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Thalamic-cortical-striatal circuitry subserving strategy set-shifting in the rat Block, Annie Eugenie 2006

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T H A L A M I C - C O R T I C A L - S T R I A T A L C I R C U I T R Y S U B S E R V I N G S T R A T E G Y S E T - S H I F T I N G IN T H E R A T by A N N I E E U G E N I E B L O C K B . A . , Reed College, 2004 A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L L M E N T F O R T H E D E G R E E O F M A S T E R O F A R T S in T H E F A C U L T Y O F G R A D U A T E STUDIES (Psychology) T H E UNIVERSITY O F BRITISH C O L U M B I A Apri l 2006 ©Annie Eugenie B lock, 2006 Abstract The mediodorsal nuclei of thalamus (MD) , prefrontal cortex (PFC) and nucleus accumbens (NAc) core form an interconnected network that may work together to subserve some forms of behavioral flexibility. The present experiments investigated the functional relationships between these regions during performance of a cross-maze based set-shifting task. In Experiment 1, transient inactivation of the M D with bilateral infusion of bupivacaine impaired set-shifting but not discrimination learning during performance of a response to visual-cue as well as on a cue to response set-shift. Similar to inactivation of the P F C , M D inactivation induced a perseverative deficit, suggesting the M D works with the P F C to disengage from a previously relevant strategy. In Experiment 2, asymmetrical inactivations of the M D on one side of the brain and P F C on the other caused a perseverative deficit in acquisition of a visual-cue discrimination on a set shift, as did asymmetrical inactivation of the P F C and the contralateral N A c core. Inactivation of the M D on one side of the brain and the N A c core contralaterally resulted in an increase in never-reinforced errors, suggesting this pathway is important for eliminating inappropriate strategies during set-shifting. These data indicate that set-shifting is mediated by a distributed neural circuit, with separate neural pathways contributing dissociable components to this type of behavioral flexibility. Abstract The mediodorsal nuclei of thalamus (MD) , prefrontal cortex (PFC) and nucleus accumbens (NAc) core form an interconnected network that may work together to subserve some forms of behavioral flexibility. The present experiments investigated the functional relationships between these regions during performance of a cross-maze based set-shifting task. In Experiment 1, transient inactivation of the M D with bilateral infusion of bupivacaine impaired set-shifting but not discrimination learning during performance o f a response to visual-cue as well as on a cue to response set-shift. Similar to inactivation of the P F C , M D inactivation induced a perseverative deficit, suggesting the M D works with the P F C to disengage from a previously relevant strategy. In Experiment 2, asymmetrical inactivations of the M D on one side of the brain and P F C on the other caused a perseverative deficit in acquisition of a visual-cue discrimination on a set shift, as did asymmetrical inactivation of the P F C and the contralateral N A c core. Inactivation of the M D on one side of the brain and the N A c core contralaterally resulted in an increase in never-reinforced errors, suggesting this pathway is important for eliminating inappropriate strategies during set-shifting. These data indicate that set-shifting is mediated by a distributed neural circuit, with separate neural pathways contributing dissociable components to this type of behavioral flexibility. i i i Table of Contents Abstract ii Table of Contents iii List of Tables iv List of Figures v Acknowledgements vi Chapter 1 1 Introduction 1 Chapter 2 , 5 Methods and Results 5 Experiment 1A 5 Experiment IB ; 13 Experiment 2 16 Chapter 3 29 Discussion 29 Conclusion 35 References 39 iv List of Tables Table 2.1. Trials per Minute: Means ± SE for all groups 13 Table 2.2 Summary of Treatments and Groups 19 V List of Figures Figure 2.1. Example of the strategy set-shifting task used in Experiment 1A and 2 22 Figure 2.2. Schematic of coronal sections of the rat brain showing the placements of the cannulae tips for all rats that received infusions of bupivacaine into the M D 23 Figure 2.3. Experiment 1 A : Inactivation of the M D disrupts shifting from a response to a visual-cue based strategy (Experiment 1A, A - C ) ...24 Figure 2.4. Experiment I B : Inactivation of the M D disrupts shifting from a visual-cue to a response strategy (Experiment I B , A - C ) 25 Figure 2.5. Schematic o f coronal sections of the rat brain showing the placements of the cannulae tips for all rats that received double cannulations 26 Figure 2.6. Experiment 2: The effects of disconnection of the M D - P F C , M D - N A c and P F C - N A c pathways on shifting from a response to a visual-cue based strategy.... 27 Figure 2.7. Experiment 2: Analysis of the type of errors committed during the acquisition of a visual-cue strategy during a strategy-shift 28 Figure 3.1. Schematic of the neural circuit involved in strategy set shifting behavior 38 Acknowledgements There are many individuals to whom I find myself indebted as I complete this thesis. I would like to begin by thanking Marie Tse for his invaluable technical expertise and friendship. And I would still be teaching rats to turn left were it not for the contributions to behavioral testing of two wonderful women, Hasina Dhanji, and Sarah Thompson. Additionally I wish to express my gratitude for the enthusiasm and guidance of my committee: Dr. Cathy Rankin and Dr. Kalina Christoff Finally, I would like to thank my supervisor Dr. Stan Floresco for his mentorship through the thesis process. 1 Chapter 1 Introduction Behavioral flexibility, the ability to adaptively vary one's behavior in response to changing environmental contingencies, is an important function of successful goal-directed behavior. The prefrontal cortex (PFC) has been implicated in different types of behavioral flexibility. Attentional set-shifting is a type of behavioral flexibility that requires an individual to disengage from a once relevant set of stimulus dimensions and begin responding to a previously irrelevant set to perform optimally. This executive process is assessed in humans using the Wisconsin Card Sort Task (WCST) , a neuropsychiatric task that is critically dependent on the integrity of the dorsolateral P F C in humans (Lombardi and others, 1999). Patients with damage to the P F C can acquire an initial sorting rule readily during performance of the W C S T , but when the rule is shifted without warning patients fail to adapt their responding to the negative feedback (Stuss and others, 2000). Strategy set-shifting is a type of behavioral flexibility that entails the complex process of inhibiting a previously relevant or rewarding strategy in favor of acquisition or activation of a newly optimal strategy. Using strategy set-shifting procedures in rodents, analogous results have been found with the medial P F C , a region homologous to dorsolateral P F C in humans (Uylings and others, 2003), repeatedly implicated as the crucial structure (Birrell and Brown, 2000; Ragozzino and others, 2003; Stefani and others, 2003; Floresco and others, 2005). In contrast, the orbital P F C in rats and primates plays an essential role in reversal learning, a simpler form of behavioral flexibility, whereas lesions to the medial P F C in rodents or the dorsolateral P F C in primates are without effect (Dias and others, 1999; Birrell and Brown, 2000; McAlonan and Brown, 2003; K i m and Ragozzino, 2005). The P F C is strongly reciprocally connected with the mediodorsal nuclei of the thalamus ( M D : Groenewegen, 1988; Conde, and others, 1995), which also plays an important role in behavioral flexibility. Functional imaging studies have revealed that performance of the W C S T causes significant activation of the M D during the receipt of negative feedback, the function of which the authors interpret as flagging the need to initiate an adaptive strategy shift (Monchi and others, 2001). Schizophrenic individuals also show deficits in strategy set-shifting (Pantelis and others, 1999) which has been linked to the observed loss of cells from the M D in schizophrenic individuals relative to controls (Popken and others, 2000). Additionally, Korsakoff's amnesiacs, whose lesions are largely centered around the M D and specifically the intramedullary lamina (Joyce, 1987) show perseverative impairments on the W C S T (Oscar-Berman and others, 2005). Indirect evidence from preclinical animal models also points to the M D as an important nucleus for regulating behavioural flexibility. Neurotoxic lesions of the M D in rats lead to an increase in perseverative-type errors during a working memory task, similar to those errors witnessed in some atypical human populations (Hunt and Aggleton, 1998). However, the importance of the M D in tasks that specifically tap into behavioral flexibility in rodents has yet to be established. Another subcortical structure that is important for behavioral flexibility is the nucleus accumbens (NAc) core. The N A c core receives a dense projection from the dorsal prelimbic and, to a lesser extent, infralimbic portions of medial P F C (Brog and others, 1993; Gabbott and others, 2005). The N A c core can indirectly affect activity in the M D via a projection to the ventral pallidum, the globus pallidus, and the substantia nigra (Zahm and Brog, 1992), and also receives a direct projection from the M D (Berendse and Groenewegen, 1990). Importantly, the N A c core has been shown to be crucial for the maintenance of new strategies as well as elimination of inappropriate response alternatives during acquisition of a strategy set-shift 3 (Floresco and others, 2006). Rats with reversible inactivation of the N A c core performing the strategy set-shift made errors that can be seen as "loss of set" errors, similar to the errors made by Parkinsonian patients performing a set-shift (Gauntlett-Gilbert and others, 1999), or rats with reversible inactivation of the dorsomedial striatum (Ragozzino and others, 2002). Interestingly, regions of the dorsal striatum in humans have been assigned dissociable roles in behavioral flexibility: the caudate and putamen do not contribute to set-shifting, but contribute to the planning and instigation of novel actions (Monchi and others, 2006). Similarly, inactivation of the shell region of the N A c had no effect on performance of a set-shift (Floresco and others, 2006). This is qualitatively different from the perseverative impairment one sees with manipulations of the P F C , indicating that these regions may play functionally distinct roles in the performance of set-shifting behavior. The data reviewed above indicate that the M D is part of a neural circuit that may contribute to behavioral flexibility functions mediated in part by the P F C and N A c core. However, the specific contribution that the M D makes to this executive function is unclear. Furthermore, given that the M D projects to both the P F C and the N A c core, it is unknown whether this nucleus interacts with one or both of these regions to mediate behaviors such as strategy set-shifting. Thus, in Experiment 1, we investigated possible differential contributions of the M D to set-shifting. Rats with reversible bilateral inactivations of the M D performed one of two strategy set-shifts: either the acquisition of an egocentric response and a subsequent shift to a visual-cue based strategy, and vice versa using a cross-maze strategy set-shifting task. In Experiment 2, we investigated possible unique contributions of the M D , N A c and P F C in the regulation of strategy set-shifting at the circuit level. We performed reversible asymmetrical disconnection lesions of the projections between M D and P F C ( M D - P F C ) , M D and N A c core 4 ( M D - N A c ) , and P F C and N A c core (PFC-NAc) to assess quality of information transfer between these regions important for strategy set-shifting. 5 Chapter 2 Methods and Results Experiment 1A Effects of Mediodorsal Thalamic Inactivation on Acquisition of a Response Discrimination and Strategy Set-Shift to a Visual-Cue To determine whether the M D mediates for learning of a response or cue discrimination, or the shift from one discrimination strategy to another, Experiment 1 examined whether inactivation of the M D via local infusion of the local anesthetic bupivacaine hydrochloride impaired acquisition or strategy set-shifting. Method Subjects. Male Long-Evans rats (Charles River Laboratories, Montreal QC) weighing 280-360g at the beginning of the experiment were the subjects. Rats were individually housed in plastic cages in a temperature controlled room (20 0 C) on a 12-hr light—dark cycle. Beginning immediately following surgery, all rats were restricted to 85% of their free-feeding weight, with free access to water for the duration of the experiment. Apparatus. A four-arm cross maze was used. The maze was made of 1.5 cm thick plywood and painted white. Each arm was 60 cm long and 10 cm wide, with 20 cm high walls on each arm and with cylindrical food wells (2 cm wide x 1 cm deep) drilled into the end of each of the arms, 2 cm from the end wall . Four removable table legs attached to the ends of each arm elevated the mazes 70 cm above the floor. Removable pieces of white opaque plastic (20 cm x 10 cm) were used to block the arms of the maze to form a " T " configuration. The maze resided in a room measuring 3.4 m x 3.4 m. 6 Surgery. Rats were anesthetized with 100 mg/kg xylazine and 7mg/kg ketamine hydrochloride, and implanted with 23 gauge bilateral stainless-steel guide cannulae into the M D (flat skull: A P : -2.9mm, M L : +/- 0.7 mm from bregma, D V : -4.9 mm from dura). The coordinates were based on the atlas of Paxinos and Watson, 1998. Four jeweler's screws were implanted surrounding the cannulae and secured in place with dental acrylic. Thirty gauge obdurators flush with the end of the guide cannulae remained in place until the rats were given infusions. Each rat was given at least 7 days to recover from surgery prior to behavioral training. During this recovery period, animals were food restricted and were handled for at least 5 min per day. Microinfus ion. Infusions into the M D were made through an injection cannula (30 gauge) that extended 0.8 mm below the guide cannulae. The injection cannulae were attached by a polyethylene tube to a 10-ul syringe. Saline or the local anesthetic 0.75% bupivacaine hydrochloride was infused at a rate of 0.5 ul/1 m 12 s by a microsyringe pump (Sage Instruments Model 341). Injection cannulae were left in place for an additional 1 min to allow for diffusion. Each rat remained in its home cage for a further 10 min period prior to behavioral testing. Ten minutes before both the response discrimination learning and the strategy set-shift, rats received a microinfusion. Each rat was assigned to one of three infusion treatment groups, (1) Day 1-saline and Day 2-saline, (2) Day 1-saline and Day 2-bupivacaine, (3) Day 1-bupivacaine and Day 2-saline. Group 1 served as the control group, Group 2 determined whether M D inactivation impaired strategy set-shifting, and Group 3 determined whether inactivation of the M D would affect the acquisition of discrimination learning or disrupt consolidation of the learning yielding behavioral differences during the strategy set-shift. Maze Familiarization Procedure. The familiarization and strategy set-shifting procedures have been described previously (Ragozzino, 2002; Floresco and others, 2006). Before the first day of familiarization to the maze, the rats were pre-exposed to 10-20 of the reward pellets they would receive in the maze. On the first day of familiarization, rats were placed in the center of the cross maze, which had each arm baited with five pellets: two in each well , and three down the length of the arm. A rat was placed in the maze and allowed to freely navigate and consume the food pellets for 15 min. If a rat consumed all 20 pellets prior to 15 min, it was removed from the maze and placed in a holding cage, the maze was rebaited with 20 additional pellets and the rat was placed back in the center of the maze. The second day of familiarization was the same as the first day, except there were only three pellets in each arm: two in each well , and one on each arm. On subsequent days of maze familiarization only 4 pellets were placed on the maze, one in each well . Additionally, a black laminated piece of poster board (9 x 20 >< 0.3 cm), which served as the visual-cue, was placed in a random arm, and rotated between rebaitings of the maze. Rats were picked up and placed at the start of an arm, allowed to traverse the arm of the maze, consume the pellet and were immediately picked up and placed at the beginning of another baited arm. Rats continued this familiarization procedure daily until they depleted the maze 4 or more times in fifteen minutes. Rats required an average of 4.58 +/-0.3 days of familiarization, (range; 3-14 days) to reach this familiarization criterion. After the rat had achieved familiarization criterion, the turn bias for the rat was determined. The white opaque. Plexiglas insert was placed at the entrance of one of the arms, forming a " T " configuration. A rat was placed in the stem arm and allowed to turn left or right to obtain a food pellet. In one of the choice arms the visual-cue insert was placed on the floor. After a rat chose an arm and consumed a food pellet, it was picked up, placed in the stem arm, 8 and allowed to make the next choice. If the rat chose the same arm as the initial choice, it was returned to the stem arm until it chose the remaining arm and consumed the food pellet. After choosing both arms, the rat was returned to the holding cage, the Plexiglas barrier and visual-cue were moved to different arms, and a new trial commenced. Thus, a trial for the turn-bias procedure consisted of entering both choice arms and consuming the food pellets. The turn that a rat made first during the initial choice of a trial was recorded and counted toward its turn bias, and the direction (right or left) that a rat turned four or more times over seven total trials was considered its turn bias. After determining the turn bias, a rat's obdurators were removed from the guide cannulae and two injection cannulae extending 0.8mm past the guide cannulae were inserted for 1 min, but no solution was injected at this time. This procedure was performed to familiarize the animal to the two infusions they would receive over the next two days of testing. Response (Experiment 1 and 3) or visual-cue (Experiment 2) discrimination training commenced on the following day. Day 1: Response Discrimination Learning. For this discrimination, the rat was required to always turn in the direction opposite its turn bias (left or right), regardless of the location of the visual-cue placed in one of the arms (See Figure 2.1). Over the course of training one of three start arms were used, discouraging the use of an allocentric spatial strategy to locate the food. On Day 1 of training, a rat was started from the arms designated west, south, and east (W, S, and E , respectively). The location of these arms relative to the spatial cues in the room was varied across animals, so that the maze was placed in one of 4 possible orientations. For every trial, the visual-cue was placed in one of the choice arms so that over every consecutive set of 12 trials it was placed an equal number of times in each choice arm, with no more than two appearances in a row in any one arm. The order of the start location for each trial, as well as the 9 position of the visual-cue, were determined pseudorandomly and taken from a preset sequence that was identical for each animal. On an individual trial, the rat was placed in the stem arm and required to make the appropriate turn in order to receive a food pellet. Between trials a rat was placed back in the holding cage on a bench adjacent to the maze. The intertrial interval was -15 sec. A rat continued to receive training trials until it reached a criterion of 10 correct consecutive choices. There was no limit on the number of trials a rat was allotted to reach this criterion. After the rat achieved this acquisition criterion, it received a probe trial; this consisted of starting the rat from the fourth arm (north, N) that was never a start arm prior to acquisition criterion. During probe trials, the visual-cue was inserted in the arm opposite to the direction that the rat was required to turn. If a rat correctly turned the same direction as was required during training, then response discrimination training was completed. If a rat made an incorrect turn, response training was continued until a rat made an additional 5 correct choices consecutively, at which time another probe trial was administered. This procedure was continued until a rat made a correct choice on the probe trial. The following measures were taken for each rat and used for data analysis: (1) trials to criterion, defined as the total number of test trials completed before a correct choice on the probe trial was made and (2) probe trials, defined as the total number of probe trials an animal required to get one correct. The total time it took to complete training was also recorded. Day 2: Shift to Visual-Cue. The acquisition of this discrimination required the animal to cease the use of a response strategy, and instead use a visual-cue based strategy to obtain food reward. The day following successful acquisition of the response discrimination, rats were trained to enter the arm that contained the visual-cue, the location of which was pseudorandomly varied in the left and right arms such that it occurred in each arm with equal frequency for every 10 consecutive set of 12 trials. The same training procedure, start arms and criteria to complete the visual-cue version were used as described in the response version. For probe trials, the visual-cue was always placed in the arm opposite that the rat had been trained to enter during response discrimination training. Six of every twelve consecutive trials required the rat to respond in this manner (i.e.; enter the arm opposite of the previously learned turn direction). A s described in previous studies (Dias and Aggleton, 2000; Ragozzino and others, 1999; Ragozzino, 2002; Floresco and others, 2006), these trials were separated into consecutive blocks of 4 trials each. Perseverative errors were scored when a rat entered the incorrect arm on 3 or more trials per block of 4 trials. Once a rat made less than three perseverative errors in a block for the first time, all subsequent errors were no longer counted as perseverative errors, because at this point the rat was choosing an alternative strategy at least half 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 entered the incorrect arm on trials where the visual-cue was placed in the same arm that the rat had been trained to enter on the previous day. For example, during training on Day 1, a rat might be required to turn left. During the shift on Day 2, a rat must now enter the arm with the visual-cue, and for half of the trials the cue was in the left arm. In this situation, a never-reinforced error was scored when a rat entered the right arm (i.e.; a choice that was not reinforced on either Day 1 or Day 2). Regressive and never-reinforced errors are used as an index of the animals' ability to respectively maintain and acquire a new strategy. 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 um sections prior to being mounted and stained with Cresyl Violet. 11 Placements were verified with reference to the neuroanatomical atlas of Paxinos and Watson (1998). Statistical Analysis. A separate mixed analysis of variance ( A N O V A ) was conducted on trials to criterion data from the response acquisition on Day 1. Trials to criterion data obtained from Day 2 were analyzed using a two-way between/within subjects A N O V A , with Treatment as the between-subjects factor and Choice Type (two levels: Correct or Error) as the within-subjects factor. When a significant main effect of Treatment was observed, multiple comparisons were conducted using Dunnett's test. Trials per minute, as well as the number of probe trials required to meet criterion were analyzed using A N O V A as well . Results Histology. Figure 2.2 illustrates the location of the cannula tips in the M D . A l l animals with placements encroaching on habenula or subthalamic nuclei were excluded. Rats receiving inactivations of the habenula were unimpaired on the strategy set-shift, though those animals with subthalamic placements generally took more trials to criterion than saline treated animals. Day 1 Response Discr iminat ion. The results for the acquisition of the response strategy on Day 1 are shown in Figure 2.3A. A one-way A N O V A showed no significant difference between groups in total trials to reach the learning criterion, (F(2, 17) = 3.11, n.s.). Additionally, there were no differences found in the number of probe trials required to reach the criterion, (F(2, 17) = 0.14, n.s.), nor were there differences in the number of trials completed per minute between groups, (F(2, 17) = 0.07, n.s.). These findings indicate that the M D does not mediate learning of a simple response discrimination. 12 Day 2 Shift to Visual-Cue. The results for the strategy set-shift to visual-cue are shown in Figure 2.3B. This analysis revealed a significant main effect of Treatment, (F(2, 17) = 14.27, p < 0.001). Additionally, there was a significant main effect of Choice type (F(\, 17) = 87.98,/? < 0.001), as rats made significantly more correct choices than errors over the course of the task. There was no Choice X Treatment interaction, (F(2, 17) = 0.36, n.s.). Dunnett's test showed that rats receiving inactivation of the M D before the strategy set-shift (n = 7) on Day 2 took significantly more trials to reach criterion than rats receiving saline (n = 7, p < 0.05), whereas rats receiving Day 1 inactivation and Day 2 saline did not differ from controls (n = 6). The number of errors committed during the strategy set-shift was analyzed separately using a mixed between-within subjects A N O V A , with Treatment as the between subjects factor and the three types of errors measured as the within subjects factors. A main effect for Treatment (F(2, 17) = 13.69,/? < 0.001) and a main effect of Error were observed, (F(2, 32) = 16.00,/? < 0.001), as well as a significant Error X Treatment interaction was observed, (F(4, 34) = 4.35,/? < 0.01), Figure 2.3C. A series of one-way A N O V A s were performed for each error type. Interestingly, there was a significant simple main effect of treatment for perseverative-type errors, (F(2, 17) = 9.56,/? < 0.005). Furthermore, there was a visually observable increase in never-reinforced errors in rats receiving inactivations of the M D , although this effect did not achieve statistical significance, (F(2, 17) = 1.65, n.s.). There was no significant effect of Treatment for regressive errors, (F(2, 17) = 0.29, n.s.). Multiple comparisons revealed that inactivation of the M D prior to strategy set-shifting increased the number of perseverative errors relative to controls (p < 0.001), though rats with Day 1 inactivations and Day 2 saline were indistinguishable from control rats (n.s.). Additional analyses of the number of probe trials required before reaching criterion and the number of trials completed per minute (See Table 13 2.1.), revealed no significant differences between groups, all (Fs < 1.02, n.s.). Thus inactivations of the M D caused an increase in perseverative errors in a manner similar to P F C manipulations (Ragozzino and others, 1999; Birrell and Brown, 2000; Floresco and others, 2006). Table 2.1. Trials per Minute: Means ± SE for all groups Experiment la Response to Cue Experiment lb Cue to Response Experiment 2 Response to Cue SS BS SB SS BS SB Saline UNI PFC-NA MD-NA MD-PFC Day 1 1.33±0.07 1.29*0.10 1.24±0.15 1.72*0.14 1.60±0.07 1.75±0.12 1.60±0.09 1.52±0.11 1.48±0.22 1.59±0.10 1.89*0.11 Day 2 1.37±0.10 1.56±0.07 1.46±0.14 1.66*0.08 1.50±0.07 1.72±0.10 1.83±0.11 1.69±0.10 2.00±0.09 1.51*0.07 2.10*0.10 Experiment IB Effects of Mediodorsal Thalamic Inactivation on Acquisition of a Visual-Cue Discrimination and Strategy Set-Shift to a Response The results from Experiment 1 indicate that inactivation of the M D does not impair acquisition of a response strategy, but does impair strategy set-shifting to a visual-cue strategy. To ensure the M D is not preferentially necessary for visual-cue discriminations, the order of the discriminations was reversed in Experiment 2. Method Visual-Cue to Response Strategy Set-Shift. For these experiments, rats were initially trained on the visual-cue version of the task on Day 1 followed by testing on the response version on Day 2. A l l other aspects of the testing procedure were identical to those described above. On the shift to the response version, the same measures were assessed as those for Experiment 1, where rats were required to shift from a response to a visual-cue strategy. 14 However, perseverative and regressive errors were analyzed from the trials in which a rat was required to turn in the arm opposite to that of the visual-cue. Ten minutes before each test day, rats received a microinfusion. Each rat was assigned to one of three treatment groups, determined by the infusion treatment administered (1) Day 1-saline and Day 2-saline, (2) Day 1-saline and Day 2-bupivacaine, (3) Day 1 -bupivacaine and Day 2-saline. Resul ts Histology. Figure 2.2 illustrates the location of the cannula tips in the M D . Slides were analyzed for the spread of the infusion. A l l animals with placements encroaching on habenula or subthalamic nuclei were excluded. Day 1 Visual-Cue Discrimination. The results for the acquisition of the visual-cue based strategy on Day 1 are shown in Figure 2.4A. A one-way A N O V A showed no significant difference between groups in total trials to reach the learning criterion, (F(2, 20) = 0.182, n.s.). . Additionally, there were no differences found in the number of probe trials required to reach the criterion, (F(2, 20) = 0.13, n.s.), nor were there differences in the number of trials taken per minute between groups, (F(2, 20) = 0.50, n.s.) or the total time to complete the discrimination, (F(2, 20) = 0.09, n.s.). These results indicate that the M D does not mediate acquisition of a simple visual-cue based discrimination. One rat receiving bupivacaine on Day 1 and saline on Day 2 was excluded from the analysis as a statistical outlier, with its trials to reach criterion on Day 2 more than 2 SD away from the mean of the group with that data point inclusive (M= 44.5, SE= 34.18: excluded rat: 108 trials). Day 2 Shift to Response. The results from the strategy set-shift to a response-based strategy are shown in Figure 2.4B. Trials to criterion were analyzed as in Experiment 1, using a 15 two-way mixed A N O V A . The analysis revealed a significant main effect of Treatment, (F(2, 20) = 4.56, p < 0.05), as well as a main effect of Choice, (F(2, 20) = 116.66, p < 0.001). Dunnett's test indicated that rats receiving inactivations of the M D before the strategy set-shift on Day 2 (n = 8) took significantly more trials to reach criterion than rats receiving saline (p < 0.01, n = 8), though rats receiving Day 1 inactivation and Day 2 saline did not differ from controls (n = l). Analysis of the errors committed during the strategy set-shift revealed an Error X Treatment interaction, (F(2, 40) = 3.29, p < 0.05, Figure 2.4C). Additionally, a main effect of Treatment, (F(2, 20) = 4.73, p < 0.05) and a main effect of Error was observed, (F(4, 40) = 31.72,/? < 0.001). Subsequent one-way A N O V A s for each error type revealed no significant treatment effect for regressive errors, (F(2, 20) = 0.57, n.s.). However, as in Experiment 1 a significant treatment effect was observed for perseverative errors, (F(2, 20) = 4.84,/? < 0.05). Pairwise comparisons indicated that this was due to an increase in perseverative errors for animals receiving M D inactivations on Day 2 relative to controls (p < 0.005), as animals with Day 1 inactivations did not differ from controls. In this experiment, we also observed a significant main effect of Treatment for never-reinforced errors for this strategy set-shift, (F(2, 20) = 3.71,/? < 0.05). Additional analyses of the number of trials completed per minute and the number of probe trials required to complete the strategy set-shift show no significant treatment effects, all Fs < 2.42, n.s. Experiment l b both confirms the results of Experiment l a and suggests a potential role for the M D in the generation of alternative strategies, as evidenced by the increase in never-reinforced errors. 16 Experiment 2 Effects of Reversible Asymmetrical Disconnection Lesions of the P F C , NAc core and M D on Acquisition of a Response Strategy and Shift to Visual-Cue. The results from Experiments 1A and IB clearly show a role for the M D in set-shifting. However, the M D sends projections to both P F C and N A c core which have been implicated in the set-shifting (Ragozzino and others, 1999; Floresco and others, 2006a; 2006b). In Experiment 2, we use an asymmetrical disconnection inactivation procedure to delineate the routes of information transfer within the thalamic-cortical-striatal circuit. Disconnection lesions are used to identify functional components of a circuit where information is transferred serially from one structure to another structure in the same hemisphere, on both sides of the brain in parallel. Furthermore, the design assumes that dysfunction wi l l result from blockade of neural activity at the origin of a pathway in one hemisphere and the termination of the efferent pathway in the contralateral hemisphere. It follows that a unilateral inactivation at either site should have no effect on behavior, as the unaffected pathway w i l l serve to compensate. A s both response to visual-cue and visual-cue to response strategy set-shifts showed a perseverative deficit with inactivations of the M D , we chose to perform only the response to visual cue strategy set-shift for Experiment 2. M e t h o d Subjects. Male Long-Evans rats (Charles River Laboratories, Montreal QC) weighing 290-3 80g at the beginning of the experiment were used. Apparatus. The apparatus was as described in Experiment l a and lb . Surgery. Three groups of rats were implanted with two sets of bilateral cannula. One group of rats was implanted with one pair of cannula in the M D and a second pair in the 17 prelimbic region of the P F C (flat skull: A P , +3.0 mm; M L , ± 0.7 mm from bregma; and D V , -2.7 mm from dura). A second group was implanted with one pair of cannula in the M D and a second pair in the N A c core. (Flat skull: A P = +1.6 mm, M L = ± 1 . 8 mm from bregma, and D V = -6.0 mm from dura). A third group of rats was implanted with one pair of cannula in the P F C and a second pair in the N A c core. Cannulation into the N A c core transects the corpus callosum, effectively eliminating any contralateral information transfer, as the disconnection procedure can only be successful with ipsilateral circuits (Floresco and others, 1999; Dunnett and others, 2005). 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 d to recover from surgery before testing. See Figure 2.5 for histological verification. Microinfus ion. A l l animals received mock infusions (injection cannula inserted with no flow) following the turn bias procedure on the last day of maze familiarization, as well as before response discrimination training on Day 1. The day after acquisition of the response discrimination, and before the set-shift on Day 2, all animals received injections in the opposite hemisphere of that which received the mock infusion the previous day. Unilateral inactivations of M D and P F C were induced with microinfusions of bupivacaine. However, for inactivations of the N A c core we used the G A B A agonists baclofen and muscimol (Sigma-Aldrich Canada, Oakville, Ontario, Canada), (75 ng/ul of each drug dissolved in saline and infused at a volume of 0.3(al). This procedure was employed because projections from brain regions that terminate in the shell of the N A c pass through the N A c core. The use of G A B A agonists would inactivate cell bodies in the N A c core, but leave fibers of passage relatively intact (McFarland and Kalivas, 2001). 18 Five groups of rats were tested, three received disconnection treatments as follows: (1) a unilateral bupivacaine infusion into the P F C in combination with bupivacaine also infused into the contralateral M D , (2) a unilateral bupivacaine infusion into the P F C in combination with a contralateral infusion of baclofen/muscimol into the N A c core, (3) a unilateral bupivacaine infusion into the M D in combination with a contralateral infusion into the N A c core. Two control groups were formed, one group received unilateral inactivations with a saline infusion into the contralateral structure as follows: (1) a unilateral infusion of baclofen/muscimol into N A c core in combination with a saline infusion into the contralateral M D or P F C (2) unilateral bupivacaine infusion into M D in combination with saline in N A c core or P F C (3) a unilateral bupivacaine infusion into the P F C in combination with a saline infusion into the contralateral N A c core or M D . Our saline control group was composed of the following combinations of asymmetrical bilateral infusions: (1) unilateral infusions of saline into both M D and contralateral N A c core. (2) infusions of saline into both P F C and contralateral N A c core. (3) unilateral infusions of saline into the M D and saline infusions into the contralateral P F C (See Table 2.2). The hemisphere (left or right) used for the injection was also counterbalanced across animals within groups. We did not include a combined ipsilateral inactivation of any structures as because numerous studies using disconnection designs have shown that ipsilateral lesions of two interconnected structures in the same hemisphere do not impair behavior relative to the effect of crossed, disconnection lesions (Olton and others, 1982; Warburton and others, 2000; Chudasama and others, 2003; Dunnett and others, 2005). Maze Famil iar iza t ion Procedure and Testing. The familiarization procedure procedure was identical to that used in Experiment l a and Experiment l b . The strategy set-shifting procedure was the response to visual-cue strategy set-shift used in Experiment l a . 19 Table 2.2 Summary of Treatments and Groups. Cannulation n Treatment Group 9 M D - B u p i PFC-Bup i Disconnection M D - P F C 3 MD-Saline PFC-Bup i Unilateral P F C 3 M D - B u p i PFC-Saline Unilateral M D 4 MD-Saline PFC-Saline Saline 7 M D - B u p i PFC-Bup i Disconnection PFC-NAc 3 PFC-Saline N A c - B a c / M u s Unilateral N A c 3 PFC-Bupi NAc-Sal ine Unilateral P F C 4 PFC-Saline NAc-Sal ine Saline 8 M D - B u p i N A c - B a c / M u s Disconnection MD-NAc 3 MD-Saline N A c - B a c / M u s Unilateral N A c 3 M D - B u p i NAc-Sal ine Unilateral M D 4 MD-Saline NAc-Sal ine Saline Results Statistical Analysis. There were no differences between saline infused rats so their data were combined to form one control group for analysis (Day 2 T T C : F(2, 9) = 0.99, n.s., n = 12). A mixed A N O V A was performed on the total trials to reach criterion during the strategy set-shift for rats with unilateral infusions, with the structure inactivated ( N A c core, P F C or M D ) as the between subjects factor. There were no differences between groups on the number of trial to reach criterion for rats receiving unilateral infusions, (PFC n = 6, N A c n = 6, M D n - 6: (F(2, 12) = 0.35, n.s.) nor was there a main effect of hemisphere of infusion ( F ( l , 12) = 1.76, n.s.), nor a Side by Group interaction, (F(2, 12) = 0.82, n.s.) so the data from all rats receiving unilateral infusions were also combined to form the second unilateral inactivation control group (Figure 2.6, insets and Table 2.2). Day 1 Response Discrimination. The results for the acquisition of the response strategy on Day 1 are shown in Figure 2.6A. These findings show no differences between groups receiving mock infusions into different regions. A one-way A N O V A showed no significant difference between groups in total trials to reach criterion for the response discrimination, (F(4, 20 49) = 0.59, n.s.). Additionally, there were no differences found in the number of probe trials required to reach the criterion, nor were there differences in the number of trials taken per minute between groups, nor the total time to complete the discrimination, all (Fs < 1.45, n.s.). Day 2 Shift to Cue. The results for the strategy set-shift to a response-based strategy are shown in Figure 2.6B. The analysis of choice type revealed a significant main effect of Treatment, (F(4, 49) = 4.82, p < 0.005), as well as a main effect of Choice, ( F ( l , 49) = 294.87,/? < 0.001). Dunnett's test indicated that rats receiving M D - P F C (n = 9) and P F C - N A c (n = 7) disconnections before the strategy set-shift on Day 2 all took significantly more trials to reach criterion than rats receiving saline (both: p •< 0.001). Although disconnection of the M D and N A c core also increased the number of trials required to reach criterion, this increase did not reach significance relative to saline (p = 0.11). Importantly, unilateral inactivations of each region did not increase the number of trials to reach criterion, as unilateral group did not differ from saline (Figure 2.6B, inset, n.s.). A mixed A N O V A with Group as the between subjects variable and the three types of Errors as levels of the within subjects factor revealed a main effect of Group, (F(4, 49) = 4.61,/? < 0.005) and a main effect of Error (F(2, 98) = 9.48,/? < 0.001, Figure 2.7). A significant Group X Error interaction was also observed, (F(8, 98) = 6.25,/? < 0.001). Subsequently, each error type was analyzed in a one-way between subjects A N O V A . Perseverative errors were found to vary by Group, (F(4, 49) = 9.12,/? < 0.001, Figure 2.7A). Dunnett's test revealed that rats receiving M D - P F C disconnections made significantly more perseverative errors than saline treated controls (p < 0.05). Similarly, the rats that received P F C - N A c disconnections also made significantly more perseverative errors (p < 0.001) relative to saline treated controls. Neither the 21 unilateral control group nor the M D - N A c disconnection group differed from controls in total perseverative errors (n.s.). However, there was no effect of Group on the number of regressive type errors, (F(4, 49) = 0.95, n.s., Figure 2.7B). The analysis of never-reinforced errors revealed an effect of Group, (F(4, 49) = 4.00, p < 0.01, Figure 2.7C). In contrast to what was observed with perseverative errors, pairwise comparisons revealed that this was due to a selective increase in never-reinforced errors for the M D - N A c disconnection group relative to saline controls (p < 0.001); all other groups were not different from saline treated rats (n.s.). Additional analyses showed an effect of Group for trials per minute taken during the strategy set-shift, (F(4, 49) = 3.189,/? < 0.05). Multiple comparisons with Tukey's H S D showed M D - P F C disconnection group was somewhat slower to complete trials than the P F C - N A c disconnection group. However, neither group differed from saline treated rats (See Table 1). Analysis of the number of probe trials required to reach criterion showed no differences between groups, (F(4, 49) = 0.59, n.s.). Visual-Cue Discrimination Figure 2.1. Example of the strategy set-shifting task used in Experiment 1A and 2. The arrows in the maze represent the correct navigation pattern to receive reinforcement. During initial response discrimination training on Day 1 (upper panels), in this example the rat was started from the south (S), west (W), and east (E) arms and always had to make a 90° turn to the right to receive food reinforcement. A black visual-cue was randomly placed in one o f the choice arms on each trial but did not reliably predict the location of food during response training. During the set-shift on Day 2 (lower panels), the rat is required to use a visual-cue discrimination strategy. Here, the rat was started from the same arms but had to always enter the arm with visual-cue, which could require either a right or left turn. Thus, the rat must shift from the old strategy and approach the previously-irrelevant cue in order to obtain reinforcement. See Methods for details. Mediodorsal Thalamus Figure 2.2 Schematic of coronal sections of the rat brain showing the placements of the cannulae tips for all rats that received infusions of bupivacaine into the M D . Brain sections correspond to the atlas of Paxinos and Watson (1998). 24 Day 1 Response Learning 100r a o 'C u •c U o 80 60 40 ea 'C 20 Day 1 TreatSaline Saline Bupivacaine Day 2 Treat: Saline Bupivacaine Saline B. Day 2 Shift to Visual Cue 100 § 8 0 h £ 60 o « 40 ' 20 0 Day 1 Treat: Saline Saline Bupivacaine Day 2 Treat: Saline Bupivacaine Saline g 25 20 15 h 10 h 5 h I Perseverative Error Type T Regressive Never-Reinforced Figure 2.3 Experiment 1 A : Inactivation of the M D disrupts shifting from a response to a visual-cue based strategy (Experiment 1A, A - C ) . Data are expressed as means +/- S E M . A , Trials to criterion on acquisition of a response discrimination on Day 1 by rats receiving infusions of saline (white, black bars) or bupivacaine into the M D (Bupi, hatched bar). Inactivation of the M D did not impair response learning. B, Trials to criterion on the shift to visual-cue discrimination strategy on Day 2 following infusions of either saline (white and hatched bar) or bupivacaine into the M D (black bar; single star denotes p<0.05 significantly different from saline control group). C , Analysis of the type of errors committed in Experiment 1A during the set-shift on Day 2. Inactivation of the M D prior to the set-shift (black bars) significantly increased the number of perseverative errors (left) but did not affect regressive (middle) and never-reinforced (right) errors (double stars denote p<0.01 significantly different from combined errors made by saline-treated rats, white bars). Inactivation of the M D prior to initial discrimination learning (hatched bars) did not alter the number of errors during the set-shift. 25 A . Day 1 Cue Learning 125 o 100 •c u o 75 50 25 Day 1 Treat:Saline Saline Bupivacaine Day 2 Treat: Saline Bupivacaine Saline B. Day 2 Shift to Response 125 C 100 o U p 75 50 25 0 Day 1 Treat: Saline Saline Bupivacaine Day 2 Treat: Saline Bupivacaine Saline 40 35 30 25 g 20 m 15 10 51-0 I i Error Type Perseverative Regressive Never-Reinforced Figure 2.4. Experiment I B : Inactivation of the M D disrupts shifting from a visual-cue to a response strategy (Experiment I B , A - C ) . A , Trials to criterion on acquisition of the visual-cue discrimination on Day 1 by rats receiving either infusions of saline (white, black bars) of bupivacaine into the M D (hatched bar). Inactivation of the M D did not impair visual-cue based learning. B, Trials to criterion on the shift to the response discrimination on Day 2 following infusions of either saline (white and hatched bars) or bupivacaine into the M D (black bar; double stars denote p<0.01 significantly different from saline control group). C , Again, M D inactivation had a significant effect on perseveration (left) and in this case, never-reinforced errors (right), but did not increase the number of regressive (middle) errors during the shift on Day 2 (double stars denote p < 0.01, single star denotes p < 0.05 significantly different from the same type of errors made by saline treated rats). 26 A. MD-PFC B. PFC-NAc C. MD-NAc Figure 2.5 Schematic of coronal sections of the rat brain showing the placements of the cannulae tips for all rats that received double cannulations. A , Location of cannulae tips (black circles) for all rats used for data analysis receiving M D - P F C cannulations. Plates are computer-generated adaptations from Paxinos and Watson (1998). Numbers beside each plate correspond to millimeters from bregma. A l l animals received infusions of either bupivacaine or saline in each hemisphere. B, Location of cannulae tips (black circles) for all rats used for data analysis receiving P F C - N A c cannulations. C , Location of cannulae tips (black circles) for all rats used for data analysis receiving M D - N A c cannulations. Brain sections correspond to the atlas of Paxinos and Watson (1998). 27 Shift to Visual Cue Saline UNI M D - P F C PFC-NAc NAc-MD Figure 2.6. Experiment 2: The effects of disconnection of the M D - P F C , M D - N A c and P F C - N A c pathways on shifting from a response to a visual-cue based strategy. Data are expressed as means +/- S E M . A , Trials to criterion on acquisition of the response discrimination on Day 1 by rats receiving mock infusions (all bars). A s there were no differences between animals receiving unilateral (UNI: M D , N A c or P F C , inset) infusions on Day 2, they were combined for analysis. B , Trials to criterion on the shift to the visual-cue discrimination on Day 2 following infusions of either saline (white bar), unilateral inactivations (UNI, gray bars), or disconnections of the M D -P F C (hatched bar), P F C - N A c (black bar), or M D - N A c (cross-hatched bar). Neither saline infusions nor unilateral inactivations impaired rats' ability to shift strategy set. However, rats receiving disconnections of the M D - P F C and P F C - N A c pathways showed significant increases in total trials to reach criterion relative to saline control group.(double stars denote p < 0.01 significantly different from saline control group). Figure 2.7. Experiment 2: Analysis of the type of errors committed during the acquisition of a visual-cue strategy during a strategy-shift. Data are expressed as means +/- S E M . A , Saline injections, unilateral inactivations (UNI) and M D - N A c disconnections did not affect the number of perseverative errors committed by rats during the strategy set-shift. However, M D - P F C and P F C - N A c disconnections resulted in a significant increase relative to saline infused controls (double stars denote p < 0.01, single starts denote p < 0.05 significantly different from saline control group). B, Neither saline injections, unilateral inactivations or disconnection treatments had any effect on regressive-type errors during the strategy-set shift. C , Only M D - N A c disconnections resulted in an increase in never-reinforced type errors relative to saline controls (double stars denote p < 0.01 significantly different from saline control group). 29 Chapter 3 Discussion The present data delineate a role for the M D , as well as describe interactions with the P F C and N A c core in the mediation of strategy set-shifting. Disconnecting the projections from the M D to the P F C served to impair the ability of rats to inhibit the previously relevant strategy or signal the need for a shift. Disconnecting the projections from the M D to the N A c core resulted in a unique deficit, where animals showed no perseverative errors, but did display a selective increase in never-reinforced errors, which indicate a failure to eliminate inappropriate response options during acquisition of the shift. Interestingly, disconnections of the P F C projection to the N A c core resulted in a perseverative deficit, suggesting an inability to suppress the use of a previously acquired strategy. Notably, no deficits were seen for unilateral inactivation of any of the targeted structures, further supporting the contention that the impairments were due specifically to functional disconnection and not due to additive effects of unilateral inactivation. Mediodorsal Thalamus Signals the Need to Shift For the first time, we demonstrate a crucial role for the M D in this form of strategy set-shifting. Reversible bilateral inactivation of the M D impaired strategy shifting from a cue to a response discrimination, as well as from a response to a cue discrimination. However, inactivation of the M D during the first strategy acquisition did not affect animals' ability to acquire either discrimination, demonstrating the M D is specifically involved in shifting between strategies, but not involved in simple discrimination learning as has been demonstrated previously (Chudasama and others, 2001). The deficit in the acquisition of the strategy shift observed in animals with bilateral inactivation of the M D was due to an increase in the total 30 number of perseverative errors, indicating a failure to disengage from a previously relevant strategy. In addition, an increase in never-reinforced type errors that indicate an inability to eliminate inappropriate response alternatives based on negative feedback. Many researchers have investigated the possibility of a role for the M D in a simpler form of behavioral flexibility, reversal learning. Tigner (1974) described a deficit in reversals of brightness and tactile discriminations with lesions of the M D and paraventricular nucleus, though no effect was seen for spatial reversals. Using a more rule-based reversal in the operant chamber, Bercochea and colleagues (1989) failed to find any effect of M D lesion on reversals. Another study found a deficit for spatial reversals, but only on those reversals subsequent to an initial reversal of stimulus-reward contingencies (Means and others, 1975). Other studies have also shown a lack of M D involvement on initial but not subsequent reversals of stimulus-reward associations (Chudasama and others, 2001). N o clear consensus emerges from these studies as to a role for the M D in this simpler form of flexibility, as its involvement seems to be highly context and stimulus dependent. However, perseverative behavior has been observed with neurotoxic lesions of the M D in other types of tasks (Hunt and Aggleton, 1998). The perseverative deficit seen with reversible inactivation of the M D is similar to what one sees after medial P F C manipulations (Birrell and Brown, 2000; Ragozzino, 2002; Floresco and others, 2006). This result was not unanticipated considering the strong reciprocal connections between the M D and the medial P F C (Conde, Maire-LePoivre and others, 1995; Gabbott, Warner and others, 2005). There have been a number of studies demonstrating that lesions and inactivations of the M D can induced similar behavioral deficits to those observed following P F C manipulations. These include radial maze tasks with a working memory component (Harrison and Mair, 1996; Floresco and others, 1999), primate 31 working memory tasks (Funahashi and others, 2004), delayed matching (Bailey and Mair, 2005), delayed-nonmatching in primates (de Zubicaray, McMahon, and others, 2001) and familiarity estimates in humans (Zoppelt and others, 2003). Thalamic activation has been linked to behavioral flexibility in normal humans during a W C S T (Monchi and others, 2001). In that study, it was observed that activation of the M D occurred specifically when the participants received negative feedback in the form of a black background on the visual interface, but not positive feedback, a light gray background. These findings suggest that the role for the M D in the W C S T is as a signal for the necessity to shift set. Interestingly, activation of the dorsolateral P F C occurred during both positive and negative feedback, indicating the P F C is critically involved in comparing online feedback with past events Monchi and others, 2001). Though both the M D inactivation in our study and P F C inactivation in others (Ragozzino and others, 1999) result in the same form of perseverative deficit, it is reasonable to hypothesize that this may result from a different underlying process as evidenced in the above study. M D - P F C Disconnections Asymmetrical bupivacaine infusions into the M D and P F C , which interrupted the flow of information between these structures, resulted in an increase in total trials to reach a criterion on a visual cue discrimination during the strategy set-shift. Analysis of the specific type of errors made by rats with this disconnection treatment indicated this deficit was due to a selective increase in perseverative errors. This perseverative deficit suggests that thalamocortical information transfer is necessary for successful disengagement from a previously relevant strategy. This further supports the contention that the effects of bilateral M D inactivation 32 observed in Experiment 1 were due to a lack of thalamocortical input to the P F C , signaling the need to shift strategies or aiding in the inhibition of the previously relevant strategy. A s reviewed above, functional imaging studies have shown that the M D has a role in signaling the need to shift strategies, based on negative feedback (Monchi and others, 2001). In the task used in the present study, this would be analogous to entering an incorrect arm during the set-shift. Based on these findings, it is reasonable to propose that following a M D - P F C disconnection the medial P F C would be deprived of informationrelated to the absence.of reward, and thus unable to begin extinguishing responding in accordance with the previously relevant strategy (Lebron and others, 2004), so a new strategy might be attempted or instantiated. Alternatively, the prelimbic P F C has been shown to have a role in maintaining an established outcome-action association in working memory for the purpose of guiding future behavior (Corbit and Balleine, 2003). In that study, rats with lesions to the prelimbic P F C were trained to differentially respond for sucrose solution and sugar pellets, one of which was subsequently experimentally devalued with a specific satiety manipulation. Lesioned rats failed to differentiate between the devalued and non-devalued reward, and globally depressed responding. These results indicate that the P F C is crucial for the incorporation of specific reward information into behavior (Corbit and Balleine, 2003). Furthermore, lesions of the M D but not the anterior thalamic nuclei reveal a similar deficit in specific devaluation (Corbit and others, 2003). When these findings are viewed in light of the present data, they suggest that the M D and the P F C must work together to suppress the use of previously established rules based on irrelevant reinforcement contingencies. P F C - N A c Disconnections 33 Asymmetrical bupivacaine infusions into the P F C and G A B A agonists into the N A c core, disconnecting the flow of information between these structures, resulted in a marked increase in performance during a strategy set-shift. A s with M D - P F C disconnections, the P F C - N A c disconnection deficit was due wholly to an increase in perseverative type errors and not regressive or never-reinforced errors, which is especially interesting in light of the role previously established for the N A c core in behavioral flexibility (Floresco and others, 2006). Specifically, bilateral inactivation of the N A c core led to an increase in regressive and never-reinforced type errors. The increase in regressive errors, but not perseverative errors indicates that the N A c core is not involved in the elimination of the inappropriate response, but instead plays and essential role in the maintenance of a new strategy. This effect was selective for the inactivation of the N A c core, as inactivation of the N A c shell during the set shift did not impair strategy shifting (Floresco and others, 2006). Regressive type errors are the result of the same operant as perseverative errors, though these errors occur later in the learning sequence when the animal is choosing the previously relevant strategy only 50% of the time or at chance levels. Thus, perseverative and regressive errors are inexorably related, as an animal can not maintain a novel strategy unless it has first shifted from the previously rewarded strategy. In the present study, disconnection of the P F C -N A c core pathway resulted in a deficit that produces an over-reliance on the previously relevant strategy, but did not increase regressive errors. This finding suggests that the P F C is unable to send updated information to the N A c core on the proximate success or failure of current strategies to the N A c core for maintenance of a novel discrimination strategy. This hypothesis is consistent with previous studies investigating the role of this pathway in the context of a 5-choice serial reaction time task (Christakou and others, 2004). In that study, disconnection of the 34 P F C - N A c pathway caused a deficit in the integration of reward information into the behavioral repertoire. Similarly, disconnections of the P F C and N A c core also severely impaired prospective foraging in a delayed-spatial win-shift radial arm maze task (Floresco and others, 1999). Taken together, these data support the contention that interactions between the P F C and N A c core play a crucial role in behaviors that require maintenance of strategies to novel reward contingencies as well as updating responding based on trial-unique reward information. These include sustained attention (Christakou and others, 2004), working memory (Floresco and others, 1999) and, as demonstrated here, strategy set-shifting. M D - N A c Disconnections Disconnecting the flow of information from the N A c core to the M D resulted in an atypical pattern of errors, distinct from that we observed in either the M D - P F C disconnection or the P F C - N A c disconnection. Specifically, we observed a selective increase in never-reinforced type errors with this disconnection, with no concomitant increase in perseverative or regressive type errors. Never-reinforced errors indicate a failure to parse out ineffective strategies. As opposed to perseverative/regressive errors, never-reinforced errors entail a choice that was incorrect during both initial discrimination training and during the shift. Rats quickly learn that the previously correct strategy is no longer appropriate and engage alternative strategies 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 turn right instead of left). Intact rats make relatively few errors of this type and learn that these strategies do not lead to reward reliably. In contrast, N A c core inactivations increased never-reinforced errors, indicating that the core also mediates the elimination of inappropriate response options, enabling the reorganization of behavior to obtain reward in an optimal manner. A s mentioned above, 35 bilateral inactivation of the N A c core results in an increase in both regressive and never-reinforced errors, supporting the contention that the N A c core is involved in the maintenance and generation of novel strategies. Thus, the present data indicate that the increase in never-reinforced errors observed in bilateral N A c core inactivation or in M D - N A c disconnection are due to a disruption of M D input to this nucleus. To our knowledge, no functional role has been established for this pathway to date. In a previous study, disconnections of this pathway did not impair working memory assessed using a response variant of the radial arm maze (Floresco and others, 1999). Thus, this pathway does not play a role in mediating working memory functions subserved by the P F C . In contrast, our data suggest this pathway plays a selective role in the context of strategy set-shifting. The increase in never-reinforced errors indicates information transfer from the M D to the N A c core is necessary for the successful elimination of inappropriate strategies based on reward feedback. These data further clarify the role for parallel functional thalamocorticostriatal circuitry involved in the overall process of strategy set-shifting. Thus, early in the set-shift, the M D sends information about the absence of reward to the P F C , where this information may be assimilated into the updated behavior via suppression of the irrelevant strategy. The P F C then signals the N A c core, which ceases instigating the strategy, i f inappropriate. A t that point, new strategies are attempted and eliminated i f inappropriate, a process facilitated by the M D - N A c pathway. When a new strategy is found, it is maintained by the P F C - N A c pathway, possibly in conjunction with other structures such as the dorsomedial striatum (Ragozzino and others, 2002, See Figure 3.1). Conclusion The present data provide insight into the pathophysiology that may underlie impairments in behavioral flexibility in diseases such as schizophrenia. There is evidence of a compromised 36 M D - P F C circuit in schizophrenia, with a reduced number of neurons appearing in the M D of schizophrenic individuals (Young and others, 2000; Dorph-Petersen and others, 2004) as well as reduced M D volume (Andreason and others, 1994). Additionally, schizophrenic individuals show deficits in tasks assessing behavioral flexibility and strategy shifting that are rooted in impaired concept formation (Pantelis and others, 1999) and often observed as perseverative responding in attentional set-shifting paradigms (Elliott and others, 1998). When viewed in light of the present findings, these data support the assertion that the deficits in behavioral flexibility observed in schizophrenia may be due to a compromised excitatory projection originating in the M D and terminating in the P F C and N A c core. Behavioral flexibility is a composite of different processes: suppression of irrelevant strategies, acquisition and generation of novel strategies, and maintenance of effective strategies. These data highlight the fact that a distributed neural circuit mediates strategy set-shifting, and clarifies the important contributions that subcortical areas make to this form of behavioral flexibility. The apparent dissociability of different phases of strategy set-shifting that we demonstrate here have been similarly described in human imaging studies using the W C S T and variants, with the P F C playing a role distinct from that of the thalamus or the striatum (Monchi and others, 2001). The present findings establish that parallel pathways originating from the M D and terminating in both the P F C and N A c core make dissociable contributions to behavior when an organism must change its behavior in response to dynamic environmental demands. Specifically, the M D projection to the P F C is important for signaling the need to shift strategies, and the projection to the N A c core important for eliminating inappropriate response strategies. Furthermore, the P F C projection to the N A c core facilitates the integration of reward information and behavioral consequences into behavioral responses geared toward the anticipation of future 37 reward. Collectively these findings further elucidate the neural circuitry that regulates strategy set-shifting and may provide important insight into the neural pathology that underlies executive dysfunction in some psychiatric disorders where an impairment in behavioral flexibility is a prominent symptom. Figure 3.1 Schematic diagram of the neural circuit involved in strategy set shifting behavior. See discussion for details. P F C , prefrontal cortex, M D , mediodorsal nuclei of thalamus, N A c , nucleus accumbens core. 39 References A l a m M N , Mal l i ck B N . 1990. 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