responded on the lever opposite to the one chosen initially. If the rat chose the same lever as the initial choice, no food was delivered, and the houselight was extinguished. This continued until the rat chose the lever opposite to the one chosen initially. After choosing both levers, a new trial commenced. Thus, one trial for the sidebias procedure consisted of responding on both levers. The lever (right or left) that a rat responded on first during the initial choice of a trial was recorded and counted toward its side bias. If the total number of responses on the left and right lever were comparable, the lever that a rat chose initially four or more times over seven total trials was considered its side bias. However, if a rat made a disproportionate number of responses on one lever over the entire session (i.e., greater than a 2:1 ratio), that lever was considered its side bias. After determining the side bias, a rat’s obdurators were removed from the guide cannulae and two injection cannulae extending 0.8 mm past the guide cannulae were inserted for 1 min, but no solution was injected. Visual-cue (Experiment 1A) or response (Experiment 1B) discrimination training commenced on the following day. 2.1.1. Experiment 1A: visual-cue to response strategy set-shifting procedure 2.1.1.1. Initial visual-cue discrimination. The visual-cue discrimination was also adapted from procedures we have employed previously using a maze-based set-shifting task [3,11,12]. For this discrimination, the rat was required to respond on the lever that had a visual-cue stimulus light illuminated above it (Fig. 1, top panels). A session began with both levers retracted and the chamber in darkness (the intertrial state). Every 20 s, a trial began with illumination of one of the stimulus lights above one of the levers. Three seconds later, the houselight was illuminated and both levers were inserted into the chamber. This procedure was employed because pilot studies revealed that rats

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Fig. 1. Diagram of the types of discriminations used in automated strategy set-shifting procedures conducted in an operant chamber. During visual-cue discrimination learning (top), rats are required to always press the lever that had a stimulus light illuminated above it. For response discrimination learning (bottom), rats are trained to always press one of the levers (e.g., left) regardless of the position of the cue light.

took substantially more trials to learn the visual-cue discrimination if illumination of the cue lights and lever insertion occurred simultaneously [7]. A response on the lever with the stimulus light illuminated above it (a correct response) resulted in the retraction of both levers and delivery of one food pellet. After food delivery, the houselights remained on for another 4 s, after which the chamber returned to the intertrial state. If the rat responded on the other lever (incorrect response), both levers retracted immediately and the chamber reverted to the intertrial state. Failure to respond on either lever within 10 s resulted in retraction of both levers, extinguishing of the houselight, and the trial was recorded as an omission. In every pair of trials, the left or right stimulus light was illuminated once, and the order within the pair of trials was random. For each trial, the lever that the animal chose and the location of the stimulus light was recorded. Trials continued until either 1) a rat had received a minimum of 30 trials and achieved criterion performance of 8 consecutive correct responses [26] or 2) after 120 trials, whichever came first. This criterion of 8 correct responses was adapted from a previous study [26], and was used to ensure that rats could learn the first discrimination in one session, in order to minimize the number of microinfusions rats would receive. Omission trials were not included in the trials to criterion measure. Upon completion of a training session, the rat was removed from the chamber and

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placed back in its home cage. If a rat did not achieve criterion on the first day of training, on the following day, it received a second microinfusion and subjected to a second, identical session of visual-cue discrimination learning. 2.1.1.2. Shift to response discrimination. The acquisition of this discrimination required the animal to cease the use of a visual-cue discrimination strategy, and instead use an egocentric spatial response strategy to obtain food reward (Fig. 1, lower panels). Here, a correct response entailed responding on the lever opposite of its side bias (left or right), regardless of the location of the stimulus light illuminated above one of the levers. As with the initial visual-cue discrimination, in every pair of trials, the left or right stimulus light was illuminated once, and the order within the pair of trials was random. Trials were given in a manner identical to initial visual-cue discrimination trials, and again, for each trial, the lever that the animal chose and the location of the stimulus light was recorded. Trials continued until either (1) a rat achieved criterion performance of 10 consecutive correct responses or (2) after 120 trials. Again, if a rat did not achieve criterion on the first day of training, the animal would receive an identical microinfusion and a session of response training on the following day. None of the rats in this study required more than 2 days of training to achieve criterion performance on either discrimination, nor did they require more than three training days in total (i.e.; received more than three microinfusions) to complete the experiment, regardless of their treatment. 2.1.1.3. Error analysis. We utilized an error analysis similar to that employed for maze-based set-shifting procedures [3,11,12,20]. Errors committed during the set-shift were broken down into three error subtypes to determine whether treatments altered the ability to either shift away from the previously learned strategy (perseverative errors) or to acquire and maintain the new strategy after perseveration had ceased (regressive or neverreinforced errors). A perseverative error was scored when a rat responded on a lever with the stimulus light illuminated above it on trials that required the rat to press the opposite lever. For example, during the set-shift, the rat may have been required to always press the left lever, a perseverative error was scored when the rat pressed the right lever when the stimulus light was illuminated above it. Eight out of every sixteen consecutive trials required the rat to respond in this manner (i.e.; press the lever opposite of the previously learned rule). In a manner similar to that described in previous studies [3,11,12,20,22] these types of trials were separated into consecutive blocks of 8 trials each. Perseverative errors were scored when a rat pressed the incorrect lever on 6 or more trials per block of 8 trials where the rat was required to press the lever that did not have the stimulus light illuminated above it. Once a rat made fewer than five perseverative errors in a block for the first time, all subsequent errors of this type were no longer counted as perseverative errors, because at this point the rat was using the original strategy less than 75% of the time. Instead, these errors were now scored as regressive errors. The third type of error, termed never-reinforced errors was scored when a rat pressed the incorrect lever on trials where the visual-cue light was illuminated above the same lever that the

rat was required to press during the set-shift. For example, during visual-cue discrimination training, rats learned to press the lever with the visual-cue light illuminated above it, regardless of its spatial position. During the shift, a rat might be required to always respond on the left lever and for half of the trials the cue was illuminated above the left lever. In this situation, a neverreinforced error was scored when a rat chose the right lever (i.e.; a choice that was not reinforced during initial discrimination training or the shift). Regressive and never-reinforced errors are used as an index of the animals’ ability to maintain and acquire a new strategy, respectively. 2.1.2. Experiment 1B: response to visual-cue strategy set-shifting procedure For this experiment, rats were initially trained on the response discrimination task followed by testing on the visual-cue version of the set-shift. All other aspects of the testing procedure were identical to those described above. On the shift to the visualcue discrimination, the same measures were assessed as those for Experiment 1A, where rats were required to shift from a visual-cue to a response strategy. However, for the set-shift, perseverative and regressive errors were analyzed from the trials in which a rat was required to respond on the lever opposite to the one it had been trained to press during initial response discrimination training. Rats received microinfusions prior to both the initial discrimination learning and the strategy set-shift. For both Experiments 1A and B, each rat was assigned to one of three infusion treatment groups: (1) initial discrimination, saline and set-shift, saline; (2) initial discrimination, saline and set-shift, bupivacaine; (3) initial discrimination, bupivacaine and set-shift, saline. The first group served as the control group, the second determined whether PFC inactivation impaired strategy set-shifting, and the third determined whether inactivation of the PFC affected the acquisition of discrimination learning that may yield behavioral differences during the set-shift. Assignment of rats to either first or second group was counterbalanced based on performance during initial discrimination learning following saline infusions. 2.2. Results 2.2.1. Experiment 1A 2.2.1.1. Visual-cue discrimination. Rats that received saline infusions prior to initial visual-cue discrimination training achieved criterion performance in ∼60 trials (n’s = 8 per group; Fig. 2A). Inactivation of the mPFC with infusions of bupivacaine (n = 8) did not affect the number of trials to achieve criterion on this discrimination. This was confirmed using a one-way ANOVA (F(2,21) = 0.02, n.s.). Only one rat in the control group and one rat that received bupivacaine infusions required a second day of training on the visual-cue discrimination. Thus, inactivation of the mPFC does not affect learning of a visual-cue discrimination strategy. 2.2.1.2. Shift to response. The results of the set-shift to the response strategy are presented in Fig. 2B. Rats in the control

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Fig. 2. Experiments 1A and B. Data are expressed as means + S.E.M. (A). Trials to criterion during initial visual-cue discrimination training by rats receiving infusions of saline (white, black bars) or bupivacaine into the mPFC (Bupi, hatched bar). (B) Trials to criterion on the set-shift to the response strategy following infusions of either saline (white and hatched bar) or bupivacaine (black bar; star denotes p < 0.05 significantly different from saline/saline). (C) Analysis of the type of errors committed in Experiment 1A during the set-shift. (D) Trials to criterion on acquisition of the response discrimination by rats receiving either infusions of saline or bupivacaine. (E) Trials to criterion on the shift to the visual-cue strategy following infusions of either saline or bupivacaine. (F) Comparison of the trials to criterion data for all saline-treated rats during visual-cue (striped bars) or response (grey bars) discrimination training for initial discrimination training (left) and for the set-shift (right). In both instances, rats required fewer trials to reach criterion when learning a visual-cue discrimination. Star denotes p < 0.05 significantly difference between discriminations.

group achieved criterion performance of 10 correct consecutive choices in ∼ 80 trials, which is similar to what we have observed for this type of shift using maze-based procedures [3,11,12]. Rats receiving inactivation of the mPFC prior to the set-shift required a substantially greater number of trials to reach criterion. Analysis of these data revealed a significant effect of Treatment (F(2,21) = 3.89, p < 0.05). Multiple comparisons with Dunnett’s test indicated that rats receiving bupivacaine infusions into mPFC prior to the set-shift required significantly (p < 0.05) more trials to achieve criterion than saline-treated rats. Furthermore, rats that received mPFC inactivation prior to initial visual-cue discrimination training did not differ from control rats. Only one rat in the control group required a second day of training on the response discrimination during the set-shift. In contrast, three of the eight rats that received mPFC inactivation prior to the set-shift required a second day of training

to achieve criterion performance. There were no differences between groups in the number of trial omission (F(2,21) = 1.11, n.s.). Analysis of error types made during the set-shift was conducted using a two-way, mixed ANOVA, with Treatment as the between subjects factor and error type as the within subjects factor (see Fig. 2C). For this preliminary analysis, we compared the total number of perseverative-like errors (both perseverative and regressive errors) to the number of neverreinforced errors committed. The analysis revealed a significant treatment × error type interaction (F(2,21) = 3.75, p < 0.05). Simple main effect analyses indicated that there were no significant differences between groups in the number of neverreinforced errors, whereas rats receiving mPFC inactivation prior to the set-shift made significantly more perseverativelike errors relative to saline-treated rats (p < 0.05). Subsequent

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one-tailed comparisons between rats in saline-bupivacaine and control group revealed a trend towards an increase in perseverative errors (t(14) = 1.36, p = 0.098) but not for regressive errors (t(14) = 0.72, n.s.). Thus, as has been observed using maze-based procedures, inactivation of the mPFC impairs strategy set-shifting from a visual-cue to response-based strategy. 2.2.2. Experiment 1B 2.2.2.1. Response discrimination. Control rats achieved criterion performance in ∼80 trials (n’s = 10 per group; Fig. 2D). Inactivation of the mPFC (n = 8) did not affect the number of trials to achieve criterion of this discrimination (F(2,25) = 0.01, n.s.). mPFC inactivation did not alter the number of trial omissions relative to saline-treated rats in the other two groups (F(2,25) = 1.39, n.s.). Only one rat in the saline-bupivacaine group required a second day of training on the initial response discrimination. Thus, inactivation of the mPFC does not affect learning of an egocentric spatial response discrimination. 2.2.2.2. Shift to visual-cue. The results of the set-shift to the visual-cue based strategy are presented in Fig. 2E. There were no trial omissions by rats in any of the groups. Rats in the control group achieved criterion performance of 10 correct consecutive choices in ∼45 trials, substantially fewer than what was observed from rats shifting from a visual-cue to response strategy in Experiment 1A. Furthermore, inactivation of the mPFC prior to the set-shift did not affect performance using this type of shift, as there were no significant difference between groups on the number of trials to achieve criterion performance (F(2,25) = 0.26, n.s.). One rat in the control group and one rat that received bupivacaine prior to initial response discrimination training required 2 days of training on the set-shift to achieve criterion performance. Of the rats that received mPFC inactivation prior to the set-shift, two rats required a second day of training to achieve criterion. However, performance in this group was highly variable, as four rats reached criterion in fewer than 20 trials, making one or a few errors. This suggests that using these procedures, this type of shift is substantially easier for rats to perform than the visual-cue to response shift used in Experiment 1A. We conducted subsequent analyses comparing the trials to criterion for both visual-cue and response tasks. Analysis of these data included all rats that received saline infusions during initial discrimination training (i.e.; control rats and rats that would receive bupivacaine infusions prior to the set-shift). As can be seen in Fig. 2F, rats trained initially on the visualcue discrimination (n = 16) required significantly fewer trials to achieve criterion performance than those animals trained on the response discrimination (n = 20; F(1,34) = 4.02, p = 0.05). A similar trend was observed when we compared the number of trials required by control rats to learn either the visualcue (n = 10) or response (n = 8) discriminations during the set-shift (F(1,16) = 4.15, p = 0.05). Thus using these procedures, rats learned visual-cue discrimination much more rapidly than response discriminations, suggesting that these training

procedures may promote a bias to using a visual-cue-based strategy. 3. Experiment 2: effects of mPFC inactivation on shifting from response to a visual-cue strategy using modified training procedures In Experiment 1, inactivation of the mPFC impaired shifting from a visual-cue to a response strategy, analogous to what has been reported using similar set-shifting tasks conducted on mazes employing shifts between these two discrimination strategies [3,11,20,26]. However using procedures in an operant chamber, mPFC inactivation did not affect shifting from a response to a visual-cue strategy, even though manipulations of the PFC have been shown to disrupt this type of shift using mazebased procedures [3,11,20,26]. Comparison of the two types of discriminations revealed that rats required fewer trials to learn a visual-cue discrimination using stimulus lights, compared to response learning, where rats must disregard the stimulus light and always respond on either the right or left lever. This suggests that the cue light may have greater salience than the spatial location of the lever when learning these discriminations. This may be attributable in part to the novelty of these stimuli, given that rats were not exposed to these light cues during pretraining. Furthermore, these findings imply that rats may have an innate bias to use this type of strategy, entailing alternate responding on each lever based on the position of a visual-cue light presented in a darkened chamber. Therefore, the response to visual-cue shift used in Experiment 1B may have been less difficult than the opposite shift used in Experiment 1A, given that rats seem to have an innate tendency to engage in a visual-cue-based strategy to begin with. Indeed, previous studies have shown that manipulation of the mPFC or interconnected brain regions can have differential effects on set-shifting, depending on the difficulty or types of shift [3,5,23]. The possibility remains that if the novelty and/or salience of the visual-cue lights were reduced throughout the course of pretraining and initial response discrimination learning, mPFC inactivation may be more effective at disrupting performance during this type of shift. This hypothesis was tested directly in Experiment 2. Here we used a similar protocol to that used in Experiment 1B, with some key modifications to our pretraining and initial response discrimination procedures. 3.1. Method 3.1.1. Pretraining The initial pressing training and subsequent training with retractable levers was conducted in a manner similar to that of Experiment 1, with two key exceptions. First, during the retractable lever pretraining, insertion of either lever into the chamber was also associated with illumination of both visualcue stimulus lights. These lights extinguished after the rat responded on the inserted lever. Second, rats were given 7–8 training sessions with the retractable levers, instead of the 5 sessions given in Experiment 1. Habituating the animals to the stimulus lights during pretraining was implemented to

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reduce the salience and novelty of the visual-cue lights during the initial response discrimination training and visual-cue set-shift. 3.1.2. Response to visual-cue strategy set-shifting procedure The initial response discrimination training and set-shifting to the visual-cue strategy was conducted in a near identical manner to that used in Experiment 1, with two key exceptions. First, rats did not receive microinfusions prior to initial response discrimination training. Second, during the initial response discrimination, rats were trained to a criterion of 12 consecutive correct choices, instead of the 8 trials used in Experiment 1. These manipulations would be expected to increase task difficulty, reflected by an increased number of trials to achieve criterion during the set-shift by control rats, compared to those in Experiment 1B. Rats were matched for performance of the initial response discrimination, and received infusions of either saline or bupivacaine into the mPFC prior to the set-shift. As in Experiment 1, if a rat did not achieve criterion on the first day of training, the animal would receive an identical microinfusion and a session of response training on the following day. 3.2. Results 3.2.1. Response discrimination Rats in this experiment (n = 25) achieved criterion performance of 12 consecutive correct choices on the response discrimination in 81 ± 5 trials. This was comparable to the number of trials required by rats in Experiment 1B to learn the same type of discrimination (84 ± 8), even though criterion in that experiment was only 8 consecutive correct choices. We extrapolated the number of trials required by rats in Experiment 2 to make 8 consecutive correct choices, to directly compare the rates of learning between both experiments. Rats exposed to the stimulus lights during pretraining required significantly fewer trials to make 8 correct consecutive choices (61 ± 4; Fig. 3A) compared to rats in Experiment 1B that had not received such pre-exposure (F(1,43) = 8.44, p < 0.01). This suggests that exposure to the visual-cue lights during pretraining decreased the novelty and salience of these stimuli. This in turn may have decreased the likelihood of rats attending to these stimuli during the initial response learning, which may have facilitated the acquisition of this strategy. Rats were matched for trials to criterion on the response discrimination (F(1,22) = 0.08, n.s.; Fig. 3B, inset), and the next day, received infusions of either saline of bupivacaine prior to the set-shift to the visual-cue discrimination. 3.2.2. Shift to visual-cue Control rats (n = 11) achieved criterion on the set-shift in ∼75 trials, which was substantially greater than control rats in Experiment 1B that were not exposed to the stimulus lights during pretraining (∼45 trials). This further supports the notion that such pre-exposure reduces the salience of these stimuli, making it more difficult for rats to attend to them when required to

Fig. 3. Experiment 2. Comparison of the trials to criterion data (8 correct consecutive responses) for all rats during initial response discrimination training in Experiment 1, where rats were not pre-exposed to the stimulus lights during pretraining (grey bar) and Experiment 2, where rats did receive such pre-exposure (white bar). Star denotes p < 0.05 significant difference between groups. (B) Trials to criterion on the response discrimination (inset) and during the set-shift to the visual-cue discrimination following infusions of saline (white bars) or bupivacaine (black bars). Star denotes p < 0.05 significant difference between groups. (C) Analysis of the type of errors committed in Experiment 2 during the set-shift.

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shift to a visual-cue strategy. As was observed in Experiment 1B, performance in the control group was highly variable. Four of the rats in this group (36%) achieved criterion in < 20 trials, making only 1–3 errors. However, another 3 rats (27%) required an additional day of training on the set-shift; one of these rats did not achieve criterion even after 240 training trials. Performance of rats receiving inactivation of the mPFC (n = 13) was also variable; 3 rats (23%) achieved criterion in < 25 trials. However, 9 of these animals (69%) required an additional day of training on the set-shift, and 5 of these did not achieve criterion even after 240 trials. An ANOVA of these data revealed that rats treated with bupivacaine prior to the set-shift required significantly more trials to achieve criterion compared to saline-treated rats (F(1,22) = 5.86, p < 0.05; Fig. 3B). These findings suggest that pre-exposing animals to the visual-cue and providing additional training trials during initial discrimination training made this response to visual-cue shift more difficult. Despite the individual variability in both groups, under these conditions, inactivation of the mPFC was effective in impairing set-shifting. Errors were analyzed in a manner similar to Experiment 1A. The analysis revealed a significant main effect of Treatment (F(1,22) = 4.41, p < 0.05), indicating that, mPFC inactivation increased the total number of errors. However, the treatment × error type interaction only approached statistical significance (F(2,21) = 4.01, p = 0.058). Inspection of Fig. 3C reveals that control rats made very few never-reinforced errors, but a similar number of both perseverative and regressive errors. Inactivation of the mPFC not only induced a disproportionate increase in the number of perseverative errors, but also moderately increased the number of regressive error as well. In contrast, manipulations of the mPFC have shown to selectively increase total perseverative errors on the maze-based set-shift tasks [11,22,26]. Thus, it appears that this particular set-shifting procedure may not be as sensitive as the maze-based procedures for detecting differential impairments in ceasing the use of an old strategy or maintenance of a new strategy. This may be due, in part, to the high degree of variability in rats performing the response to visual-cue shift. 4. Experiment 3: effects of mPFC inactivation on response reversal learning The strategy set-shifting task used in the present study required rats to cease attending to a previously relevant stimulus dimension (e.g., lever position) and attend to a newly relevant stimulus (e.g., the visual-cue) in order to obtain reward. However, given that the same stimuli were used during the initial discrimination and during the set-shift, it may be argued that this task also assessed a form of reversal learning. In this regard, lesions or inactivation of the mPFC typically do not impair reversal learning using a variety of different training protocols and stimuli [2,4,22, but see 6]. Nevertheless, we conducted another experiment assessing the effects of mPFC inactivation on learning of a response reversal. A lack of an effect in this experiment would suggest that the impairments in behavioral flexibility observed in Experiment 1 were not attributable to a disruption of processes related to reversal learning.

4.1. Method 4.1.1. Pretraining The pretraining protocols were identical to those used for Experiment 1 (i.e.; 5 days of retractable lever training, no stimulus light presentation during pretraining). 4.1.2. Response discrimination training and reversal The initial response discrimination was conducted in a manner identical to that used for Experiment 1B, except that rats did not receive microinfusions prior to initial response training. For the initial discrimination, rats were trained to press the lever opposite of their side bias (left or right), regardless of the position of the visual-cue light, to a criterion of 8 consecutive correct choices. On the following day, animals were trained on a reversal of this discrimination. Here, the position of the reinforced lever was reversed. Training trials continued until a rat achieved criterion performance of 10 consecutive correct choices. Rats were matched for performance of the initial response discrimination, and received infusions of either saline or bupivacaine into the mPFC prior to the reversal. 4.2. Results Rats achieved criterion performance on the initial response discrimination in 87 ± 7 trials (Fig. 4A), which was comparable to rats in Experiment 1B. All of the rats reached criterion in one training session. Animals were matched for performance and on the subsequent day, received infusions of saline (n = 7) or bupivacaine (n = 7) prior to reversal learning. As can be observed in Fig. 4B, inactivation of the mPFC did not affect reversal learning of the response discrimination, where rats were now required to cease responding on one lever and press the opposite lever (F(1,12) = 0.087, n.s.). Rats in both groups required a substantially greater number of trials (∼115) to achieve criterion performance of 10 consecutive correct choices during the reversal. One of the rats in the saline group and two of the rats in the bupivacaine group required a second training session to acquire the reversal discrimination. There were no differences in the number of trial omissions between groups (F(1,12) = 1.61, n.s.). Thus, inactivation of the mPFC does not impair learning of an egocentric spatial reversal. Representative placements of all acceptable infusion for rats in Experiment 1 are presented in Fig. 5. Rats with placements that are asymmetrical, located rostral in the medial orbital cortex, or ventral in the infralimbic cortex were excluded from the data analysis. Five of these rats received bupivacaine infusions prior to the set-shift in Experiment 1A or Experiment 2. These animals were generally unimpaired on the task, compared to the other rats in the respective groups that had accurate placements. 5. Discussion The present study utilized a strategy set-shifting task conducted in an operant chamber employing similar rule shifts used in maze-based set-shifting tasks [3,11,12,20,22]. In general, control rats acquired the visual-cue discrimination much

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Fig. 4. Experiments 3. (A). Trials to criterion during initial response discrimination training by rats that would receive infusions of saline (white) or bupivacaine (black bars) into the mPFC prior to reversal training. (B) Trials to criterion during the reversal of the response discrimination following infusions of either saline or bupivacaine.

more rapidly that the response discrimination, suggesting that rats may have a pre-existing bias to using the former strategy. Inactivation of the mPFC did not impair the initial discrimination learning of either strategy, but did impair shifting from a visualcue to a response-based strategy (Experiment 1A), consistent with previous findings [22]. Interestingly, PFC inactivations did not impair performance when rats were required to perform the easier response to visual-cue set-shift (Experiment 1B). Yet, in Experiment 2, when rats were pre-exposed to the visual-cue lights during pretraining, thereby reducing the salience of these stimuli, inactivation of the mPFC did impair performance on this type of shift. Furthermore, similar inactivations did not impair reversal learning of a response discrimination. Thus, in accordance with previous findings, the present data further support the notion that the mPFC plays a selective role in facilitating shifts between rules, strategies or attentional sets, but is not essential for shifting between different stimulus-reward associations within a particular stimulus dimension [2,4,22]. These findings demonstrate the utility of this automated procedure for assessing set-shifting functions mediated by the mPFC. They also provide

Fig. 5. Histology. Schematic of coronal sections of the rat brain showing the placements of the cannulae tips for all rats in Experiment 1. Brain sections correspond to the atlas of Paxinos and Watson [19].

important new information, indicating that relative salience of the particular stimuli used in these discriminations (i.e.; lights vs spatial location) can have a direct impact on whether or not neural activity in the mPFC is required for shifting between strategies. 5.1. Comparisons with other set-shifting tasks As noted previously, it is now well established that lesions or inactivation of the mPFC impairs performance on tasks that engage more complex forms of behavioral flexibility. For example, Birrell and Brown [2] showed that lesions of the mPFC induce a selective impairment in extradimensional shifts of attention, when rats were required to change their discrimination strategy from one stimulus dimension to a previously

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irrelevant dimension (e.g., shifting from texture to odor discriminations). In that same study, rats with lesions of the mPFC learned a reversal of either a texture or odor discrimination as readily as sham lesions animals. In a similar vein, lesions, inactivations or pharmacological manipulations of the mPFC also impair shifts between different discrimination strategies similar to the ones used in the present study, but not reversal learning within a particular stimulus dimension [4,11,22,26]. In contrast to the above-mentioned findings, lesions of the OFC impairs reversal learning, but leaves set-shifting intact [14,15,17,24]. With respect to the present study, the fact that inactivation of the mPFC impaired strategy set-shifting, but not reversal learning further supports the notion in an attempt to solve this type of set-shifting task, rats do not appear to use a cognitive strategy entailing a simple change in stimuli valence within a particular dimension. Rather, it is more likely that rats solve this task by taking a fundamentally new approach, which entails attending to particular components of complex stimuli [14,15,30]. Thus, rats likely engage in extradimensional shifts of attention when solving this task, redirecting attention to a previously irrelevant stimulus dimension (i.e.; egocentric spatial location instead of proximal visual-cue). There were a number of similarities in how rats learned visual-cue and response discrimination rules in an operant chamber, compared to what we have observed previously using maze-based procedures. For example, using both procedures, we and others have observed that rats typically require fewer training trials to achieve criterion on a visual-cue discrimination versus a response discrimination [3,11,12,20]. In the present study, rats required ∼60 trials to learn the visual-cue strategy, and ∼85 trials to learn the response strategy, which is comparable to what we have reported previously using a maze-based task [11,12]. We have also observed that rats tend to require fewer trials to achieve criterion on the response-to visual-cue set-shift than the converse type of shift conducted on a maze, also consistent with the present findings. There are a number of reasons why rats may learn a visual-cue discrimination more readily that a response discrimination. First, rats generally have an innate bias to using an alternation strategy [8], and the visualcue discrimination requires rats to alternate between either one of two levers or maze arms, with the correct choice indicated by the presence of a proximal visual-cue. In the present study, this bias may have be augmented during pretraining, where rats were trained to press one of the two levers, the location of which alternated from trial to trial. Furthermore, during discrimination training, the stimulus lights were illuminated 3 s before the houselight and extension of the levers. This would be expected to attract the attention of the rat to that location of the chamber, increasing the probability of the rat pressing that particular lever. A key difference between the present findings and those obtained using a maze-based set-shifting task is that inactivation of the mPFC had differential effects on strategy set-shifting, depending on the type of shift used. In Experiment 1A, mPFC inactivation impaired shifting from a visual-cue to a response strategy, whereas in Experiment 1B, similar inactivations were without effect. In contrast, we and others have shown that manipulations of the mPFC are equally effective at impairing both

types of shifts conducted on a cross maze [11,20,26]. However, there have been a number of reports in the literature showing that manipulations of the PFC can cause differential effects on set-shifting, depending on the difficulty of the particular shift. Ragozzino et al. [23] demonstrated that inactivation of the mPFC produced an enduring impairment in the ability of rats to shift from using an allocentric spatial to visual-cue search strategy on a “cheeseboard” task. However, when rats were required to shift from using a visual-cue to a spatial strategy, similar inactivations of the PFC produced only a transient impairment. In a similar vein, dopamine depletions of the lateral PFC in marmosets impaired the initial acquisition of an attentional set, resulting in improved performance during an extradimensional shift, but only in animals performing a more difficult “lines to shape” shift [5]. In that study, monkeys tended to show a general bias towards responding to ‘shape’ exemplars as compared to ‘line’ exemplars during discrimination learning and extradimensional shifts, resembling the present findings that rats learned a visual-cue rule more readily than a response rule. The notion that task difficulty has an influence on whether or not PFC manipulations can affect set-shifting is supported by findings from human patients with frontal lobe damage performing the Wisconsin Card Sorting task. In these studies, reducing task difficulty by alerting subjects to an impending category shift dramatically improves performance [16,27]. In Experiment 1B of the present study, control rats required substantially fewer trials to successfully complete the response to visual-cue shift, compared to those rats performing the opposite shift, suggesting that rats found the former shift substantially easier to perform. As discussed above, this is likely due to the fact that rats generally learned the visual-cue discrimination faster, regardless if it was during initial discrimination training or the set-shift. Indeed, a number of rats in Experiment 1B achieved criterion on the visual-cue discrimination after making only one or no errors. Thus, the lack of effect of mPFC inactivations on this shift may simply be attributable to the fact that this was an easier shift for the animals to perform, given that many rats appear to have had a pre-existing bias to using the visual-cue lights to guide their choice behavior under these conditions. Thus, the present data, in combination with the above-mentioned findings indicate that when devising tasks for animals that require shifts between discrimination strategies, one must take into account potential preexisting biases animals may have towards using one type of strategy over another. In Experiment 2, we sought to make the response to visualcue shift more difficult, using two distinct manipulations. First, rats were pre-exposed to the stimulus lights during the pretraining phase. The rationale behind this manipulation was that pre-exposure to these stimuli would render them less salient during discrimination learning. It is well established that preexposure to a stimulus prior to associative learning can impede subsequent learning about that stimulus, a phenomenon referred to as latent inhibition [28,29]. This pre-exposure was effective at reducing the salience of these stimuli during initial response discrimination training. Rats learned the response discrimination in fewer trials compared to animals in Experiment 1B, presumably because they were less likely to use the cue lights to guide their behavior. Second, we increased the number of trials to criterion

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required during initial response discrimination training, which would further augment the association between the particular lever and reinforcement. These manipulations were successful in making the shift to a visual-cue strategy more difficult, in that control rats required more trials to achieve criterion performance during the set-shift compared to rats in Experiment 1B that were not subjected to these conditions. Moreover, following these changes to our training protocols, inactivation of the mPFC did impair performance during the set-shift. It is important to note that performance in both groups was still highly variable, with some animals in each group making very few errors during the set-shift, while others were unable to achieve criterion after 240 trials. Taken together, these findings suggest that the involvement of the rodent mPFC in strategy shifting is critically dependent on the nature and inherent difficulty of the shift the animal is required to perform. Furthermore, our findings that the visual-cue to response shift yielded more reliable and less variable data indicate that this shift may be better suited for future studies using these procedures to investigate the roles of other brain systems in the mediation of this form of behavioral flexibility. 5.2. Analysis of the types of errors An inherent advantage to these types of strategy set-shifting tasks conducted on a maze or in an operant chamber is that a detailed analysis of the types of errors rats make can distinguish between different types of impairments in this form of behavioral flexibility. Thus, early in the set-shift, rats persist in using the previously relevant, but now incorrect strategy (e.g., approach the visual-cue, instead of always make a “left” response). On half of the trials, rats still obtain food using this strategy (i.e.; when the cue is in the left position), whereas on the remaining trials, they do not. Early in the set-shift, these types of errors are scored as perseverative, and are used as an index of how readily animals are able to inhibit the use of the now incorrect strategy. As training progresses, rats start to disengage from the previously relevant strategy. When rats start to use the new strategy on less than 75% of trials where using the previous strategy would be incorrect, (3 out of 4 trials using maze-based procedures, or 6 out of 8 trials in the present study) these types of errors are no longer scored as perseverative. This is because at this point, the animal is attempting a new strategy on 40–50% of trials. Instead, these errors are now scored as “regressive” and serve as an index of the ability to maintain a novel strategy once perseveration has ceased. Rats can also make a third type of error on this task, when they make a response that was incorrect during both initial discrimination training and during the shift (e.g., making a “right” response when the visual-cue is in the “left position). These are termed “never-reinforced” errors and are used as an index of how quickly animals are able to parse out ineffective strategies. During the set-shift, intact rats learn quickly that the previously correct strategy is no longer appropriate and engage alternative strategies until they find the optimal solution. Never-reinforced errors may be interpreted as an attempt to use alternative strategies, perhaps a reversal of the previously acquired rule (e.g., always make a “right” response

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instead of “left”, or always make a response away from the cue). Previous studies using a maze-based set-shifting task in combination with this type of error analysis have revealed that separate neural circuits related to the mPFC make distinct contributions to these dissociable components of behavioral flexibility. Thus, inputs from the mediodorsal thalamus and the dopamine system converging in the mPFC plays a role in suppressing the use of a previously relevant strategy, as disruptions in this circuitry induces a selective increase in perseverative errors [3,11,22]. In contrast, inactivation of striatal outputs of the PFC, including the dorsomedial striatum and the nucleus accumbens core, do not induce perseveration, but instead increase regressive errors, suggesting that these regions facilitate the maintenance of new strategies [12,21]. Moreover, disconnection of thalamic inputs to the nucleus accumbens increases the number of neverreinforced errors, without altering perseverative or regressive errors, suggesting that this circuit facilitates the acquisition of a novel strategy [3]. In the present study, inactivation of the mPFC increased the overall number of “perseverative type” errors (i.e.; both perseverative and regressive), without affecting the number of never-reinforced errors, as has been reported using maze-based set-shifting tasks [3,11,26]. This suggests that the strategy-setshifting task used in the present study can be used to dissociate the role of particular brain systems in the suppression of previously relevant strategies and the acquisition of novel strategies. Yet, although we observed an increase in perseverative errors, mPFC inactivation also increased the number of regressive errors as well. This is in contrast to what has been reported previously using maze-based set-shifting procedures, where inactivations or pharmacological manipulations of the mPFC selectively increase perseverative but not regressive errors [11,20,22]. It is notable that the total number of errors made by saline-treated rats in Experiments 1A and 2 were similar in their magnitude and variability to what we have observed previously using mazebased procedures (between 20 and 30 total errors) [3,11,12,14]. However, in the present study, control rats tended to make more perseverative errors than regressive errors, whereas using a maze-based protocols, rats typically make a similar number of both types of errors [3,11,12,14]. Although the reasons for these discrepancies are unclear, it may be related to the differences in the relative complexity of the environments in which rats are performing these tasks. On the maze, rats are typically released from different spatial locations on each trial. During the set-shift, once perseveration has ceased, rats may attempt to use an allocentric spatial strategy to solve the task. In the operant chamber, the animal remains in the same basic spatial orientation from trial to trial, providing the rat with a fewer number of strategies to choose from during the shift relative to those available on the maze. This may increase the likelihood of rats regressing back to the original strategy during the shift, rather than attempting novel ones. The slight differences in the pattern of errors induced by mPFC manipulations using these two procedures suggest that the task used in the present study may not be as sensitive in delineating the respective roles of different brain systems in the suppression of previously strategies versus

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the maintenance of novel strategies. Nevertheless, the fact that inactivation of the mPFC did lead to perseverative deficits on this task suggests that this procedure does have utility in examining the neural basis of behavioral flexibility mediated by the mPFC. To summarize, the present study demonstrates that inactivation of the mPFC impairs strategy set-shifting using a novel, automated procedure, thereby confirming the validity of this task. Furthermore, the present results also reveal that the relative difficulty of the type of shift rats are required to perform has a direct impact on whether or not the mPFC contributes to this form of behavioral flexibility. Impairments in set-shifting are a characteristic cognitive deficit observed in a number of neuropsychiatric disorders, including schizophrenia, depression and attention deficit disorder [1,18,31]. As preclinical animal models represent a crucial first step in the drug discovery process [13], the procedures described here may serve as a practical and efficient means for assessing the efficacy of experimental pharmacological approaches intended to treat impairments in this type of executive functioning. Acknowledgments This work was supported by a Discovery Grant from the Natural Science and Engineering Research Council of Canada and a Young Investigator Award from the National Alliance for Research on Schizophrenia and Depression (to SBF). SBF is a Canadian Institutes of Health Research New Investigator and a Michael Smith Foundation for Health Research Scholar. References [1] Austin MP, Mitchell P, Goodwin GM. Cognitive deficits in depression: possible implications for functional neuropathology. Br J Psychiatry 2001;178:200–6. [2] Birrell JM, Brown VJ. Medial frontal cortex mediates perceptual attentional set-shifting in the rat. J Neurosci 2000;20:4320–4. [3] Block AE, Dhanji H, Thompson-Tardif SF, Floresco SB. Thalamicprefrontal cortical-ventral striatal circuitry mediates dissociable components of strategy set shifting. Cereb Cortex 2007;17:1625–36. [4] Boulougouris V, Dalley JW, Robbins TW. Effects of orbitofrontal, infralimbic and prelimbic cortical lesions on serial spatial reversal learning in the rat. Behav Brain Res 2007;179:219–28. [5] Crofts HS, Dalley JW, Collins P, Van Denderen JC, Everitt BJ, Robbins TW, et al. Differential effects of 6-OHDA lesions of the frontal cortex and caudate nucleus on the ability to acquire an attentional set. Cereb Cortex 2001;11:1015–26. [6] De Bruin JP, Feenstra MG, Broersen LM, Van Leeuwen M, Arens C, De Vries S, et al. Role of the prefrontal cortex of the rat in learning and decision making: effects of transient inactivation. Prog Brain Res 2000;126:103–13. [7] Dhanji H, Block A, Floresco SB. Inactivation of the prefrontal cortex impairs flexible responding using an automated attentional set-shifting procedure. Soc Neurosci Abst, 35th Annual Meeting; 2005. Program No. 412.10. Abstract Viewer/Itinerary Planner, Washington, DC [online]. [8] Dias R, Aggleton JP. Effects of selective excitotoxic prefrontal lesions on acquisition of nonmatching- and matching-to-place in the T-maze in the rat: differential involvement of the prelimbic-infralimbic and anterior cingulate cortices in providing behavioural flexibility. Eur J Neurosci 2000;12:4457–66. [9] Dias R, Robbins TW, Roberts AC. Dissociable forms of inhibitory control within prefrontal cortex with an analog of the Wisconsin Card Sort Test:

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