The Effect of Rat Anterior Cingulate Inactivation on Cognitive Flexibility

Transcription

1 Behavioral Neuroscience Copyright 2007 by the American Psychological Association 2007, Vol. 121, No. 4, /07/$12.00 DOI: / The Effect of Rat Anterior Cingulate Inactivation on Cognitive Flexibility Michael E. Ragozzino and Suzanne Rozman University of Illinois at Chicago The present experiment investigated the effects of muscimol injections into the rat dorsal anterior cingulate on the acquisition and reversal learning of a 4-choice odor discrimination. Long Evans rats were trained to dig in cups that contained distinct odors. In the odor discrimination, one odor cup contained a cereal reinforcement in acquisition whereas a different odor cup contained a cereal reinforcement in reversal learning. The other 2 odor cups were never associated with reinforcement. Bilateral infusions of the gamma aminobutyric acid A agonist muscimol did not impair acquisition of the odor discrimination but impaired reversal learning in a dose-dependent manner. During reversal learning, dorsal anterior cingulate inactivation did not lead to perseveration but selectively increased errors to the odor cups that were never reinforced. These findings suggest that the dorsal anterior cingulate supports learning when conditions require a shift in choice patterns and may enhance cognitive flexibility by decreasing interference of irrelevant stimuli. Keywords: prefrontal cortex, muscimol, rat, learning, attention A large body of research indicates that separate rat prefrontal cortex subregions differentially contribute to learning, memory, and other cognitive functions (for a review, see Dalley, Cardinal, & Robbins, 2004; Uylings, Groenewegen, & Kolb, 2003). Differential involvement of prefrontal cortex areas has been observed in tests that examine sustained attention, working memory, memory consolidation, and task switching (Birrell & Brown, 2000; Chudasama et al., 2003; Delatour & Gisquet-Verrier, 2000; Dias & Aggleton, 2000; Haddon & Killcross, 2006; Joel, Doljansky, & Schiller, 2005; Ostlund & Balleine, 2005; Ragozzino, Kim, Hassert, Minniti, & Kiang, 2003; Ragozzino, Wilcox, Raso, & Kesner, 1999; Rudebeck, Walton, Smyth, Bannerman, & Rushworth, 2006). In the case of task switching, several studies indicate that lesions or drug infusions into the orbitofrontal cortex impair reversal learning, which requires a shift in a specific choice pattern (Bohn, Giertler, & Hauber, 2003; Chudasama & Robbins, 2003; Ferry, Lu, & Price, 2000; McAlonan & Brown, 2003; Schoenbaum, Nugent, Saddoris, & Setlow, 2002). However, these same manipulations of the orbitofrontal cortex do not impair extradimensional shifts in which a subject must inhibit the use of one strategy and learn a new strategy (McAlonan & Brown, 2003). In contrast, other experiments indicate that the medial prefrontal cortex is not critical for a shift between choice patterns as assessed by reversal learning but is critical when conditions require an extradimensional shift (Birrell & Brown, 2000; Ragozzino, Detrick, & Kesner, 1999; Ragozzino, Kim, Hassert, Minniti, & Kiang, 2003; Ragozzino, Wilcox, et al., 1999). Some studies investigating the medial prefrontal cortex in behavioral flexibility indicate that the prelimbic area, in particular, may be an area Michael E. Ragozzino and Suzanne Rozman, Department of Psychology, University of Illinois at Chicago. Correspondence concerning this article should be addressed to Michael E. Ragozzino, Department of Psychology, University of Illinois, 1007 West Harrison Street, Chicago, IL mrago@uic.edu critical for a shift in strategies. Specifically, experiments that have found an extradimensional shift deficit following a drug infusion into the medial prefrontal cortex have had cannula concentrated in the prelimbic area (Floresco, Magyar, Ghods-Sharifi, Vexelman, & Tse, 2006; Ragozzino, 2002; Ragozzino, Detrick, et al., 1999; Ragozzino et al., 2003; Ragozzino, Wilcox, et al., 1999; Stefani, Groth, & Moghaddam, 2003; Stefani & Moghaddam, 2005). Furthermore, a previous study found that tetracaine infusions dorsal to the prelimbic area in the dorsal anterior cingulate do not impair a shift between a place and a visual cue strategy but that infusions into the prelimbic area do impair such a shift (Ragozzino, Wilcox, et al., 1999). Taken together, the findings indicate that the orbitofrontal cortex and prelimbic area are two prefrontal cortex areas that may differentially contribute to learning when conditions require a shift in strategies or response patterns. Although several studies have demonstrated that the rat orbitofrontal cortex and prelimbic areas are important for task switching, few studies have shown that the rat anterior cingulate plays a role in task switching. As stated above, dorsal anterior cingulate inactivation does impair a shift between a place and a visual cue strategy (Ragozzino, Wilcox, et al., 1999). These results are comparable to those of other experiments in which aspiration lesions of the anterior cingulate were shown not to impair a shift between a tactile and a visual cue strategy (Doar, Finger, & Almli, 1987) and large cingulate lesions that included anterior and posterior portions of the cingulate cortex were shown not to affect a shift from a place to a visual cue strategy in a water maze task (Sutherland, Whishaw, & Kolb, 1988). Other studies have also investigated whether the anterior cingulate plays a role in reversal learning. Several studies have found that anterior cingulate lesions do not impair object or visual cue reversal learning (Becker, Olton, Anderson, & Breitinger, 1981; Bussey, Muir, Everitt, & Robbins, 1997; Li & Shao, 1998), spatial reversal learning (Becker et al., 1981; Joel, Tarrasch, Feldon, & Weiner, 1997; Meunier et al., 1991; Warburton, Aggleton, & Muir, 1998), or odor reversal learning (Chaillan, Marchetti, Delfosse, Roman, & Soumireu- 698

2 ANTERIOR CINGULATE AND REVERSAL LEARNING 699 Mourat, 1997). Thus, comparable to studies involving an extradimensional shift, various experiments have also found that anterior cingulate lesions do not affect reversal learning. Despite several studies not finding a reversal learning impairment following anterior cingulate lesions, a study by Gemmell and O Mara (1999) found that lesions of the anterior cingulate and prelimbic areas do not impair acquisition of a place discrimination but do impair place reversal learning. In their study, three different arms were used as choice locations along with three different start locations. This contrasts with most studies, in which a reversal learning deficit was not observed in situations where a rat had to choose between two possible locations. One possibility is that the use of three choice arms required a greater level of discrimination among stimuli that involved activation of the anterior cingulate to optimally shift choice patterns in this task. On the basis of Pavlovian conditioning paradigms, the anterior cingulate has been proposed to be critical for discriminating among multiple stimuli that have similar features (Cardinal et al., 2003). Other experiments using discrimination tests have demonstrated that anterior cingulate lesions do not impair acquisition using one pair of stimuli but do impair acquisition using eight pairs of stimuli (Bussey et al., 1997). Furthermore, anterior cingulate lesions have been found not to impair a conditional discrimination when one lever is used but to impair performance when rats are switched to using two levers (Winocur & Eskes, 1998). Taken together, the findings suggest that the anterior cingulate may be critical for learning when conditions involve discriminating among multiple stimuli or a shift in response patterns when several alternatives are available. Because several experiments have found that anterior cingulate lesions did not impair two-choice reversal learning but may be critical when conditions require discriminating among multiple stimuli, the present study investigated whether inactivation of the dorsal anterior cingulate affects the acquisition and/or reversal learning of a four-choice odor discrimination. In this task, a rat is required to dig in one of four odor cups for a food reinforcement in acquisition. During reversal learning the rat must choose a different odor cup, with two of the odor cups never reinforced. As judged by the number of trials to reach criterion, the four-choice odor discrimination is more difficult to learn in both the acquisition and reversal learning sessions compared with a two-choice odor discrimination (Kim & Ragozzino, 2005; Ragozzino et al., 2003). If the dorsal anterior cingulate is important for learning and/or a shift in choice patterns when conditions require discrimination among multiple stimuli, then dorsal anterior cingulate inactivation should impair the acquisition and/or reversal learning of a four-choice odor discrimination. Subjects Method Male Long Evans rats (Charles River Laboratories; Raleigh, NC) weighing g at the beginning of the experiment served as subjects. Rats were housed individually in plastic cages (26.5 cm wide 50.0 cm long 20.0 cm high) located in a temperature-controlled room (24 C) that was maintained at 20% 40% humidity. The rats were kept on a 12-hr light dark cycle (lights on at 0700). All rats were food restricted to maintain their weight at approximately 85% of their ad libitum weight but had free access to water throughout the experiment. Apparatus A rectangular-shaped maze made of 0.6-cm black plastic was used in both experiments. The floor of the maze was 76 cm 50 cm. The maze was placed on a table that was elevated 75 cm above the floor. The two side walls were each 76 cm 32 cm. The front and back walls were each 50 cm 32 cm. Surgery Rats received atropine sulfate (0.2 ml of a 250 g/ml solution, administered intraperitoneally) 10 min before the general anesthetic (sodium pentobarbital, 50 mg/kg ip). A midsagittal incision was made, and the scalp was retracted. Each rat received a bilateral implant of a 5-mm stainless steel guide cannula (22 gauge; Plastics One, Roanoke, VA) aimed toward the dorsal anterior cingulate area rostral to the genu of the corpus callosum. The stereotaxic coordinates were 2.0 mm anterior to bregma, 1.7 mm lateral to the midline, and 2.0 mm ventral to dura. The incisor bar was lowered to below horizontal zero. The coordinates were based on the atlas of Paxinos and Watson (1996). The cannulas were inserted at a 15 angle. Four jeweler s screws were placed in the skull surrounding the cannulas. The cannulas were secured in place with dental acrylic (Plastics One). Stylets were secured in the guide cannulas after the dental acrylic dried. After surgery, rats received ground rat chow mixed in water for 1 day. Pretraining Procedure Rats were allowed 7 10 days to recover from surgery before the pretraining procedure was started. One day after surgery, rats were food restricted to 85% of their original ad libitum weight. Rats were handled for 10 min per day until pretraining began. On the first day of pretraining, four round stainless steel bowls (9.0 cm in diameter and 3.2 cm deep) were each filled with 100 g of sterile sand and placed at the back wall of the maze. The bowls were kept 5 cm apart. One half piece of Kellogg s Froot Loops cereal was placed on top of the sand in each of the four bowls. A rat was placed at the opposite end of the maze and allowed to navigate to the bowls and consume the cereal pieces. After a rat had consumed all of the cereal pieces it was placed in a holding cage. Each sand bowl was baited again, and the rat was returned to the maze. Beginning with this trial, the pieces were increasingly buried under the sand until they were completely buried, so that the rat had to dig in the sand to retrieve the cereal. Each pretraining session lasted 15 min. When a rat had retrieved completely buried cereal pieces for 2 consecutive days, it was given 1 final day of pretraining. In this final pretraining session, the number of bowls containing a cereal piece varied from one to four. The trials in which one, two, three, or four bowls contained a cereal piece were randomly presented across the session. The pieces were placed pseudorandomly such that cereal was buried an equal number of times in each bowl for the session. A rat was allowed to dig until it found each of the pieces. After the final pretraining session, a rat s stylets were removed from the guide cannulas, and an injection cannula was

3 700 RAGOZZINO AND ROZMAN inserted in each guide cannula for 1 min. No solution was injected; this procedure was performed to prevent clogging of the microinfusion on test days. Behavioral testing was started the next day. Microinfusion Five min before each test session, rats received a bilateral injection into the anterior cingulate through an inner cannula (28 gauge) that extended 1.0 mm below the guide cannula. The inner cannula was attached by a polyethylene tube (PE 20) to a 10- l Hamilton syringe. The syringe was driven by a microinfusion pump with solutions infused in a volume of 0.25 l per side for 2 min. The inner cannula was left in place for 1 min after completion of the infusion to allow for diffusion. Rats received either the gamma aminobutyric acid A agonist muscimol (0.05, 0.2, or 0.5 g per side) or saline. Odor Discrimination Test Procedure Each rat was tested across 2 consecutive days. The first day involved the acquisition phase, and the second day involved the reversal learning phase. In the test procedure, each bowl contained 100 g of sand with 1.25 g of either cinnamon, cumin, curry, or nutmeg mixed in. Because some of the spices differ in color, each spice was mixed in the sand such that it was not visually detectable on the surface. During the acquisition phase, one spice was designated the positive odor and the other spices were designated the negative odors. One half piece of cereal was always buried in the bowl containing the positive odor. The bowls were randomly switched among spatial locations across trials. A rat was allowed to dig in only one bowl per trial. A rat reached criterion when it made 10 consecutive correct choices. Thus, a session was not terminated until a rat had achieved a criterion of 10 consecutive correct trials. In the reversal learning phase, one of the three negative odors became the positive odor. Thus, a positive odor in acquisition became a negative odor in reversal learning, along with the other two odors. Therefore, in this procedure, two of the odors were never associated with reinforcement during either the acquisition or the reversal learning phase. The same learning criterion was used as in the acquisition session. The spices used for the positive odor in acquisition and the positive odor in reversal learning were pseudorandomly chosen such that each spice was used in each condition a similar amount among the rats. The positive odors for acquisition and reversal learning were paired as follows: cumin cinnamon, cumin nutmeg, curry cinnamon, and curry nutmeg. Pairs were counterbalanced such that an odor from each pair served as the positive odor on acquisition and the positive odor on reversal learning a similar number of times. Group assignment was determined by the treatment administered before the test session: (a) acquisition saline and reversal learning saline (n 6); (b) acquisition saline and reversal learning muscimol 0.05 g (n 6); (c) acquisition saline and reversal learning muscimol 0.2 g (n 6); (d) acquisition saline and reversal learning muscimol 0.5 g (n 6); (e) acquisition muscimol 0.2 g and reversal learning saline (n 6); and (f) acquisition muscimol 0.5 g and reversal learning saline (n 6). Group 1 served as the control group; Groups 2, 3, and 4 were used to determine whether inactivation with muscimol impaired reversal learning in a dose-dependent manner; and Groups 5 and 6 were used to determine whether a muscimol injection impaired acquisition of the odor discrimination. An analysis of the error pattern was carried out for the reversal learning phase. In particular, errors were divided into three categories: perseverative, regressive, and distracter. To measure perseverative errors, we separated the trials into consecutive blocks of four trials each. Perseveration was defined as a rat initially digging in the odor cup that was reinforced during acquisition for three or more trials in a four-trial block. Thus, a rat had to choose the previously reinforced choice on the majority of trials for the block to be counted as perseveration. This criterion is similar to that used in previous studies (Kim & Ragozzino, 2005; Ragozzino, Kim, et al., 2003). Once a rat had chosen the previously reinforced odor cup fewer than three times in a block, these errors were no longer counted as perseverative but were now counted as regressive. This measured the ability to maintain a new correct choice or begin to make different choices after the initial shift from the previously correct choice. Distracter errors were those in which a rat dug in either of the two odor cups that had never been reinforced during either acquisition or reversal learning. Because a rat could not know beforehand that these choices were incorrect during reversal learning, errors were counted only after the first time one of these odor cups was chosen. The total number of subsequent choices of both odor cups was the distracter error score. Histology After behavioral testing, rats received a lethal dose of sodium pentobarbital followed by a 0.5- l injection of 2.5% Chicago blue stain through each guide cannula. As in previous experiments (Kim et al., 2005; Ragozzino et al., 2003), the stain was used to highlight the location of the cannula tip. Rats were perfused intracardially with 0.9% saline followed by a 4% formaldehyde solution. Brains were removed and stored in a 4% formaldehyde solution. The brains were frozen and cut into coronal sections (40 m) on a cryostat. The sections were mounted on slides, dried, and examined to ascertain the location of the cannula tips. The brain sections were later stained with cresyl violet to allow observation of any gross structural changes in the brains infused with muscimol compared with those infused with saline. Statistical Analysis An analysis of variance (ANOVA) was used to determine whether trials to criterion differed significantly among the groups in the acquisition and reversal learning phases. ANOVAs were also used to assess differences in perseveration, regressive, and distracter errors among the groups. Newman Keuls post hoc tests were used to determine significant differences between groups when there was an overall group effect. Histology Results The locations of the cannula tips in the dorsal anterior cingulate are shown in Figure 1. The histological analysis indicated that the injection tips were located mainly in the dorsal anterior cingulate cortex rostral to the genu of the corpus callosum. As determined

4 ANTERIOR CINGULATE AND REVERSAL LEARNING 701 trials to reach criterion in acquisition. The analysis showed there was not a significant difference in trials to criterion among the groups, F(5, 30) 0.66, p.05. Figure 2B illustrates the results on odor reversal learning. In reversal learning, the difference in trials to criterion among the groups was significant, F(5, 30) 9.31, p.01. Newman Keuls tests revealed that the groups that received either muscimol 0.2 or muscimol 0.5 g in reversal learning required significantly more trials to achieve criterion compared with rats that received saline ( ps.01) or rats that received muscimol 0.05 g (ps.01). Furthermore, the group that received muscimol 0.05 g was comparable to the salinetreated groups in number of trials needed to complete reversal learning ( ps.05). An analysis of the errors made during reversal learning (see Figure 3) revealed there was not a significant difference among the groups in the number of perseverative errors, F(5, 30) 0.942, p.05. There was a trend toward significance in regressive error differences among the groups, F(5, 30) 2.42, p.059. The analysis showed a significant difference, though, for distracter errors among the groups, F(5, 30) 5.04, p.01. Newman Keuls tests indicated that the groups that received muscimol 0.2 or 0.5 g during reversal learning committed significantly more distracter errors than saline-treated rats ( ps.05). Discussion Figure 1. Placement of cannula tips in the anterior cingulate for rats included in the behavioral analyses. The location of the placements ranged from 1.7 to 3.2 mm anterior to bregma. The number of circles does not match the total number of cannula tips for rats included in the behavioral analyses because some cannula placements overlapped to such a large extent that a single circle represents more than one cannula tip placement. The rat brain sections were modified from The Rat Brain in Stereotaxic Coordinates (3rd ed.; Figures 7 10 and 12) by G. Paxinos and C. Watson, 1996, San Diego, CA: Academic Press. Copyright 1996 by Elsevier. Adapted with permission. using the atlas of Paxinos and Watson (1996), cannula were found in the dorsal anterior cingulate ranging from 3.2 mm to 1.7 mm anterior to bregma. The distribution of cannula placements was comparable among the groups. The data from 3 rats were excluded from the behavioral analyses because of a cannula misplacement. All of these rats had cannula placements lateral to anterior cingulate in the primary motor cortex. Behavioral Testing The results on acquisition of the four-choice odor discrimination are shown in Figure 2A. All groups required approximately The present findings indicate that dorsal anterior cingulate inactivation does not impair acquisition of a four-choice odor discrimination but does impair four-choice reversal learning in a dose-dependent manner. This reversal learning deficit contrasts with findings from several previous studies in which anterior cingulate lesions did not impair reversal learning in two-choice discrimination tests (Becker et al., 1981; Bussey et al., 1997; Chaillan et al., 1997; Joel et al., 1997; Meunier et al., 1991; Warburton et al., 1998). However, the present results are comparable to the study in which lesions of the anterior cingulate and prelimbic areas impaired place reversal learning in a three-choice discrimination (Gemmell & O Mara, 1999). The reversal learning deficit observed in the present study is unlikely due to muscimol inactivating the prelimbic area, as well as the anterior cingulate area. This is because a previous study found that inactivation of the prelimbic area does not impair performance on the acquisition or reversal of a four-choice odor discrimination using the same procedure as the present study (Ragozzino et al., 2003). However, prelimbic inactivation does impair an extradimensional shift, but dorsal anterior cingulate inactivation does not impair an extradimensional shift (Ragozzino, Detrick, et al., 1999; Ragozzino, Wilcox, et al., 1999). Thus, the reversal learning deficit observed in the present study is likely principally due to inactivating the dorsal anterior cingulate region. A previous experiment examined the effects of intracranial muscimol infusions at different brain sites and with various infusion volumes (Edeline, Hars, Hennevin, & Cotillon, 2002). The study involved measuring diffusion of labeled muscimol, as well as multiunit recordings from areas distant to the infusion site. The autoradiography findings indicated that muscimol diffused some distance from the injection site and was larger than previously reported (Martin, 1991). Although this method does not provide a functional measure and diffusion can vary greatly on the basis of

5 702 RAGOZZINO AND ROZMAN Figure 2. A: Mean ( SEM) trials to criterion in acquisition of a four-choice odor discrimination following bilateral infusions of saline (SAL) or muscimol (MUS; 0.2 or 0.5 g) into the anterior cingulate. Treatment with muscimol did not affect acquisition. B: Mean ( SEM) trials to criterion in reversal learning of a four-choice odor discrimination following bilateral infusions of saline or muscimol (0.05, 0.2, or 0.5 g) into the anterior cingulate. Treatment with muscimol at 0.2 and 0.5 g significantly increased trials to criterion compared with saline controls. The treatment received in each phase is shown in bold. **p.01 for difference from saline controls. volume, concentration, and infusion method, the results raise the possibility that the muscimol effect observed in the present study was due, at least in part, to inactivation in areas outside of the dorsal anterior cingulate. However, though there was almost certainly some diffusion of the drug into surrounding areas, results from various studies suggest that muscimol diffusion into other prefrontal subregions or other frontal areas does not affect behavioral performance in the same manner. As discussed above, a dissociation has been observed between prelimbic inactivation and dorsal anterior cingulate inactivation in the four-choice odor reversal learning test (Ragozzino et al., 2003). It is important to note that the injection volume used to inactivate the prelimbic area in that study was 0.5 l, double what was used in the present study. Furthermore, muscimol infusions into the ventral and lateral orbitofrontal cortex impaired four-choice odor reversal learning but led to a somewhat unique error pattern compared with that observed following dorsal anterior cingulate inactivation (Kim & Ragozzino, 2005). Moreover, in experiments in which drug infusions into either the prelimbic or the orbitofrontal cortex led to behavioral deficits, injection of an effective dose of the drug medial and/or lateral to the intended infusion site did not result in a behavioral deficit (Ragozzino, 2002; Ragozzino et al., 2003; Kim & Ragozzino, 2005). In a comparable manner, 2 rats in the present study had placements in the ventral primary motor cortex but did not show a reversal learning deficit despite receiving either a 0.5- or a 0.2- g muscimol infusion. Therefore, the reversal learning impairment was unlikely due to a significant spread to and inactivation of other prefrontal cortex subregions.

6 Figure 3. Mean ( SEM) number of errors of each of three types in reversal learning after bilateral infusions of saline (SAL) or muscimol (MUS) into the anterior cingulate. A: There was no significant difference among the groups on perseverative errors. B: There was no significant difference among the groups in regressive errors. C: Muscimol at 0.2 and 0.5 g significantly increased the number of distracter errors compared with the other treatment groups. The treatment received in the reversal-learning phase is shown in bold for each group. * p.05 for difference from all other treatment groups.

7 704 RAGOZZINO AND ROZMAN Despite the evidence arguing against the reversal learning impairment arising from diffusion into prefrontal areas ventral to the dorsal anterior cingulate or more distal frontal areas, other experiments that have reported deficits following dorsal anterior cingulate lesions have often involved damage to the medial precentral area (Dias & Aggleton, 2000; Haddon & Killcross, 2006; Ragozzino & Kesner, 2001). Thus, the reversal learning impairment observed in the present study could be possibly due, at least in part, to inactivation of the medial precentral area, as well as the dorsal anterior cingulate. Although muscimol injections into the dorsal anterior cingulate impaired four-choice reversal learning in a dose-dependent manner, the same doses that were effective in impairing reversal learning did not affect initial learning of the odor discrimination. Thus, the anterior cingulate does not appear to play a more general role in discrimination learning. Furthermore, the selective impairment also suggests that the reversal learning deficit observed following anterior cingulate inactivation is not due to an alteration in motor or motivational processes. Because the present study demonstrated that dorsal anterior cingulate inactivation impairs reversal learning in a four-choice discrimination whereas several previous studies have shown that anterior cingulate lesions do not impair reversal learning in twochoice discriminations, these findings may suggest that the number of stimuli required to be discriminated among is the critical factor that engages the anterior cingulate to enable a shift in choice patterns. However, Joel et al. (1997) trained rats in an eight-arm radial maze to discriminate among four arms that contained a food reinforcement and four arms that did not contain a food reinforcement. Anterior cingulate lesions did not impair acquisition or reversal learning in this test. Therefore, the absolute number of stimuli to be discriminated among is unlikely to be the sole factor that determines when the anterior cingulate is recruited in conditions that require behavioral flexibility. However, unclear is why differences in reversal learning following manipulation of the anterior cingulate were observed in the Weiner et al. study compared with the present experiment. Specific differences in the procedures used may have contributed to the different pattern of results. For example, half of the arms were reinforced in the Weiner et al. radial-arm maze task, whereas only one fourth of the choices were reinforced in the present study. In addition, rats had to reverse in a single session in the present study, but in the radial-arm maze task rats were tested across multiple sessions, during which possible recruitment of other brain areas involved in memory consolidation may have influenced reversal learning performance. Still another possibility is that the anterior cingulate lesions produced by Weiner and colleagues did not involve the anterior cingulate to the same extent as the muscimol infusions in the present study. As discussed above, several studies have found that dorsomedial prefrontal cortex lesions centered in the anterior cingulate do not impair two-choice reversal learning. The present experiment found that when the number of choices was increased to four, but still only one choice was reinforced, muscimol infusions targeted at the dorsal anterior cingulate impaired reversal learning. These findings may be consistent with previous proposals that the anterior cingulate is important for control of response conflict (Haddon & Killcross, 2006; Williams, Mohler, & Givens, 1999). In an experiment by Haddon and Killcross (2006), rats learned visual and auditory conditional discrimination in two different settings. Subsequently, an auditory stimulus and a visual stimulus were presented simultaneously. In some trials, the two stimuli had been conditioned to elicit the same response. In other trials, the stimuli had been conditioned to elicit different responses. In the trials that involved a response conflict, control rats used the contextual cues to discriminate between stimuli, whereas rats with anterior cingulate lesions had reduced ability to use contextual cues. In contrast, anterior cingulate lesions did not impair acquisition of a biconditional discrimination or affect performance in trials when the combined stimuli were conditioned to elicit the same response (Haddon & Killcross, 2006). Thus, switching to a combined presentation of stimuli alone did not affect performance, but when the two stimuli were associated with opposite responses, anterior cingulate lesions affected the response selection process. The four-choice reversal learning test used in the present study may represent another situation that requires greater control for response selection in that the task not only requires inhibiting a previously learned choice and selecting a new choice but also requires not selecting two other choices that were never reinforced. The impairment observed in acquisition of a delayed match-tosample strategy following lesions of the dorsal anterior cingulate may also indicate that this frontal area supports learning in rats when conditions require control over response conflict (Dias & Aggleton, 2000). This is because rats commonly exhibit a bias toward using a nonmatch-to-sample strategy or spontaneously alternating (Dember & Fowler, 1958); thus, when rats are required to learn a match-to-sample strategy, the condition may demand greater control of response selection and inhibit their bias of displaying a nonmatch-to-sample strategy. This study also revealed that anterior cingulate lesions led to perseveration of using a nonmatch-to-sample strategy early in testing. In the present study, dorsal anterior cingulate inactivation did not affect perseveration but led to an increase in distracter errors. Taken together, the findings suggest that manipulations of the anterior cingulate can lead to deficits in situations involving response conflict but, depending of the specific behavioral conditions, may lead to various error patterns. During reversal learning, dorsal anterior cingulate inactivation increased the number of choices rats made of the two odor cups that had never been reinforced during acquisition or reversal learning. The increase in distracter errors suggests that the dorsal anterior cingulate may be critical for reducing interference of distracting stimuli when conditions require a shift in choice patterns. The results are comparable to those of a previous study that found that anterior cingulate lesions impair trace conditioning, but only when a distracter stimulus is presented during the delay (Han et al., 2003). The selective increase in distracter errors following dorsal anterior cingulate inactivation contrasts with orbitofrontal cortex inactivation, in which a reversal learning deficit in the four-choice odor discrimination led to a significant increase in perseverative and regressive errors in addition to distracter errors (Kim & Ragozzino, 2005). Taken together, the results suggest that the dorsal anterior cingulate may play a more specific role in supporting a shift in choice patterns than the orbitofrontal cortex. If the anterior cingulate supports cognitive flexibility when conditions support a switch in choice patterns by reducing interference of irrelevant stimuli, as opposed to inhibiting responses to previously relevant stimuli, then this may be one explanation for why

8 ANTERIOR CINGULATE AND REVERSAL LEARNING 705 previous studies did not find an impairment with anterior cingulate inactivation on two-choice reversal learning tests because in these tests, only an inhibition of the previously relevant choice is required. An alternative possibility is that dorsal anterior cingulate inactivation led to an increase in distracter errors because muscimol injections into the anterior cingulate impaired memory retrieval. In this case, dorsal anterior cingulate inactivation may have impaired rats ability to recall that two of the odor cups that were not reinforced during reversal learning were also not reinforced during acquisition. This memory retrieval deficit would lead rats that received the higher doses of muscimol during reversal learning to choose the two odor cups that were never reinforced more frequently. Previous studies indicate that the anterior cingulate is important for memory retrieval (Frankland, Bontempi, Talton, Kaczmarek, & Silva, 2004; Frankland et al., 2006). However, these studies indicate that anterior cingulate inactivation does not impair memory retrieval for information learned 1 day earlier but does impair retrieval for more remote memories (Frankland et al., 2004). Furthermore, if anterior cingulate inactivation produces a general memory retrieval deficit, then there should also be an effect on perseverative and regressive errors. More specifically, with a memory retrieval deficit, anterior cingulate inactivation should actually lead to a decrease in perseverative and regressive errors, as inactivation should impair recall of the previously reinforced choice. However, muscimol infusions into the anterior cingulate did not affect perseverative or regressive errors compared with errors made by control rats. Thus, the pattern of results is not consistent with the notion of anterior cingulate inactivation leading to a general deficit in memory retrieval. In summary, the present study demonstrates that muscimol infusion into the dorsal anterior cingulate dose-dependently impairs four-choice odor reversal learning but not acquisition of the four-choice discrimination. The reversal learning deficit following dorsal anterior cingulate inactivation led to a significant increase in distracter errors. The findings suggest that the dorsal anterior cingulate enables a shift in choice patterns by decreasing interference of irrelevant stimuli but not the inhibition of previously reinforced choices. References Becker, J. T., Olton, D. S., Anderson, C. A., & Breitinger, E. R. (1981). Cognitive mapping in rats: The role of the hippocampal and frontal system in retention and reversal. Behavioural Brain Research, 3, Birrell, J. M., & Brown, V. J. (2000). Medial frontal cortex mediates perceptual attentional set-shifting in the rat. Journal of Neuroscience, 20, Bohn, I., Giertler, C., & Hauber, W. (2003). Orbital prefrontal cortex and guidance of instrumental behaviour in rats under reversal conditions. Behavioural Brain Research, 143, Bussey, T. J., Muir, J. L., Everitt, B. J., & Robbins, T. W. (1997). Triple dissociation of anterior cingulate, posterior cingulate and medial frontal cortices on visual discrimination tasks using a touchscreen testing procedure for the rat. Behavioral Neuroscience, 111, Cardinal, R. N., Parkinson, J. A., Marbini, H. D., Toner, A. J., Bussey, T. J., Robbins, T. W., & Everitt, B. J. (2003). Role of the anterior cingulate cortex in the control over behavior by Pavlovian conditioned stimuli in rats. Behavioral Neuroscience, 117, Chaillan, F. A., Marchetti, E., Delfosse, F., Roman, F. S., & Soumireu- Mourat, B. (1997). Opposite effects depending on learning and memory demands in dorsomedial prefrontal cortex lesioned rats performing an olfactory task. Behavioural Brain Research, 82, Chudasama, Y., Passetti, F., Rhodes, F. E. V., Lopian, D., Desai, A., & Robbins, T. W. (2003). Dissociable aspects of performance on the 5-choice serial reaction time task following lesions of the dorsal anterior cingulate, infralimbic and orbitofrontal cortex in the rat: Differential effects on selectivity, impulsivity and compulsivity. Behavioural Brain Research, 146, Chudasama, Y., & Robbins, T. W. (2003). Dissociable contributions of the orbitofrontal and infralimbic cortex to Pavlovian autoshaping and discrimination reversal learning: Further evidence for the functional heterogeneity of the rodent frontal cortex. Journal of Neuroscience, 23, Dalley, J. W., Cardinal, R. N., & Robbins, T. W. (2004). Prefrontal executive and cognitive functions in rodents: Neural and neurochemical substrates. Neuroscience & Biobehavioral Reviews, 28, Delatour, B., & Gisquet-Verrier, P. (2000). Functional role of rat prelimbic infralimbic cortices in spatial memory: Evidence for their involvement in attention and behavioural flexibility. Behavioural Brain Research, 109, Dember, W. N., & Fowler, H. (1958). Spontaneous alternation behavior. Psychological Bulletin, 55, Dias, R., & Aggleton, J. P. (2000). 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. European Journal of Neuroscience, 12, Doar, B., Finger, S., & Almli, C. R. (1987). Tactile visual acquisition and reversal learning deficits in rats with prefrontal cortical lesions. Experimental Brain Research, 66, Edeline, J. M., Hars, B., Hennevin, E., & Cotillon, N. (2002). Muscimol diffusion after intracerebral microinjections: a reevaluation based on electrophysiological and autoradiographic quantifications. Neurobiology of Learning and Memory, 78, Ferry, A. T., Lu, X. C. M., & Price, J. L. (2000). Effects of excitotoxic lesions in the ventral striatopallidal thalamocortical pathway on odor reversal learning: Inability to extinguish an incorrect response. Experimental Brain Research, 131, Floresco, S. B., Magyar, O., Ghods-Sharifi, S., Vexelman, C., & Tse, M. T. L. (2006). Multiple dopamine receptor subtypes in the medial prefrontal cortex of the rat regulate set-shifting. Neuropsychopharmacology, 31, Frankland, P. W., Bontempi, B., Talton, L. E., Kaczmarek, L., & Silva, A. J. (2004, May 7). The involvement of the anterior cingulate cortex in remote contextual fear memory. Science, 304, Frankland, P. W., Ding, H. K., Takahashi, E., Suzuki, A., Kida, S., & Silva, A. J. (2006). Stability of recent and remote contextual fear memory. Learning & Memory, 13, Gemmell, C., & O Mara, S. M. (1999). Medial prefrontal cortex lesions cause deficits in a variable-goal location task but not in object exploration. Behavioral Neuroscience, 113, Haddon, J. E., & Killcross, S. (2006). Prefrontal cortex lesions disrupt the contextual control of response conflict. Journal of Neuroscience, 26, Han, C. J., O Tuathaigh, C. M., van Trigt, L., Quinn, J. J., Fanselow, M. S., Mongeau, R., et al. (2003). Trace but not delay fear conditioning requires attention and the anterior cingulate cortex. Proceedings of the National Academy of Sciences, USA, 100, Joel, D., Doljansky, J., & Schiller, D. (2005). Compulsive lever pressing in rats is enhanced following lesions to the orbital cortex, but not to the basolateral nucleus of the amygdala or to the dorsal medial prefrontal cortex. European Journal of Neuroscience, 21, Joel, D., Tarrasch, R., Feldon, J., & Weiner, I. (1997). Effects of electrolytic lesions of the medial prefrontal cortex or its subfields on 4-arm

9 706 RAGOZZINO AND ROZMAN baited, 8-arm radial maze, two-way active avoidance and conditioned fear tasks in the rat. Brain Research, 765, Kim, J., & Ragozzino, M. E. (2005). The involvement of the orbitofrontal cortex in learning under changing task contingencies. Neurobiology of Learning and Memory, 83, Li, L., & Shao, J. (1998). Restricted lesions to ventral prefrontal subareas block reversal learning but not visual discrimination learning in rats. Physiology & Behavior, 65, Martin, J. H. (1991). Autoradiographic estimation of the extent of reversible inactivation produced by microinjection of lidocaine and muscimol in the rat. Neuroscience Letters, 127, McAlonan, K., & Brown, V. J. (2003). Orbital prefrontal cortex mediates reversal learning and not attentional set shifting in the rat. Behavioural Brain Research, 146, Meunier, M., Jaffard, R., & Destrade, C. (1991). Differential involvement of anterior and posterior cingulate cortices in spatial discriminative learning in a T-maze in mice. Behavioural Brain Research, 44, Ostlund, S. B., & Balleine, B. W. (2005). Lesions of medial prefrontal cortex disrupt the acquisition but not the expression of goal-directed learning. Journal of Neuroscience, 25, Paxinos, G., & Watson, C. (1996). The rat brain in stereotaxic coordinates (3rd ed.). San Diego, CA: Academic Press. Ragozzino, M. E. (2002). The effects of dopamine D1 receptor blockade in the prelimbic infralimbic areas on behavioral flexibility. Learning & Memory, 9, Ragozzino, M. E., Detrick, S., & Kesner, R. P. (1999). Involvement of the prelimbic infralimbic areas of the rodent prefrontal cortex in behavioral flexibility for place and response learning. Journal of Neuroscience, 19, Ragozzino, M. E., & Kesner, R. P. (2001). The role of rat dorsomedial prefrontal cortex in working memory for egocentric responses. Neuroscience Letters, 308, Ragozzino, M. E., Kim, J., Hassert, D., Minniti, N., & Kiang, C. (2003). The contribution of the rat prelimbic infralimbic areas to different forms of task switching. Behavioral Neuroscience, 117, Ragozzino, M. E., Wilcox, C., Raso, M., & Kesner, R. P. (1999). Involvement of rodent prefrontal cortex subregions in strategy switching. Behavioral Neuroscience, 113, Rudebeck, P. H., Walton, M. E., Smyth, A. N., Bannerman, D. M., & Rushworth, M. F. (2006). Separate neural pathways process different decision costs. Nature Neuroscience, 9, Schoenbaum, G., Nugent, S. L., Saddoris, M. L., & Setlow, B. (2002). Orbitofrontal lesions in rats impair reversal but not acquisition of go, no-go odor discriminations. NeuroReport, 13, Stefani, M. R., Groth, K., & Moghaddam, B. (2003). Glutamate receptors in the rat medial prefrontal cortex regulate set-shifting ability. Behavioral Neuroscience, 117, Stefani, M. R., & Moghaddam, B. (2005). Systemic and prefrontal cortical NMDA receptor blockade differentially affect discrimination learning and set-shift ability in rats. Behavioral Neuroscience, 119, Sutherland, R. J., Whishaw, I. Q., & Kolb, B. (1988). Contributions of cingulate cortex to two forms of spatial learning and memory. Journal of Neuroscience, 8, Uylings, H. B., Groenewegen, H. J., & Kolb, B. (2003). Do rats have prefrontal cortex? Behavioural Brain Research, 146, Warburton, E. C., Aggleton, J. P., & Muir, J. L. (1998). Comparing the effects of selective cingulate cortex lesions and cingulum bundle lesions on water maze performance by rats. European Journal of Neuroscience, 10, Williams, J. M., Mohler, E. G., & Givens, B. (1999). The role of the medial prefrontal cortex in attention: Altering predictability of task difficulty. Psychobiology, 27, Winocur, G., & Eskes, G. (1998). Prefrontal cortex and caudate nucleus in conditional associative learning: Dissociated effects of selective brain lesions in rats. Behavioral Neuroscience, 112, Received December 21, 2006 Revision received February 19, 2007 Accepted March 5, 2007