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Alterations in executive functioning induced by repeated amphetamine exposure Whelan, Jennifer M. 2008

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ALTERATIONS IN EXECUTIVE FUNCTIONING INDUCED BY REPEATED AMPHETAMINE EXPOSURE  by Jennifer M. Whelan B.Sc. Honors, University of Western Ontario, 2006  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in The Faculty of Graduate Studies (Neuroscience) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) June 2008  © Jennifer M. Whelan, 2008  ABSTRACT Chronic exposure to psychostimulants such as amphetamine (AMPH) can induce long-term disruptions in cognition via actions on prefrontal cortex dopamine. Previous work has shown that two types of executive functions, set shifting and working memory (WM), are disrupted by AMPH sensitization and that these cognitive domains are impaired in schizophrenics and stimulant abusers. We assessed the effects of AMPH sensitization on behavioural flexibility using a cross-maze set shifting task and a WM task using the delayed spatial win-shift (SWSh) task in Long Evans (LE) and Sprague Dawley (SD) rats. Rats were exposed to an AMPH sensitization regimen (15 AMPH or saline injections: 1-5 mg/kg every 2nd day, increasing the dose by 1 mg every 3rd injections) following habituation on the mazes. In experiment 1, LE and SD rats were initially trained on a visual cue discrimination. During the set shift, rats were required to shift from the previously acquired visual-cue-based strategy to a response strategy (e.g.; always turn left, ignore the visual cue). For the reversal, rats were trained to reverse their turn direction. AMPH treatment did not impair learning of the initial cue discrimination in either strain. However, AMPH treated rats learned the response discrimination faster than controls during the set shift and AMPH treated LE rats were faster than controls to reach acquisition criterion during the response reversal. AMPH treatment neither impaired nor improved reversal learning in SD rats. In experiment 2, rats were tested on the SWSh task in which spatial information acquired during a training phase was used 30 minutes later during the testing phase in order to retrieve food pellets on the maze. In this task, AMPH treated rats were faster to reattain criterion than control rats. Correlational analysis further revealed that AMPH sensitized rats that required more days to reach criterion before AMPH treatment (i.e. slow learners) tended to make more errors during re-acquisition of the memory task. Viewed collectively, these results  ii  suggest that chronic AMPH treatment can enhance behavioural flexibility and WM assessed in this manner. However, repeated AMPH exposure may have exacerbated pre-existing cognitive deficits in slow learning rats.  iii  TABLE OF CONTENTS  Abstract................................................................................................................................................ii Table of Contents ...............................................................................................................................iv List of Figures.....................................................................................................................................vi Acknowledgements .......................................................................................................................... vii 1. Introduction.....................................................................................................................................1 2. Materials and Methods................................................................................................................11 2.1. Experiment 1: Set shifting on the cross-maze .......................................................................11 2.1.1. Subjects.............................................................................................................................11 2.1.2. Apparatus..........................................................................................................................11 2.1.3. Maze habituation procedure ............................................................................................11 2.1.4. Amphetamine sensitization .............................................................................................13 2.1.5. Set shifting procedure ......................................................................................................14 2.1.6. Visual cue discrimination................................................................................................14 2.1.7. Strategy set shift to a response discrimination...............................................................16 2.1.8. Reversal of the response discrimination.........................................................................16 2.2. Experiment 2: Working memory on the radial arm maze ....................................................18 2.2.1. Subjects.............................................................................................................................18 2.2.2. Apparatus..........................................................................................................................18 2.2.3. Delayed spatial win-shift (SWSh) task, initial training.................................................18 2.2.4. Amphetamine sensitization .............................................................................................20 2.2.5. Retraining .........................................................................................................................20 2.3. Challenge dose of amphetamine.............................................................................................21 2.4. Data analysis............................................................................................................................21 3. Results ............................................................................................................................................22 3.1. Experiment 1: Set shifting on the cross-maze .......................................................................22 3.1.1. Visual cue discrimination................................................................................................22 3.1.2. Strategy set shift to a response discrimination...............................................................22 3.1.3. Reversal of the response discrimination.........................................................................23 3.2. Experiment 2: Working memory on the radial arm maze: ...................................................27 3.3. Locomotor activity..................................................................................................................30 4. Discussion.......................................................................................................................................32 4.1. Behavioural Flexibility ...........................................................................................................32 4.2. Working Memory....................................................................................................................41 4.3. AMPH sensitization and executive function: relation to schizophrenics ............................43 4.4. AMPH sensitization and executive function: relation to stimulant abusers........................44  iv  5. Conclusions....................................................................................................................................47 References ..........................................................................................................................................48 Appendices ……………………………………………………………………………………...57  v  LIST OF FIGURES Figure 1  Example of the attentional set shifting task used in Experiment ………………. 15  Figure 2  Diagram of the delayed spatial win-shift (SWSh) eight-arm radial-maze task ....17  Figure 3  Experiment 1: The effects of AMPH sensitization on a visual cue discrimination, set shift to a response discrimination and response reversal discrimination …... 19  Figure 4  Total number of errors between AMPH and saline treated rats on the set shift discrimination ………………...………………………………………………... 21  Figure 5  Number of probe trials needed to complete the response reversal discrimination ………………………………………………………………….. 24  Figure 6  The effects of repeated AMPH or saline treatment on number of days to reach criterion on the SWSh task …………………………………………………….. 25  Figure 7  Analysis of the number of errors during re-testing on the SWSh task postinjections ……………………………………………………………………...... 26  Figure 8  Analysis of the relationship between days to reach criterion pre-injections and errors committed post-injections during re-testing on the SWSh task ………… 27  Figure 9  The effects of an AMPH challenge dose on locomotor activity in rats sensitized to AMPH ………………………………………………………………………….. 30  vi  ACKNOWLEDGEMENTS I would like to thank Dr. Stan Floresco for his supervision and guidance for this study. I would also like to thank Maric Tse, Desirae Haluk, Sarvin Ghods-Sharifi, Jennifer St. Onge and Lawrence Bau for their valuable help with this study.  vii  1. INTRODUCTION Repeated exposure to psychostimulant drugs such as amphetamine (AMPH) can lead to long term behavioural and neural adaptations, many of which are related to changes in the mesolimbic and striatal dopamine (DA) systems (Featherstone et al., 2007; Paulson and Robinson, 1995). One of the most prominent behavioural changes induced by repeated, intermittent administration of drugs like AMPH is locomotor sensitization in response to a challenge dose of the drug (Stefani and Moghaddam, 2002; Russig et al., 2005; Paulson and Robinson, 1995; Peleg-Raibstein and Feldon, 2008). These behavioural changes are associated with alterations in receptor expression, neuronal excitability and morphology in a number of DA terminal regions including the nucleus accumbens and prefrontal cortex (PFC) (Stefani and Moghaddam, 2002). Earlier studies on the neural basis of sensitization focused on AMPHinduced changes in the nucleus accumbens. Thus, repeated treatment with AMPH results in altered morphology of neurons in the nucleus accumbens, including increases in length and density of dendritic spines and an increase in number of branched spines on medium spiny neurons (Robinson and Kolb, 1997). As well, AMPH treatments result in increased DA levels in the nucleus accumbens (Warburton et al., 1996; Cadoni and Di Chiara, 2007). However, more recent studies have focused on how repeated AMPH exposure can induce neurophysiological alterations in the PFC, including altered patterns of synaptic activity in the PFC (Robinson and Kolb, 1997). Furthermore, AMPH sensitization reduces PFC DA turnover (as measured by DOPAC/DA) in the dorsolateral PFC of primates (Castner et al., 2005) and leads to changes in DA levels in different subregions of the medial PFC in rats (Steketee, 2003). These alterations in PFC DA neurotransmission may mediate some of the behavioural effects associated with repeated AMPH treatment (Steketee, 2003; Hedou et al., 2001).  1  Neuronal changes associated with chronic AMPH administration have been implicated in the development of drug addiction and cognitive deficits related to addiction (Stefani and Moghaddam, 2002; Wyvell and Berridge, 2001). A predominant theory of drug addiction focuses on the incentive-sensitization theory in which the positive-incentive value of addictive drugs increase with drug use (Robinson and Berridge, 1993). Essentially, the pleasurable effects associated with obtaining the drug (the ‘wanting’ aspect) becomes sensitized whereas tolerance may build to the reinforcing value of the drug (taking/’liking’ the drug) (Wyvell and Berridge, 2001). AMPH sensitization can lead to increased cue-triggered craving of a drug, where sensitized rats will excessively choose sucrose when it becomes a reward-related cue. Even when rats are in a drug-free state following sensitization, there is increased motivation and ‘wanting’ for the sucrose reward in the presence of a cue (Wyvell and Berridge, 2001). These studies show that repeated AMPH exposure can be used to model this ‘wanting’ aspect of psychostimulant abuse that is highly relevant to the propensity of addiction in people that take psychostimulant drugs. Drug-induced DA release after repeated AMPH administration has been shown to correlate positively with drug-induced wanting and novelty seeking and leads to increased DA release (Leyton, 2007). Furthermore, manipulations that increase nucleus accumbens DA overflow may also influence drug-taking behaviours, in that the sensitization of midbrain DA neuronal activity through drug-induced manipulations is related to increased selfadministration of psychostimulant drugs including AMPH (Vezina, 2004). In light of these considerations, the AMPH sensitization model has provided important insight into the behavioural and neural changes that may be associated with drug addiction. Repeated AMPH treatments have also been proposed to model the positive symptoms and cognitive deficits associated with schizophrenia. Repeated AMPH abuse in humans can lead  2  to a psychotic-like state that resembles the positive symptoms of schizophrenia (Featherstone et al., 2007). Accordingly, low doses of AMPH worsen psychotic symptoms in patients with schizophrenia (Laruelle et al., 1999), and imaging studies have implicated an overactive striatal DA system that underlies these symptoms (Laruelle et al., 1996). In contrast, studies using PET have revealed increases in PFC D1 receptor density and binding in people with schizophrenia (Abi-Dargham, 2003; Hirvonen et al., 2006). Other studies suggest there may be contrasting levels of cortical DA in people with schizophrenia, leading to a prefrontal hypodopaminergic state (Weinberger et al., 1988) compared with increased levels of DA in the nucleus accumbens. Furthermore, it is now commonly believed that decreased prefrontal DA may give rise to cognitive deficits associated with the disorder (Davis et al., 1991). In this regard, although the positive symptoms of schizophrenia have been studied in animal models of the disorder, less is known about how these models may resemble cognitive impairments observed in schizophrenia (Floresco et al., 2005). These cognitive deficits are negatively associated with measures of functional outcome (Featherstone et al., 2007; Green, 1996) and antipsychotic treatments used for schizophrenic patients are typically not effective in alleviating cognitive impairments observed in this disorder (Featherstone et al., 2007; Meltzer and McGurk, 1999; Velligan and Miller, 1999, Floresco et al., 2005). Therefore, the establishment of predictive animal models of these cognitive impairments in schizophrenia would facilitate preclinical research designed to assess novel pharmacological treatment that may alleviate these cognitive deficits. AMPH sensitization has been shown to disrupt a number of forms of simpler forms of learning that are also impaired in schizophrenia. These behaviours include prepulse inhibition (PPI) and latent inhibition (LI). Schizophrenic patients show reduced PPI compared to control subjects (Featherstone et al., 2007) and show disrupted LI in some cases (Baruch et al., 1988),  3  but not others (Swerdlow et al., 1996). However, there exist some controversies as to whether AMPH sensitization in animals disrupts PPI: larger dosing regimens (1-10 mg/kg) produced decreases in PPI (Peleg-Raibstein et al., 2006a), whereas lower doses (1-5 mg/kg) tended to result in no change in PPI (Murphy et al., 2001; Russig et al., 2003, 2005). Similarly, LI was disrupted in rats following acute injections of AMPH (Feldon and Weiner, 1992, Warburton et al., 1994) and AMPH sensitization has been shown to disrupt LI in tasks using conditioned taste avoidance (Murphy et al., 2001) and conditioned taste aversion learning (Tenn et al., 2005b). Yet, LI was not disrupted when the AMPH injection regimen was altered (i.e. higher dose of AMPH) (Peleg-Raibstein et al., 2006b). Thus, it appears that disruptions in PPI and LI by repeated AMPH exposure depended on the injection regimen, indicating that varying injection procedures could account for the differential outcomes (i.e. intermittent versus continuous injections and dose used). More recent studies have begun to examine the cognitive deficits associated with schizophrenia and stimulant abuse, which include deficits in reasoning and problem solving and working memory (Weinberger and Gallhofer, 1997; Ornstein et al., 2000; Featherstone et al., 2007; Ersche et al., 2006; Gonzalez et al., 2007). Behavioural flexibility is one cognitive domain that has been shown to be impaired in these disorders. Laboratory tests such as the Wisconsin Card Sort Task (WCST) and the intra-/extra-dimensional set shifting task (IDS/EDS) have been used to assess frontally-mediated executive functioning in people with schizophrenia (Chudasama and Robbins, 2006). Schizophrenic patients display perseverative deficits on the WCST, which is indicative of impairments in cognitive flexibility, mediated by the frontal lobes (Weinberger et al, 1988; Stratta et al., 1997; Laws, 1999). Another type of test used to assess cognitive set shifting is the CANTAB attentional set shifting task (ID/ED) (Chudasama and  4  Robbins, 2006; Ornstein et al., 2000). The extra-dimensional shift stage (EDS) of the task is equivalent to a category shift in the WCST where participants have to learn that the previously irrelevant dimension (e.g.; lines) is now relevant and the previously relevant dimension (e.g.; shapes) is now irrelevant to correctly complete the task (Ornstein et al., 2000). Similar to schizophrenics, chronic AMPH abusers are impaired on the EDS task (Ornstein et al., 2000). However, in another study by the same group, Ersche et al. (2006) assessed set shifting ability in chronic AMPH abusers using a modified three-dimensional-ID/ED task, which involves stimuli that comprise three different dimensions (number, shape and colour), making it a more difficult task. In contrast to the previous study, chronic AMPH abusers were not impaired on the 3DIDED task, however they did make numerically more errors on the EDS task compared to controls (Ersche et al., 2006). Similarly, stimulant abusers were not impaired on the WCST (Grant et al., 2000). These studies demonstrate that schizophrenics consistently show impairments in set shifting, whereas stimulant abusers display less consistent impairments on these tasks. Another executive function that has been reported to be impaired in both schizophrenics and stimulant abusers is working memory. Patients with schizophrenia have been shown repeatedly to be impaired on a variety of different forms of working memory tasks (Castner et al., 2004; Weinberger and Gallhofer, 1997). Recent-onset schizophrenic patients are impaired on a verbal n-back test, where they have significantly longer reaction times on the test compared to healthy controls (Daban et al., 2005). In keeping with these findings, PET analysis revealed elevated D1 receptor binding potential in the dorsolateral PFC of schizophrenics which was shown to be a predictor of poor performance on the n-back task (Abi-Dargham et al., 2002). Working memory function in stimulant abusers has not been investigated as thoroughly as it has  5  been in schizophrenic patients. A recent study reported that abstinent methamphetamine abusers display deficits in working memory on a delayed non-match to sample task (Gonzalez et al., 2007). As well, abstinent methamphetamine abusers showed modest deficits in short-term memory and these deficits were associated with decreases in striatal DA transporter binding potential (McCann et al., 2008), suggesting that psychostimulant abuse can have long-lasting effects on memory function that persist after abstinence of drug use. The above mentioned findings suggest that stimulant abusers sometimes display impairments in set shifting and working memory and there are inconsistent results across studies. However the underlying causes for these deficits are not fully understood. Specifically, it is difficult to ascertain whether the impairments in executive function are due to exacerbations of pre-existing deficits that contribute to addiction, or due to the neural alterations caused by repeated drug taking. There is evidence that pre-existing deficits may contribute to drug-taking behaviour (Ersche et al., 2006). For example, adolescent groups that display neurobehavioral disinhibition (indexed by indicators of poor executive cognitive functioning, emotional regulation and behaviour control) were more likely to develop substance abuse disorder as an adult (Tarter et al., 2003). As well, weaknesses in executive functions such as response inhibition, response speed and symbol-digit modalities were found to contribute to the risk of developing alcoholism (Nigg et al., 2004). As it is not clear whether executive functioning deficits occur as a result of drug-taking or whether pre-existing impairments in cognition lead to drug-taking behaviour, animal models may serve as a useful tool in elucidating the underlying neuroadaptations associated with drug abuse. As noted previously, AMPH sensitization appears to disrupt some aspects of DA transmission in the PFC, which may be an underlying cause of the cognitive deficits observed in  6  schizophrenia and stimulant abusers. The dopaminergic basis of set shifting and working memory has been studied extensively in animals. For example, studies using a cross-maze procedure, requiring animals to shift between visual-cue or a response discrimination strategy have been used to study the role of PFC DA in mediating cognitive flexibility in rats. Blockade of either D1 or D2 receptors impairs set shifting from a response to a visual-cue discrimination strategy and vice versa, causing a selective increase in perseverative errors (Ragozzino, 2002; Floresco et al., 2006b). Furthermore, catecholamine (DA and noradrenaline) depletions within the frontal cortex of marmosets disrupt the ability to acquire an attentional set, whereas DA depletion in the caudate nucleus does not impair set shifting (Crofts et al., 2001). Additional correlational evidence that PFC DA plays a critical role in behavioural flexibility comes from microdialysis studies, where the magnitude of increase of PFC DA is related to the rapidity with which rats shift between discrimination rules (i.e. larger increases in DA are associated with faster shifting to the second discrimination rule) (Stefani and Moghaddam, 2006). Other studies have used a rodent version of the IDS/EDS task, where animals have to solve a series of discriminations based on dimensions of the stimuli (either texture or odour). During this task, rats initially discriminate between bowls based on the relevant dimension (odour, texture or the medium that fills the bowl) and continue to perform a series of discriminations including the EDS where rats have to discriminate bowls based on a new stimulus dimension in order to receive a food reward (Birrel and Brown, 2000). Birrel and Brown (2000) observed that lesions of the medial PFC disrupt performance on the EDS set shift, whereas lesions of the orbital PFC disrupt performance during the reversal discrimination (a switch within the same stimuli dimension) (McAlonan and Brown, 2003). Similar deficits in set shifting have been shown in monkeys with damage to the PFC (Dias et al., 1996; Dias et al.,  7  1997). Fletcher et al. (2005) used an AMPH sensitization regimen in rats to disrupt PFC DA and found that rats were impaired on both the set shift and reversal discriminations, indicating alterations in the medial and orbital PFC, respectively. Impairments in set shifting were alleviated by infusion of a D1 agonist into the medial PFC, which may have compensated for the DA hypofunction and restored performance on the EDS task (Fletcher et al., 2005). Based on these studies, it can be hypothesized that AMPH sensitization disrupts DA signalling and alters DA levels in the PFC. These alterations in DA could lead to impairments in set shifting and reversal learning, which are dependent on intact DA levels in the medial and orbital PFC, respectively. DA D1 receptor activity is a crucial factor mediating working memory processes (Seamans et al., 1998). D1 antagonism in the prelimbic region of the PFC leads to impairments on the delayed spatial win-shift (SWSh) task, a task used to assess working memory, specifically executive control of memory to guide action in rodents (Seamans et al., 1998). As well, an optimal level of DA D1 receptors mediates working memory performance, such that blockade or over-stimulation of D1 receptors can impair performance on delayed response tasks (Floresco and Magyar, 2006; Floresco and Phillips, 2001). Similarly, DA receptor antagonists produce impairments on delayed response tasks in monkeys (Sawaguchi and Goldman-Rakic, 1991), whereas systemic administration of low doses of DA receptor agonists enhance performance on working memory tasks (Arnsten et al., 1994; Cai and Arnsten, 1997). In light of neurophysiological data indicating that repeated AMPH treatments may disrupt PFC DA function (including D1 receptors), it is possible that these treatments may lead to impairments in working memory. However, studies of repeated AMPH treatment in rats have failed to show impairments in working memory during a delayed-alternation task and during a delayed non-  8  match to position task (Stefani and Moghaddam, 2002; Featherstone et al., 2008). As well, rats undergoing chronic testing on a delayed-alternation T-maze task in the presence of AMPH showed tolerance to working memory impairments, resulting in similar performance to control rats (Shoblock et al., 2003). It is important to note, however, that working memory assessed using delayed alternation procedures using a T-maze task is not disrupted by infusions of a D1 antagonist into the PFC (Romanides et al., 1999). This suggests that this task may not be appropriate to investigate D1-mediated working memory processes, which may explain why repeated amphetamine treatments did not produce impairments on the task. In contrast, chronic methamphetamine treated rats were impaired on the delayed SWSh task, suggesting that chronic methamphetamine treatment induces impairments in working memory (Nagai et al., 2007). However, the task they used was slightly different from other procedures in that they tested all rats on the SWSh task 1, 4, 7 and 14 days after the last METH treatment, but separate groups of rats were tested at one of three delay time point (5, 30, 60 minutes) after the training phase. As well, they relate their findings to disrupted kinase activity in the hippocampus that may be contributing to methamphetamine-induced impairments in working memory (Nagai et al., 2007). The effects of chronic AMPH on working memory are less consistent, which may be attributable to the types of tasks used to assess working memory in rodents, and need further analysis. The purpose of the present study is to assess the effects of repeated AMPH on set shifting and working memory performance on maze-based tasks. Although past research has looked at the effects of chronic AMPH on set shifting (Fletcher et al., 2005), the current study will use a cross-maze to assess cognitive flexibility, permitting analysis of specific types of problems in set shifting and reversal learning. The way in which we score errors we can determine whether rats are having trouble learning/maintaining a new rule or if they cannot stop using the old rule,  9  indicating possible impairments in the medial and orbital PFC. As well, since AMPH sensitization may disrupt PFC DA function, it is possible that this manipulation may disrupt working memory processes. We will also analyze performance on the maze pre- and postAMPH treatments in order to access whether pre-existing deficits may be related to working memory performance. In order to control for possible differences in rat strain, we tested both Long Evans (LE) and Sprague Dawley (SD) rats. It was hypothesized that a sensitizing regimen of AMPH may disrupt PFC DA function which would lead to cognitive deficits in tasks that require optimal levels of DA in the PFC, including set shifting and working memory. In the first experiment, we used a sensitizing regimen of AMPH (1mg/kg increasing dose to 5mg/kg) in rats and tested them on the cross-maze to assess set shifting. In the second experiment, a separate group of well-trained rats were subjected to an identical sensitizing regimen and tested on the delayed SWSh task, which assesses working memory processes that are dependent on PFC D1 receptor activity.  10  2. MATERIALS AND METHODS 2.1. Experiment 1: Set shifting on the cross-maze 2.1.1. Subjects Male Long Evans (LE) (n= 16) and male Sprague Dawley (SD) (n= 15) rats (Charles River Laboratories, Montreal, Canada) weighing 250-300 grams at the beginning of the experiment were used. We used both strains of rats to develop a comprehensive model of the effects of AMPH sensitization on executive functions. Previous studies have used SD rats for sensitization paradigms (Gruen et al., 1999), and others have studied the effects of repeated AMPH on cerebral neurophysiology, including AMPA receptors (Lu and Wolf, 1999) in this strain of rat. In contrast, LE rats are usually studied in relation to behavioural outcomes (Ragozzino et al., 1999; Floresco et al., 2006). Rats were individually housed in plastic cages in a temperature controlled room (20 oC) on a 12-h light-dark cycle. All rats were restricted to 85% of their free-feeding weight, with water available ad libitum. 2.1.2. Apparatus A four-arm cross-maze was used, 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 table legs attached to the ends of each arm elevated the maze 70 cm above the floor. Removable pieces of white opaque plastic (20 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 x 3.4 meters. 2.1.3. Maze habituation procedure Prior to maze habituation, rats were given 10-20 of the reward pellets (Bioserv, Frenchtown, NJ) they would receive in the maze. On day one of habituation, rats were placed in  11  the centre of the cross-maze, which had each arm baited with 5 reward pellets: 2 in each well and 3 down the length of the arm. A rat was placed in the maze to explore and consume the food pellets for 15 minutes. If the rat consumed all 20 of the food pellets prior to 15 minutes, the rat was removed from the maze and placed in a holding cage then the maze was re-baited with 20 additional pellets before placing the rat back into the centre of the maze. Day two of habituation was the same as day 1, except that 3 pellets were placed in each arm: 2 in each well and 1 on each arm. Subsequent days of maze habituation were the same as day one and two, but only 4 pellets were placed on the maze: 1 in each well. During habituation, a black and white striped laminated piece of poster board (9 x 20 x 0.3 cm) was used as the visual cue and was placed in a random arm and rotated during rebaitings of the maze. Rats were placed at the start of one arm, allowed to move down the arm and consume the food pellet, then were immediately picked up and put at the start of another baited arm. This procedure continued daily until rats consumed all pellets four or more times within 15 minutes. It took rats approximately 5 days to reach habituation criterion. On the final day of habituation, once rats achieved criterion, the turn bias for each rat was determined. A white opaque Plexiglas insert was placed at the entrance of one of the arms, forming a ‘T’ configuration and the laminated visual cue was placed in one of the choice arms. Rats were released from the stem arm and turned either left or right to obtain the food reward. Once the rat chose an arm and consumed the food pellet, it was picked up and released again form the stem arm until it consumed the food pellet from the remaining arm. Once the rat consumed food pellets from each arm, it was returned to the holding cage and the Plexiglas insert and visual cue were moved to different arms of the maze, then a new trial began. Thus, in order to complete a turn bias trial, rats had to consume food pellets from both of the choice arms. The  12  turn that a rat made first during the initial choice of a trial was recorded and counted as its turn bias and the turn bias (either left or right) that occurred 4 out of the 7 trials was considered the turn bias for that rat. 2.1.4. Amphetamine sensitization Two days after habituation, rats were counter-balanced based on their locomotion data before being assigned to either the AMPH or saline group and they received an intraperitonial (i.p.) injection of either D-amphetamine (Sigma-Aldrich) or saline (0.9 % sodium chloride) every second day. The AMPH dose increased from 1-5 mg/kg increasing the dose by 1 mg every 3rd injections. This sensitization regimen has previously been shown to produce a sensitized locomotor response to a challenge dose of AMPH (Fletcher et al., 2005). Five minutes after each injection, rats were placed in operant chambers that recorded their locomotion data for 30 minutes then returned to their home cages. The operant chambers (30.5 cm x 24 cm x 21 cm; Med-Associates, St. Albans, VT, USA) were enclosed in sound-attenuating boxes. Boxes were equipped with a fan to provide ventilation and to mask extraneous noise. Each chamber was illuminated by a single 100- mA house light located in the top-center of the wall. Four infrared photobeams were mounted on the sides of each chamber 3 cm above the grid floor, and another photobeam was located in the food receptacle. Locomotor activity was measured as the number of photobeam breaks that occurred during a session. Past research using these chambers indicates that with this positioning of the photobeams, beam breaks are not a reliable index of sniffing or other stereotypies that may be induced by stimulant drugs, but do reliably reflect ambulatory locomotion through the chambers. All experimental data were recorded by an IBM personal computer connected to the chambers via an interface.  13  2.1.5. Set shifting procedure Two weeks after the last injection, rats were trained on the cross maze to complete three tasks: visual cue discrimination, strategy set shift to a response discrimination, and reversal of the response discrimination (see Figure 1).  Visual cue discrimination  Set shift to a response discrimination  Response reversal discrimination  Figure 1 Example of the attentional set shifting task used in Experiment 1: The arrows in the maze represent the correct navigation pattern to receive food reinforcement. During the visual cue discrimination (1st panel), the rat is required to always enter the arm with the visual cue, which could either be in the left or right arm. During the set shift, the rat has to always turn left to obtain a food reward, regardless of the visual cue placement (2nd panel). Thus, the rat must shift from the old strategy and attend to the previously irrelevant response strategy in order to obtain reinforcement. During the reversal, the rat has to always turn right in order to obtain a food reward (3rd panel). Thus, the rat must reverse their previous response strategy in order to obtain reinforcement. 2.1.6. Visual cue discrimination Rats were trained to go down an arm where the visual cue was placed in order to obtain a sugar pellet reward. The visual cue was placed randomly in the left or right arms with equal frequency. Over the course of training, animals were released from one of three arms (excluding the north arm) in order to prevent learning of strategies based on spatial cues. During all training days, the maze was placed in one of four spatial orientations randomly across animals, in order that the west, south and east (W, S, E) arms were varied with respect to the visuo-spatial cues in  14  the room. The location of the visual cue was pseudo randomly varied in the left and right arms such that it occurred in each arm with equal frequency for every consecutive set of 12 trials. A trial began when a rat was released from a stem arm (W, S or E) and made a choice on the maze: either go down the arm with the visual cue to obtain the food reward (correct choice) or down the opposite arm where there was no food reward (incorrect choice). Between each trial, rats were removed from the maze and put in their holding cages while the maze was re-baited and the visual cue was moved to a different arm, based on the preset sequence described above. 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, however rats were only tested to a maximum of 40 trials per day (if a rat was approaching the 40 trial mark but made consecutive correct choices it was given subsequent trials beyond 40 until it made an error). Once the rat reached criterion, it was given a probe trial where it was released from the north arm of the maze. During the probe trial, the rat had to correctly go down the arm with the visual cue in order to obtain the food reward. If the rat made an incorrect choice, visual cue training was continued until the rat made five consecutive correct choices then another probe trial was administered. Once the rat correctly completed the probe trial, the visual response training was complete and the rat was returned to its holding cage until it began the next maze task the following day. The following measures were taken for each rat and used for data analysis on all three discriminations: (1) acquisition criteria, defined as the number of trials required to make 10 correct choices in row (2) trials to criterion, defined as the total number of test trials completed before a correct choice on the probe trial was made (10 correct choices in a row plus the number of test trials to complete the probe) and (3) probe trials, defined as the total  15  number of probe trials an animal required to get one correct. The time it took to complete training was also recorded. 2.1.7. Strategy set shift to a response discrimination During the second maze task, rats were required to learn a new rule in order to obtain a food reward: a rat had to always turn in the opposite direction of its turn bias (either left or right), regardless of where the visual cue was placed, to obtain the reward. 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. The order of the start location for each trial as well as the position of the visual cue was determined pseudo randomly and taken from a preset sequence that was identical for each animal. The training procedure and measures needed to reach criterion were similar to those described in the visual cue discrimination procedure. However, the probe trial differed in that the visual cue was placed in the opposite arm from where the rat needed to turn in order to obtain a food pellet (i.e. if the rat needed to always turn left to obtain a food pellet, the visual cue would be placed in the right arm). Once a rat chose the correct arm during the probe trial, it was finished the set shift to response discrimination and returned to the holding cage. Again, similar to the cue discrimination, 40 trials per day were administered until a rat reached criterion and successfully completed the probe trial. 2.1.8. Reversal of the response discrimination The day after completing the set shift to response discrimination, rats were trained on the reversal response discrimination. For this task, rats had to turn in the opposite direction from what they had previously learned in the set shift discrimination to obtain a food reward (i.e. if the rat learned to turn left to obtain a reward during the set shift then during the reversal the rat had to turn right in order to obtain a reward). Again, for every trial, the visual cue was placed in one  16  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. The order of the start location for each trial as well as the position of the visual cue was determined pseudo randomly and taken from a preset sequence that was identical for each animal. As well, the training procedure and measures needed to reach criterion were similar to those described in the visual cue discrimination procedure. The same guidelines were used to reach criterion and complete the probe trial as described in the set shift response section. Errors committed during the three maze tasks were divided into three categories: perseverative, regressive and never-reinforced. Perseverative errors occurred when rats were unable to shift away from the previously learned strategy, for example rats continued to turn in the direction of the visual cue (old rule) when they were supposed to turn left based on the response discrimination (new rule) and ignore the cue. Six of every 12 consecutive trials required the rat to respond using the new rule (i.e. turn left or right, ignoring the visual cue). Perseverative errors were scored when a rat entered the incorrect arm on three or more trials per block of four trials. Perseverative errors ceased when a rat made less than three perseverative errors in a block of four trials, indicating that the rat was choosing an alternative strategy at least half of the time. Regressive and never-reinforced errors occurred when the rat was not able to maintain the new strategy, once perseverative errors had ceased. Never-reinforced errors occurred when the rat entered an arm that was not reinforced during either the visual cue discrimination or the response discrimination. Regressive and never-reinforced errors are used as an index of the animals’ ability to maintain and acquire a new strategy, respectively.  17  2.2. Experiment 2: Working memory on the radial arm maze 2.2.1. Subjects Male Long Evans (LE) (n= 16) and male Sprague Dawley (SD) (n= 14) rats (Charles River Laboratories, Montreal, Canada) weighing 250-300 grams at the beginning of the experiment were used. Rats were individually housed in plastic cages in a temperature controlled room (20oC) on a 12-h light-dark cycle. All rats were restricted to 85% of their freefeeding weight, with water available ad libitum. 2.2.2. Apparatus An eight-arm radial maze was used for all working memory experiments. The maze had an octagonal centre platform 40 cm in diameter connected to eight equally spaced arms, each measuring 50 x 9 cm wide, with a cylindrical food cup at the end. Removable pieces of white opaque plastic (9 x 13 cm) were used to block the arms of the maze. The maze was elevated 40 cm from the floor and was surrounded by numerous extra maze cues (e.g. posters, cupboards, the door, and the experimenter), in a room which was illuminated with overhead fluorescent lights. 2.2.3. Delayed spatial win-shift (SWSh) task, initial training Rats were initially habituated to the radial arm maze for two days. Following habituation, training trials were given once daily and consisted of a training phase and testing phase, separated by a delay. Before the training phase began, four arms on the maze were randomly blocked, according to a list of randomly-generated four arm sequences. Food pellets (Bioserv, Frenchtown, NJ) were placed in the food cups of the four remaining, open arms. During the training phase, rats had 5 minutes to retrieve the food pellets from the four open arms of the maze. When the rats completed the training phase they were returned to their home cage for a 5 minute delay period. Following the delay, rats were put back on the maze for the test  18  phase. During the test phase, all of the arms were open, but only the previously blocked arms contained food pellets (see Figure 2). Once again, rats had a maximum of 5 minutes to retrieve the pellets from the four food cups.  DELAY 30 min Blocked arm Baited arm Training Phase  Test Phase  Figure 2 Diagram of the delayed spatial win-shift (SWSh) eight-arm radial-maze task. The delayed SWSh task consists of a training and a test phase. During the training phase, 4 of the 8 arms on the maze are randomly blocked and the 4 remaining open arms are baited. After the rat has retrieved the 4 food pellets from the open arms, it is removed from the maze for a delay (5 or 30 minutes). Following the delay, the rat is placed back in the maze for the test phase. The rat must remember which arms were previously blocked and enter those arms in order to retrieve the food reward.  The initial delay between the training and test phase was 5 minutes. An error was scored when rats went down an un-baited arm. After rats achieved criterion on the maze, in which rats consumed all four food pellets, while making less than or equal to one error for two consecutive days, the delay time was increased to 30 minutes. Rats were then trained until they achieved criterion performance for the 30 minute delay (same procedure as used for 5 minute delay). After a rat made criterion, it was given an injection of either saline or AMPH the next day. Assignment to a treatment group was counterbalanced so that number of days to criterion in both groups were numerically equal (each treatment group had an equal number of ‘fast’ and ‘slow’ learners). Individual rats took between 8-28 days to reach criterion. The latencies to reach the food cup of the first arm visited and to complete the phase were also recorded. Once rats 19  reached criterion performance on the 30 minute delay, they received repeated injections (i.p.) of either saline or AMPH in a manner identical that described in Experiment 1. 2.2.4. Amphetamine sensitization After rats achieved criterion on the training task, they were assigned to receive an injection (i.p.) of either D-amphetamine (Sigma-Aldrich) or saline (0.9 % sodium chloride) every second day. The AMPH and saline groups were counter-balanced to ensure equal numbers of rats that were ‘fast’ versus ‘slow’ learners on the maze (i.e. ‘fast’ learners achieved criterion on the maze in fewer days than the ‘slow’ learners) and similar pre-injection locomotion counts before injections commenced. The AMPH dose increased from 1-5 mg/kg at a rate of 1mg/kg every 3rd injections. Five minutes after each injection, rats were placed in operant chambers that recorded their locomotion data for 30 minutes then were returned to their home cages. 2.2.5. Retraining Two weeks after the last injection, rats were tested on the radial arm maze task using the same procedure as during the training task with a 30 minute delay. All rats were tested for a minimum of 12 days. If a rat took less than 12 days to re-attain criterion performance of making one or no errors for two consecutive days, testing continued until the rat had reached the 12 day mark. Conversely, if a rat did not attain criterion performance within the 12 days of training, it continued to receive training sessions until it did re-achieve criterion performance. The following measures were taken for each rat and used for data analysis: (1) days to re-achieve criterion performance, defined as consumption of all four food pellets, while making less than or equal to one error for two days of testing in a row (2) number of errors, defined as number of total errors committed over the testing days.  20  2.3. Challenge dose of amphetamine For both experiments 1 and 2, after the completion of behavioural testing, AMPH sensitized and saline treated rats were tested for sensitization of locomotor activity to a challenge dose of AMPH approximately 2-4 weeks after the final injection of the sensitization regimen. Rats were injected (i.p.) with 1 mg/kg of AMPH, followed by a 5 minute delay then were placed in the operant chambers. Rats’ locomotor activity was recorded for 30 minutes (measured as photobeam breaks). 2.4. Data analysis A 2 x 3 mixed between/within-subjects ANOVA was conducted to evaluate whether a sensitizing AMPH regimen had an effect on visual cue discrimination, set shifting and reversal learning in LE (n= 16) and SD (n= 15) rats (measured as number of trials to reach criterion). Also, the number and types of errors committed during the set shifting task were analyzed using a 2 x 3 repeated measures analysis of variance, with treatment as the between subjects factor and the three types of errors (perseverative, regressive and never-reinforced) as the within-subjects factors. The number of probe trials needed to complete the discriminations was also analyzed using a 2 x 2 repeated measures analysis of variance. For the working memory task, a 2 x 2 mixed between/within-subjects ANOVA was conducted to evaluate whether repeated AMPH treatment would affect days to criterion during re-testing after injections (measured as number of days to reach criterion) and number of total errors during re-testing in LE and SD rats. As well, a Pearson’s r correlation was calculated to assess the relationship between number of days to reach criterion pre-injections and number of errors committed post-injections.  21  3. RESULTS 3.1. Experiment 1: Set shifting on the cross-maze A 2 x 3 mixed between/within-subjects ANOVA was conducted to evaluate whether a sensitizing AMPH regimen had an effect on visual cue discrimination, set shifting and reversal learning in LE (n= 16) and SD (n= 15) rats (measured as number of trials to reach criterion). There was a significant main effect of treatment (F1, 27 = 4.50, P < 0.05). There was no main effect of strain, nor was there a significant strain x treatment interaction (all F < 0.13, not significant [NS]). Most importantly, there was a significant discrimination x treatment interaction (F2, 54 = 5.02, P < 0.01), indicating that AMPH and saline treated rats were different in the number of trials it took to learn some discriminations and not others. There was not a significant discrimination x strain interaction (F2, 54 = 1.49, NS), however the discrimination x strain x treatment interaction approached significance (F2, 54 = 2.95, P = 0.06) and will be discussed in the following paragraphs. 3.1.1. Visual cue discrimination There were no significant differences in number of trials to reach criterion between AMPH and saline treated rats, across both LE and SD rat strains, (F1, 81 = 0.40, NS) (Figure 3a). Additionally, there was no significant difference in the number of probe trials needed to complete the discrimination between AMPH and saline treated rats, across both strains (all F < 1, NS). Therefore, repeated AMPH treatment did not significantly disrupt acquisition of a visual cue discrimination. 3.1.2. Strategy set shift to a response discrimination Simple main effect analysis revealed a significant effect of treatment for the set shift to the response discrimination (F1, 81 = 12.24, P < 0.005), where AMPH treated rats achieved  22  criterion on the set shift (10 correct choices in a row plus the probe trial) in fewer trials than saline rats (Figure 3b). Further inspection of Figure 3b shows that AMPH sensitization improved learning of the set shift response in both strains of rats, although the improvement was more prominent in SD rats. Subsequent analysis of data obtained from the set shift revealed that there was a significant difference between AMPH and saline treated rats for total number of errors (treatment: F1, 27 = 6.32, P < 0.05), where AMPH treated rats made fewer errors overall compared to saline treated rats (Figure 4). There was no effect of strain nor was there a significant strain x treatment interaction for total number of errors committed (all F < 1.86, NS). Since there were no significant interactions, we did not analyze specific error types. These findings show that AMPH treatment is not selective for one error type. Additionally, there was not a significant difference in number of probe trials needed to reach criterion for either treatment group, across both strains during the set shifting task (all F < 1.34, NS). 3.1.3. Reversal of the response discrimination A one-way ANOVA showed no significant difference between AMPH and saline treated rats for trials to criterion on the response reversal, across both strains of rats (F1, 81 = 1.60, NS). When the data from each strain group was analyzed separately, there were no significant differences on the response reversal for SD rats (F1, 81 = 0.02, NS). However, as mentioned previously the discrimination x strain x treatment interaction approached significance (F2, 54 = 2.95, P = 0.06). Further exploration into this effect revealed that AMPH treated LE rats were faster to reach acquisition criterion (criterion of 10 correct consecutive choices, before the probe trial) during the response reversal (F1, 14 = 6.82, P < 0.05) (Figure 3c). However, analysis of the number of probe trials needed to reach criterion for the response reversal task revealed a  23  significant strain x treatment interaction (F1, 27 = 5.74, P < 0.05). This was due to the fact that LE rats that were subjected to AMPH sensitization required significantly more probe trials to complete the reversal task (Figure 5). Thus, even though sensitized LE rats were able to learn the reversal rule more quickly, they had difficulty in applying the rule to a new start location. In contrast, AMPH sensitization did not increase the number of probe trials required to complete the reversal task for SD rats (F1, 27 = 5.74, NS). Error analysis revealed no significant difference between treatment groups for perseverative and regressive errors, across both strains of rats (F1, 14  = 0.53, NS). Neither was there a significant difference of treatment, across both strains of rats  for errors committed towards or away from the cue (F1, 27 = 0.51, NS). Thus, AMPH treatment improved learning acquisition of a response reversal discrimination in LE, but not SD rats. There was a significant effect of strain for latency to complete all three discriminations (F1, 27 = 5.81, P < 0.05), in that LE rats completed more trials per minute during the discriminations than SD rats (LE completed 2.04 +/- 0.1 trials per minute whereas SD completed 1.55 +/- 0.1 trials per minute). Thus, LE rats displayed faster response latencies relative to SD rats, independent of drug treatment. LE  SD  a 150 150  100 50  Trials to Criterion  120  0  Amph sensitized  Saline  90  60  30  0  Amph Sensitized  Saline  24  b  150 100 50 0  Amph sensitized  Saline  150  Trials to Criterion  120  90  60  30  0  Amph Sensitized  c  Saline  150  100  50  0  AMPH sensitized  Saline  Figure 3 Experiment 1: The effects of AMPH sensitization on a visual cue discrimination, set shift to a response discrimination and response reversal discrimination. The data are expressed as means +/- SEM . (a) Trials to criterion (10 correct choices in a row plus the number of test trials to complete the probe) for the visual cue discrimination. There was no significant difference between AMPH and saline treated rats and there was no significant difference between LE (hatched bar) and SD rats (filled bar) rats (inset graph). (b) Trials to criterion for the set shift from a visual cue to a response. AMPH treated rats (black bar) took significantly less trials to reach criterion compared to saline treated rats (white bar). ** P < 0.005. AMPH sensitization improved learning of the set shift response in both strains of rats, most prominently in SD rats (inset graph). (c) Trials to reach acquisition criteria for the response reversal discrimination. There was no significant difference between AMPH and saline treated rats for trials to criterion on the response reversal. However, AMPH treated LE rats took less trials to reach acquisition criterion compared to saline rats. ** P < 0.05  25  Number of Total Errors  60 50 40 30 20 10 0  Amph Sensitized  Saline  Figure 4 Total number of errors between AMPH and saline treated rats on the set shift discrimination. AMPH treated rats made significantly less errors overall compared to saline treated rats. ** P < 0.05.  Number of Probe Trials  3  2  1  0  Amph Sensitized  Saline  Figure 5 Number of probe trials needed to complete the response reversal discrimination. AMPH rats took significantly more probe trials to complete the response reversal discrimination compared to saline treated rats. ** P < 0.05. 26  3.2. Experiment 2: Working memory on the radial arm maze: A 2 x 2 mixed between/within-subjects ANOVA was conducted to evaluate whether a chronic AMPH regimen affected working memory performance in LE and SD rats on the SWSh task. The dependent variables were days to criterion during re-testing and number of total errors committed over 12 days of re-testing. Analysis of the days to re-achieve criterion data revealed a significant main effect of treatment (F1, 15 = 5.15, P < 0.05). This was due to the fact that AMPH treated rats were faster to re-attain criterion than saline treated rats (Figure 6). As well, there was a significant main effect of strain (F1, 15 = 4.65, P < 0.05), suggesting that LE rats were faster to re-attain criterion (mean: 6.8 days +/- 1.03) on the SWSh task than SD rats (mean: 9.8 +/- 1.10). There was not a significant strain x treatment interaction (F1, 15 = 0.56, NS). Thus, following a 7 week period of no training, AMPH treated rats were faster to re-learn the task relative to saline treated rats.  Repeated Saline  Repeated AMPH  20  Number of Days  16  12  8  4  0  Pre-Treatment  Post-Treatment  Figure 6 The effects of repeated AMPH or saline treatment on number of days to reach criterion on the SWSh task. There was no significant difference between AMPH (black bar) and saline (white bar) treated rats in number of days to reach criterion pre-treatment (left bars). In contrast, AMPH treated rats took significantly fewer days to reach criterion compared to saline treated rats after treatment (right bars). ** P < 0.05. 27  Errors were collapsed over 2 day blocks. We analyzed the errors committed at baseline, before injections commenced, and errors post-injections for a total of 12 testing days. There was no significant difference in number of errors during re-testing between AMPH and saline treated rats (F1, 26 = 0.81, NS) (Figure 7), nor was there a significant difference in number of errors between LE and SD rats (F1, 26 = 0.23, NS). There was a significant difference between blocks of days (F6, 156 = 10.31, P < 0.001), indicating that performance improved on the task when rats had greater exposure to the maze with subsequent days of training. Overall, AMPH treatment did not affect the number of errors committed on the working memory task during re-testing, compared to saline treated rats.  Repeated AMPH  Repeated Saline 4  2  7 week break  Errors  3  1  0  Pre- 1 treatment  2  3  4  5  6  Post-treatment Blocks of 2 days Figure 7 Analysis of the number of errors during re-testing on the SWSh task post-injections. AMPH and saline treated rats did not significantly differ in the number of errors committed during re-testing.  28  As rats in each group were allowed to learn the task at their own rate, we were able to conduct a correlational analysis on how the rate of learning prior to treatment was related to performance after treatment. Specifically, we assessed the relationship between number of days to reach criterion during the initial learning phase, before injections, and number of errors committed during re-testing after injections. Only errors committed during the last 6 days were analyzed, when performance of all the rats was stable on the maze. Prior to the 6 day mark, some rats did not move consistently on the maze. For saline treated rats, there was no significant correlation between days to reach criterion pre-treatment and number of errors post-treatment (r = - 0.12, NS). In contrast, there was a strong positive correlation (r = 0.69) for AMPH treated rats, that was significantly different from zero, (P < 0.02; Figure 8), indicating that AMPH treated rats that were slow to learn the task to begin with (i.e.; more days to reach criterion) made more errors during re-testing following repeated AMPH treatment. Furthermore, the correlations between saline and AMPH treated rats were also significantly different (P < 0.05). In general, saline treated rats showed no strong correlation between how they performed before or after treatment. In contrast, however, AMPH rats that were slower to learn the task initially displayed poorer performance on the task following repeated AMPH treatments, relative to those rats that acquired the task in fewer training trials.  29  LE  Errors after Treatment  Repeated Saline  SD  Repeated AMPH  20  20  r= -0.12 15  15  10  10  5  5 10  15  20  25  30  Days to Criterion Slow Fast learner learner  r= 0.69 10  15  20  25  30  Days to Criterion Slow Fast learner learner  Figure 8 Analysis of the relationship between days to reach criterion pre-injections and errors committed post-injections during re-testing on the SWSh task. For saline-treated rats, there was no significant correlation between days to reach criterion pre-treatment and number of errors post-treatment (left graph). In contrast, for AMPH treated rats there was a strong positive correlation that was significantly different from zero (P < 0.02) (right graph). AMPH rats that took more days to reach criterion pre-injections tended to make more errors post-injections.  3.3. Locomotor activity To assess whether AMPH treatment had a sensitizing affect on locomotor activity, rats were tested for locomotor response to a challenge dose of 1 mg/kg AMPH following approximately 2-4 weeks after behavioural testing. A 2 x 2 x 2 analysis of variance revealed a significant main effect of treatment (F1, 53 = 4.70, P < 0.05), indicating that AMPH treated rats showed an increase in locomotor activity when treated with a challenge dose of AMPH compared to saline rats (Figure 9). As well, there was a significant main effect of strain (F1, 53 = 36.19, P < 0.001), suggesting that LE rats had a greater increase in locomotor activity (mean: 1827.91 beam breaks +/- 103.01) compared to SD rats (mean: 1115.93 +/- 64.78) when treated with a challenge dose of AMPH. Similarly, there was a significant main effect of task (F1, 53 = 6.37, P < 0.05), where the set shift rats showed a greater increase in locomotor activity (mean:  30  1630.58 +/- 108.14) compared to the working memory rats (mean: 1343.57 +/- 104.57), possibly due to the fact that the working memory rats received the challenge dose approximately 5 weeks later in the experiment than set shifting rats, thus they were older and possibly less active. Additionally, there was no significant interaction between any of the between subjects factors (treatment, strain, test) (all F < 1, NS). These data confirm that repeated exposure to AMPH did yield a sensitized locomotor response induced by an acute injection of AMPH.  1800 1620 1440  Beam Breaks  1260 1080 900 720 540 360 180 0  Amph Sensitized  Saline  Figure 9 The effects of an AMPH challenge dose on locomotor activity in rats sensitized to AMPH. Locomotor activity was assessed 2-4 weeks after behavioural testing. Beam breaks were measured for 30 minutes after an acute challenge dose of AMPH (1 mg/kg). AMPH treated rats had significantly increased locomotor activity (measured as beam breaks) compared to saline treated rats. ** P < 0.05  31  4. DISCUSSION The present study was designed to assess how repeated AMPH exposure affects two executive functions that are dependent on intact DA transmission in the PFC. We observed that AMPH sensitized rats required fewer trials to learn the set shift response compared to saline treated rats. Furthermore, LE AMPH rats also required fewer trials to reach acquisition criterion during reversal learning. However, these rats required more probe trials to reach criterion on the response reversal, compared to saline rats, suggesting that AMPH treated rats could learn the new rule but had difficulty applying the rule in a new start arm. Similarly, AMPH treated rats took fewer days to re-attain criterion on the working memory task compared to controls, but there were no differences between groups in the total number of errors made over 12 days of retraining. Correlational analyses further revealed that AMPH sensitized rats that required more days to reach criterion before AMPH treatment (i.e. slow learners) tended to make more errors during re-acquisition of the memory task. This suggests that these slower learning rats may have been more vulnerable to the deleterious effects that repeated AMPH exposure may exert on working memory functions. Overall, AMPH treated rats learned the set shift and reversal tasks more quickly and were faster to reach criterion on the working memory task. However, there was an important strain difference in reversal learning, in that LE rats acquired the task in fewer trials than SD rats, but these same rats took more probe trials to successfully complete the task. In contrast, AMPH sensitization did not affect reversal learning acquisition nor the number of probe trials required to complete the task for SD rats. 4.1. Behavioural Flexibility The improvements on the set shift for the AMPH rats is surprising in light of the fact that previous studies have mainly found impairments in set shifting in rats that are sensitized to  32  AMPH (Fletcher et al., 2005; Featherstone et al., 2008). However, these studies used a rodent version of the IDS/EDS task, which may account for the different outcomes between their studies and our findings. For this task, rats discriminate between odour and texture and undergo multiple discriminations: simple discriminations, intradimensional shift, extradimensional shift and 3 separate reversals. Rats that were subjected to an AMPH sensitization regimen identical to the one in the present study were impaired on the reversals and the EDS (set shift) component of the task. In the present experiment, we in fact observed the opposite effect: AMPH rats were improved on both the set shift and the reversal. One key difference between the IDS/EDS task and the cross maze strategy shifting task used in the present study is that in the former, rats are required to discriminate the same type of stimulus dimension (e.g. odour) for the first five discriminations (i.e. exemplars associated with odour are always correct for the simple discriminations, reversal 1 and 2 and the IDS). During the EDS stage, the relevant stimulus dimension changes (i.e. if odour was the correct dimension previously, now texture is the correct dimension). This procedure, where the initial dimension is used for the first 5 discriminations, would be expected to cause rats to form a strong association with the initial discrimination as the correct dimension to follow in order to complete the task successfully. In contrast, during the cross-maze task, the relevant dimension (i.e.; visual cue) is only correct during one discrimination. Under these conditions, it would be expected that rats would form a relatively weaker association between the relevant stimulus and the reward, which may facilitate shifting to the next discrimination. From these data, it is apparent that chronic AMPH does not cause a general impairment in behavioural flexibility. Instead, the effects of repeated AMPH treatment on this form of executive functioning appears to depend on the type of task used to assess set shifting.  33  AMPH sensitization did not disrupt learning of a reversal discrimination compared to saline rats and led to improvements in acquisition of the reversal response for LE rats. When the orbital PFC is inactivated, rats are impaired in reversal learning on the ID/ED task adapted for rats (McAlonan and Brown, 2003) and on maze based set shifting tasks (Ghods-Sharifi et al., 2008; Kim and Ragozzino, 2005). Furthermore, rats treated with chronic AMPH are impaired in reversal learning on the IDS/EDS task compared to saline rats (Fletcher et al., 2005; Featherstone et al., 2008). In the present experiment, however, AMPH treated rats were not impaired in reversal learning, so AMPH sensitization does not consistently impair OFC-mediated reversal learning. The differences between the present findings and Fletcher et al.’s (2005) study could be due to the difference in age of the rats between the two studies or differences in task difficulty. However, previous studies have found improvements in reversal learning in rats repeatedly treated with AMPH during training. In experiments conducted by Weiner et al. (1986 a, b, c), repeated AMPH treatment improved performance on a two-choice simultaneous brightness discrimination reversal task. In all three experiments, AMPH treatments were given during the initial discrimination and continuously throughout the sessions, including the reversal. Rats were trained to correctly respond to the light stimulus in order to receive reinforcement and once they reached criterion they were trained on the reversal, where the dark side became the reinforcing stimulus. AMPH treatments facilitated reversal learning in all of the experiments (Weiner et al., 1986 a, b, c). These results are similar to the present findings in that AMPH treated rats were not impaired on the reversal discrimination and LE AMPH rats reached acquisition criterion on the reversal task in fewer trials than saline treated rats. The similar results may be due to the fact that AMPH treated rats do not form a stable association between the original reinforcing stimulus and the reward, which facilitates a rapid switch in response to  34  the newly reinforced stimulus (Weiner et al., 1986 c). Although LE AMPH treated rats in the present study were able to reach acquisition criterion more quickly than saline rats, they required more probe trials to complete the reversal discrimination. This suggests that AMPH treated rats had difficulty applying the new rule in a new start arm. The findings in the present study are in direct contrast to Fletcher et al.’s (2005) findings for both the set shift and reversal learning discriminations. Fletcher et al. (2005) only used SD rats, but in the present study we used both LE and SD rats, which could account for the different outcomes. During the set shifting task, we found that AMPH treated rats completed the set shift in less trials than saline rats, regardless of strain. Although there was not a significant effect of strain, further inspection of Figure 3b shows that AMPH treatments improved learning on the set shift more prominently in SD rats compared to LE rats. The differences in performance between the SD rats in Fletcher et al.’s study and the SD rats in the present study could be due to the age of the rats when they first were exposed to AMPH. Rats in the present study weighed between 250-300 grams at the start of the experiment, whereas rats in Fletcher et al.’s study weighed between 175-200 grams at the beginning of the experiment. This would mean that the latter rats were adolescents when they were receiving AMPH treatments and these AMPH treatments were affecting brain development during a formative period. Performance on a set shifting task can be affected by age which has been shown in a study where aged LE rats (27-28 months old) were impaired on a set shift task compared to young rats (4-5 months old) (Barense et al., 2002). This provides an example of age as a contributing factor to impairments in set shifting. Perhaps adolescent rats were more sensitive to the deleterious effects of AMPH than adult rats, resulting in impaired performance on the set shift compared to the rats in the present study.  35  The present results also differ from Featherstone et al.’s (2008) EDS/IDS set shift findings, although results are similar to Featherstone et al.’s strategy-shifting results. For the strategy-shifting experiment, rats initially learned to locate a food reward based on either a place or response strategy in a plus maze and once they made criterion, they had to learn to shift to the alternate strategy (either place or response). AMPH treated rats were not impaired on the set shift for this task. However, AMPH sensitized rats were impaired on the EDS component and third reversal of the set shifting task (Featherstone et al., 2008). Contrasting results between the present experiment and Featherstone et al.’s results could again be due to procedural differences. We habituated animals first then gave them injections of either AMPH or saline, followed by training on the maze, whereas Featherstone et al. (2008) sensitized rats then habituated them to the maze (strategy-shift) or trained them to dig in bowls for food (attentional set-shifting task). Perhaps habituating the rats to the maze before they received injections helped to familiarize the rats to the new environment and reduced any anxiety-related confounding factors during testing. Furthermore, it is notable that the same group of researchers (Featherstone et al., 2008 and Fletcher et al., 2005) did not obtain consistent results across studies. Although they used identical dosing regimens, they found contrasting results in terms of set shifting impairments on the strategy-shift and attentional set-shifting tasks. As well, in the Fletcher et al. study AMPH treated rats were impaired on all three reversals and on the EDS, whereas in the Featherstone et al. study AMPH treated rats were only impaired on the EDS and the last reversal, and rats in both studies underwent identical AMPH dosing regimens. Thus, AMPH sensitization does not consistently impair set shifting across multiple types of tasks. During the set shifting task, LE and SD AMPH rats did not significantly differ in number of trials to reach criterion. Strain does not seem to be a contributing factor to differences in set  36  shifting in the present experiment, as neither the strain x treatment nor the strain x discrimination interactions were significant. Previous studies assessing the effects of AMPH sensitization on set shifting have not compared strains within the same study and have only used SD rats (Featherstone et al., 2008; Fletcher et al., 2005). Although in the present study SD rats were generally slower to complete the trials during the set shift compared to LE rats, this did not affect overall performance in terms of trials to reach criterion on the set shift. The effects of AMPH sensitization on set shifting seem to be consistent, regardless of rat strain. In terms of reversal learning, AMPH sensitized LE rats made acquisition criterion in fewer trials than saline treated rats, but had difficulty applying the rule to a new start arm, whereas AMPH treated SD rats did not differ in number of trials to reach criterion compared to saline rats. Few studies have looked at differences in reversal learning ability between different strains of rats, however in one study SD rats took longer to acquire a lever-pressing response in an autoshaping task compared to LE rats and this strain of rat performed better on a two-object discrimination task compared to SD rats. In contrast SD rats were more accurate in choosing a platform on a swim maze task than LE rats (Andrews et al., 1995). The authors of the study propose that differences in performance on the tasks between the strains of rats could be due to variations in baseline activity levels, anxiety levels, and in baseline attentional levels (Andrews et al., 1995). Perhaps LE rats in the present study were able to initially learn the reversal rule more quickly due to increased attention and less anxiety on the maze compared to SD rats. However, SD rats were well-handled prior to any testing so anxiety levels between the two strains of rats should not have significantly differed. Other studies have assessed the effects of DA agonists on PPI in SD and LE rats (Swerdlow et al., 2005; Swerdlow et al., 2007) and found that SD rats were more sensitive than LE rats to the PPI-disruptive effects of both the direct D1/D2 agonist apomorphine and AMPH  37  (Swerdlow et al., 2005). As well, PPI was disrupted in SD rats that were injected subcutaneously or intra-cerebrally in the nucleus accumbens core with AMPH, but not in LE rats (Swerdlow et al., 2007). From these studies it appears that SD rats are more sensitive to the deleterious effects of AMPH on PPI. In the present study, we do not know the mechanism underlying differences between rat strains in set shifting and reversal learning. However, based on the current results LE rats are sensitive to the beneficial effects of AMPH on reversal learning compared to SD rats. It was surprising that AMPH sensitized rats were not impaired on the set shift and reversal discriminations since these tasks are dependent on intact PFC DA functioning. AMPH sensitization reduces PFC DA activity (Castner et al., 2005; Hedou et al., 2001), and there is evidence that D1 receptor function is reduced (Peterson et al., 2006). As well, chronic cocaine administration results in a decrease in DA D2 mRNA in the medial and orbital PFC and a decrease in D2 receptor protein in the medial PFC (Briand et al., 2008), further supporting the notion that repeated psychostimulants disrupt PFC DA signalling. With respect to set shifting, blockade of either D1 or D2 receptors in the PFC prior to the set shift impairs performance (Ragozzino, 2002; Floresco et al., 2006b), suggesting that activity at both of these receptors is crucial for mediating shifts between different strategies. However, there are instances where disruptions in PFC DA transmission has been reported to improve set shifting (Roberts et al., 1994). Monkeys with 6-hydroxydopamine (6-OHDA) lesions induced prior to any behavioural training were tested on an EDS/IDS attentional set shifting paradigm. Under these conditions, lesions of DA terminals in the PFC results in improved performance on the EDS (Roberts et al., 1994). In the same study, the authors also observed that depletion of DA in the PFC resulted in increased extracellular DA levels in the striatum, suggesting that improvements in set shifting could be mediated by depressed PFC DA function and elevated striatal DA function (Roberts et  38  al., 1994). Since we observed similar behavioural outcomes using AMPH sensitization, it is possible that repeated AMPH may have decreased PFC DA activity, while simultaneously increasing DA levels in the striatum, leading to the enhanced performance observed on the set shift seen in the present experiment. Another important point to consider is whether DA manipulations are administered relative to initial learning of a discrimination and the set shift component of the task. In the Floresco et al. (2006b) study, rats were trained on the initial discrimination in a drug-free state then were infused with either DA agonists or antagonists before the set shifting discrimination. In contrast, Roberts et al. (1994) lesioned monkeys two weeks before testing on the IDS/EDS task commenced, so DA levels were disrupted during all phases of the IDS/EDS task. A similar phenomenon to that found in the Roberts et al. (1994) study may underlie the improvements observed here, in that perturbations in DA signalling induced by AMPH treatments prior to any discrimination learning may have impaired the ability for these rats to form a stable attentional set, allowing them to switch more quickly to the next discrimination. In addition to its effect on the PFC, AMPH sensitization affects other DA terminal regions such as nucleus accumbens. The effects of AMPH on the nucleus accumbens has been well documented (Di Chiara et al., 2004; Vezina, 2004; Young et al., 2005) and these studies suggest an association between drug taking and increases in DA in the nucleus accumbens (Di Chiara et al., 2004; Vezina, 2004), and potential disruptive effects of AMPH on LI as a function of DA release in the nucleus accumbens (Young et al., 2005). In this regard, the nucleus accumbens core has also been shown to be critically important for set shifting functions, as inactivations of this nucleus results in impairments in cognitive flexibility, mainly due to disruptions in the acquisition and maintenance of a new strategy (Floresco et al., 2006a). In  39  contrast, inactivation of the nucleus accumbens shell before initial discrimination learning leads to improved performance on the set shift. In that study, the authors argued that disruptions to the nucleus accumbens shell may have impaired latent inhibition of learning, where rats were unable to learn to ignore irrelevant stimuli, which in turn would facilitate the ability to attend to those stimuli during the shift (Floresco et al., 2006a). There is a vast literature demonstrating that disruption of the nucleus accumbens shell leads to impairments in latent inhibition (Weiner, 2003; Weiner et al., 1996; Gal et al., 2005). Latent inhibition is a phenomenon that results from pre-exposure to a non-reinforced stimulus (i.e. a tone) that affects conditioning performance on a subsequent learning task that involves the same stimulus (again, the tone). Animals that are exposed to a non-reinforcing stimulus during pre-exposure perform poorly during a conditioning stage where the stimulus is now paired with a reward because the animals learned to ignore the irrelevant stimuli during the initial pre-exposure. In contrast, animals with impaired latent inhibition will perform better during the conditioning stage because they did not ignore the irrelevant stimuli during pre-exposure (Weiner, 2003). Studies show that AMPH sensitization disrupts latent inhibition (Tenn et al,. 2005; Weiner, 2003; Weiner et al., 1996), most likely as a result of enhanced DA activity. It is thought that repeated AMPH treatment disrupts the nucleus accumbens by altering the activity of the mesocorticolimbic DA systems (Tenn et al., 2005), whereby a challenge dose of AMPH increases shell DA levels (Cadoni and Di Chiara, 2007). This increase in DA exerts a tonic inhibitory effect on shell neurons, resulting in impaired latent inhibition (Weiner, 2003). With respect to the present study, this disruption in latent inhibition could facilitate shifting between strategies using the cross-maze task. It is possible that improvements in set shifting in the present study may be attributable in part to a failure to ignore stimuli that do not reliably predict reinforcement (e.g.; turn direction), which would enhance  40  learning about the relevance of these previously irrelevant stimuli during the set shift. Therefore, another explanation for the observed improvements in the AMPH treated rats in the present experiment may be due to a disruption in latent inhibition, allowing these rats to shift their attention to the previously irrelevant stimuli more quickly. 4.2. Working Memory The finding that AMPH-treated rats were not impaired on the working memory task and even showed improvements is consistent with previous studies that have also assessed AMPH sensitization and working memory. Stefani and Moghaddam (2002) found that AMPH sensitized rats were not impaired on a delayed alternation task and Shoblock et al. (2003) also found that chronically treated AMPH rats displayed tolerance to the working memory impairments exhibited during initial testing. It is important to consider that these studies both used T-maze based delayed alternation tasks, which do not appear to require D1 receptor activity in the PFC, unlike the SWSh task (Floresco and Magyar, 2006; Romanides et al., 1999). Yet, in the present study AMPH sensitization once again did not produce impairments on the working memory task, suggesting that these effects are consistent, regardless of the type of task used during testing. Similar to our results, Featherstone et al. (2008) found that AMPH sensitization did not impair performance on a working memory task. In contrast, AMPH sensitization in monkeys produces impairments on a working memory task (Castner et al., 2005), providing evidence that the effects of AMPH sensitization on working memory performance is not consistent across species and is task-dependent. Previous studies have shown that D1 antagonism in the PFC leads to impairments on working memory tasks (Seamans et al., 1998; Floresco and Magyar, 2006). However, our results demonstrate that chronic AMPH treatment does not impair working memory performance on the  41  SWSh task. Therefore, it seems that chronically administered AMPH may not disrupt D1 receptor function to the same extent that a D1 antagonist would, as evidenced by the lack of impairments on the working memory task. After repeated AMPH treatment, there is a reduction in voltage-sensitive sodium current density in medial PFC neurons, so D1 receptor function is reduced (Peterson et al., 2006). Since it has been shown that an optimal level of D1 receptor function/activation in the PFC is needed for intact working memory performance (Peterson et al., 2006; Seamans et al., 1998; Floresco and Magyar, 2006), perhaps AMPH sensitization did not alter the optimal D1 range enough to cause impairments in working memory. As well, its been shown that experimenter-administered cocaine has no effect on D1 receptor sensitivity in the PFC (Nogueira et al., 2006), so perhaps the experimenter-administered AMPH in this study did not affect D1 receptors in the PFC and this may have contributed to the lack of impairments in working memory. Despite the lack of impairment in working memory induced by repeated AMPH, there was a significant correlation between original rates of learning before drug treatments and number of errors post-treatment. It is tempting to speculate that this effect may be related to basal levels of PFC DA activity. Perhaps rats that were ‘slow learners’ prior to treatment had lower basal levels of D1 receptor activity. In this instance, chronic AMPH would be expected to exert a more disruptive effect on the D1 receptors, further reducing signalling, which could lead to impaired performance in working memory post-treatment. In contrast, rats that were ‘fast learner’ rats may have had basal D1 signaling that was at a normal or even slightly higher level, so that any disruptions in D1 receptor activity induced by chronic AMPH may not have been sufficient to disrupt performance on the SWSh task during re-testing. Nevertheless, the present data, in addition to previous findings indicate that, this domain of cognition appears to be robust  42  against the deleterious effects of AMPH sensitization. Yet, it seems that rats that are naturally slow learners to begin with may be more susceptible to disruptions in working memory following repeated AMPH, possibly due to an exacerbation of pre-existing deficits in cognition 4.3. AMPH sensitization and executive function: relation to schizophrenics Impairments in set shifting tasks assessed with either the WCST or the IDS/EDS task, has shown to be one of the most reliable impairments in executive functioning observed in schizophrenia (Weinberger et al, 1988; Stratta et al., 1997; Laws, 1999; Pantelis et al., 2004 and 1999). However, in the present study, AMPH treated rats were not impaired on the set shift or reversal learning, and actually learned the set shift discrimination more quickly than control rats. This would suggest that perhaps AMPH sensitization does not represent a valid model of the set shifting deficits seen in schizophrenic patients. As well, impairments in set shifting are consistently found in schizophrenic patients, regardless of the type of task used to assess cognitive flexibility, whereas animal tests of set shifting are not as reliable and produce contrasting results. The studies conducted by Fletcher et al. (2005) and Featherstone et al. (2008) produced differing results (impairments on the EDS set shift, but no impairments on the strategy set shift), even though rats went through identical dosing regimens and the same group of researchers conducted the experiments. Therefore, AMPH sensitization in rats does not induce a reliable deficit in set shifting. Patients with schizophrenia exhibit impairments in working memory (Castner et al., 2004; Park et al., 1999; Goldman-Rakic, 1994; Weinberger and Gallhofer, 1997) and working memory impairment is the most consistently observed type of cognitive deficit in schizophrenics (Castner et al., 2004). AMPH treated rats in our study were not impaired on the working memory task and their performance on the task improved compared to control rats, in that they  43  took fewer days to reach criterion during re-testing on the SWSh task. It is hypothesized that there is a decrease in D1 signaling in the dorsolateral PFC in patients with schizophrenia and this may be correlated with impaired cognitive function (Castner et al., 2004). Although AMPH sensitization may also decrease the action of PFC D1 receptors (Peterson et al., 2006), the effects of reduced DA on working memory in humans and rats may not be directly comparable. Since schizophrenic patients are consistently found to be impaired in working memory tasks, yet studies of AMPH sensitized rats do not lead to impairments in working memory, it seems clear that this is may not be the most appropriate approach to model schizophrenia-like impairments in this domain of executive functioning. 4.4. AMPH sensitization and executive function: relation to stimulant abusers Impairments in set shifting have not been observed as consistently for stimulant abusers as with schizophrenics. Chronic AMPH abusers have been found to be impaired on the EDS task (Ornstein et al., 2000), whereas other studies have found no impairments on an EDS task (Ersche et al., 2006) or on the WCST in polysubstance abusers (including stimulants) (Grant et al., 2000). The cross maze task is a rodent version of the WCST, where rats have to shift attentional set from visual cue to response, similar to the WCST where participants have to shift between different sorting rules (cards are sorted based on colour, type of figure or number of figures). An important similarity between the two tasks is that the relevant stimulus dimension is only correct for one particular discrimination, so when shifting to a subsequent rule, a new stimulus dimension is associated with reward. The present results are consistent with Ersche et al. (2006) and Grant et al.’s (2000) findings as the AMPH-treated rats were not impaired on any of the discriminations of the behavioural flexibility task. This suggests that there may be other factors besides exposure to drugs contributing to the deficits in set shifting.  44  There have been a few reports that chronic stimulant abusers display impairments in working memory. Methamphetamine abusers were impaired on the delayed non-match to sample working memory task and made significantly more errors compared to controls and even other types of drug abusers, including alcoholics (Gonzalez et al., 2007; Bechara and Martin, 2004). In the present study, we did not observed any impairments in working memory following repeated AMPH treatment, although we did observe a positive correlation between the initial rates of learning prior to drug treatment and the number of errors made post treatment. A possible explanation for the discrepancy in results between AMPH sensitized rats and chronic stimulant abusers may be that substance abusers use multiple types of drugs. Although they report methamphetamine as their drug of choice, most likely they use other types of drugs as well, so it is difficult to isolate the specific effects of methamphetamine on working memory (Gonzalez et al., 2007). Although the present finding suggest that AMPH sensitized rats show improvements on a working memory task, it is important to look at the correlational findings more closely. The ‘fast learner’ rats made fewer errors post-treatment, whereas the ‘slow learner’ rats made more errors post-treatment. This suggests that these slower rats may have been more susceptible to the deleterious effects that repeated AMPH exposure may exert on working memory functions. The chronic stimulant abusers in the studies by Gonzalez et al. (2007) and Bechara and Martin (2004) may also have pre-existing cognitive deficits which may contribute to their drug-taking behaviour and subsequent working memory impairments. Based on the findings that AMPH treated rats that were slow learners before treatments made more errors during re-testing, it could be hypothesized that cognitive deficits observed in stimulant abusers may be due to an exacerbation of pre-existing problems. There are correlations between poor executive cognitive functioning in adolescence and later development of substance abuse  45  disorder (Tarter et al., 2003; Nigg et al., 2004), which suggests a possible influence of preexisting deficits in cognition leading to later impairments associated with chronic drug use. Chronic AMPH treatment may only adversely affect working memory performance in rats with pre-existing conditions that contributed to slower learning during initial training on the task. This would explain why ‘slow learners’ made more errors after AMPH treatment. Evidence for pre-existing deficits related to later stimulant abuse is sparse, so more research needs to be done in order to assess cognitive function pre- and post- drug treatment in animals. This could provide further insight into the role pre-existing deficits play in developing cognitive deficits associated with drug addiction.  46  5. CONCLUSIONS The current study assessed behavioural flexibility and working memory in AMPH sensitized rats. AMPH treated rats were not impaired in either the set shift task or in the working memory task and actually displayed improved performance in both tasks compared to control animals. These results suggest that AMPH sensitization may not replicate impairments in behavioural flexibility and working memory observed in schizophrenic patients, and thus may not be a valid model of the cognitive deficits associated with this disorder. Interestingly, rats that were slower to learn the working memory task before treatment tended to make more errors after AMPH treatment during re-testing, suggesting that AMPH sensitization may be exacerbating preexisting deficits in working memory. Other domains of cognition, such as attention and decision making, may be better suited to model the cognitive impairments associated with schizophrenia and drug addiction and lead to more comprehensive models of the disorders.  47  REFERENCES Abi-Dargham A (2003) Probing cortical dopamine function in schizophrenia: What can D1 receptors tell us? World Psychiatry 2:166-171. Abi-Dargham A, Mawlawi O, Lombardo I, Gil R, Martinez D, Huang Y, Hwang DR, Keilp J, Kochan L, Van Heertum R, Gorman JM, Laruelle M (2002) Prefrontal dopamine D1 receptors and working memory in schizophrenia. J Neurosci 22:3708-3719. 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