Approach-Avoidance Conflict for Sucrose and Footshock Pairing in Cocaine-Sensitized Rats

Transcription

1 Approach-Avoidance Conflict for Sucrose and Footshock Pairing in Cocaine-Sensitized Rats by David Nguyen A thesis submitted in conformity with the requirements for the degree of Master of Arts Department of Psychology University of Toronto Copyright by David Nguyen 2013

2 Approach-Avoidance Conflict for Sucrose and Footshock Pairing in Cocaine-Sensitized Rats Abstract David Nguyen Master of Arts Department of Psychology University of Toronto 2013 Repeated administration of psychostimulant drugs induces a long-term state of sensitization in the mesolimbic dopamine system. This hyperdopaminergic state is associated with enhanced reward-seeking behaviors. Such aberration of incentive motivational processing is suggested to facilitate the initiation and maintenance of compulsive drug-taking behaviors. A defining characteristic of addiction is the persistence to pursue drug reinforcement despite negative consequences associated with administration. Thus, it is likely that addicts frequently experience states of motivational conflict to both seek and avoid the drug. The present study investigated the effects of repeated cocaine exposure on goal-seeking behaviors in rats, utilizing conflict paradigms wherein positive and negative incentive motivations were simultaneously evoked. Here it was shown that cocaine-experienced rats displayed both enhanced approach and avoidance behaviors, depending upon the conditions put forth in each paradigm. The results contribute to elucidating the consequences of drug administration upon basic motivational processes that may influence compulsive drug-taking behaviors. ii

3 Table of Contents 1. Introduction Neurochemical and Behavioral Consequences of Drug Exposure Cross-Sensitization for Natural Reinforcers Drug-Induced Alterations upon Motivation to Avoid Aversive Stimuli The Present Study Methods Cocaine Drug Administrations Elevated Plus Maze Stimuli of Motivational Value Experiment Experiment 2a Experiment 2b Results Experiment Experiment 2a Experiment 2b Discussion Repeated-Cocaine Exposure Attenuates Learning for Aversive Associations Repeated Cocaine-Exposure Facilitates Reward Seeking Cocaine-Experienced Rats Displayed Enhanced Avoidance in the Presence of Clearly Defined Boundaries Conclusion and Future Directions 47 iii

4 List of Figures Figure 1 56 Figure 2 57 Figure 3 58 Figure 4 59 Figure 5 60 Figure 6 61 Figure 7 62 Figure 8 63 Figure 9 64 Figure Figure Figure Figure Figure Figure Figure Figure Figure iv

5 List of Diagrams Diagram 1 74 Diagram 2 74 Diagram 3 75 Diagram 4 75 v

6 1 1 Introduction The rewarding and motivational properties of drugs of abuse have long been postulated to share the same neural substrates utilized by natural reinforcers such as food, water, and sex (Di Chiara, 1995; Koob, 2009; Wise, 1989). Early electrical brain stimulation studies have revealed that stimulation of certain brain regions elicited reinforcing effects in the stimulated animal (Olds & Milner, 1954). Specifically, animals would self-stimulate at a higher rate per minute in areas of the medial forebrain bundle connecting the ventral tegmental area (VTA) to the basal forebrain, indicating low reward thresholds at these sites (Koob; 2009; Olds & Olds, 1965; Wise, 1989). It was later revealed that noradrenergic and dopaminergic receptor antagonists were effective at blocking the elevated rate of medial forebrain bundle self-stimulation, indicating a key role for monoaminergic activity in the reinforcing effects of the electrical stimulation. (Esposito, Faulkner, & Kornestky, 1979; Fouriezos & Wise, 1975; Rolls, Kelly, & Shaw, 1974). Further, drugs of abuse such as cocaine have been shown to lower the reward threshold in the medial forebrain bundle, suggesting that the reinforcing properties of drugs of abuse are elicited at least in part by monoaminergic activity within this site (Esposito, Motola, & Kornetsky, 1978; Kornetsky & Esposito, 1979). Nearly all addicting drugs function by increasing extracellular dopamine (DA) concentrations in the nucleus accumbens (NAc), suggesting a common mechanism by which these drugs exert their reinforcing effects (Di Chiara & Imperato, 1988). Lesions of the NAc attenuate the rewarding effects of intravenous amphetamine, while destruction of DA terminals in the NAc attenuates intravenous cocaine self-administration in rats (Pettit, Ettenberg, Bloom, & Koob, 1984; Taylor & Robbins, 1986). Further, rats effectively learn to lever-press for microinjections

7 2 of amphetamine into the NAc, and develop a conditioned place preference (CPP) for locations in which they received microinjections of either amphetamine or DA itself (Carr & White, 1983; Chevrette Stellar, Hesse, & Markou, 2002; Hoebel, Monaco, Hernandez, & Aulisi, 1983). Expanding upon these classic findings, a growing body of research has since shown that druginduced activation of dopaminergic reward circuits leads to a cascade of neuroadaptations, eventually giving rise to aberrant forms of learning that contribute to the development and sustenance of pathological drug-taking and reward-seeking behaviors (Koob & Volkow, 2010; Wheeler & Carelli, 2009). Repeated exposure to psychostimulants, such as cocaine and amphetamine, is particularly effective at inducing a state of sensitization characterized by enhanced excitation of VTA DA neurons in response to both the drug and stimuli predictive of drug availability (Bradberry, 2007; Kalivas & Stewart, 1991; Luscher & Malenka, 2011; Mameli & Luscher, 2011; Smith, Lobo, Spencer, & Kalivas, 2013). The consequential increase of NAc extracellular DA has been postulated to augment the motivational value of the drug, and also enable drug-associated stimuli to trigger a state of intense craving and increased motivation to seek out the reinforcing effects of the drug (Everitt, Dickinson, & Robbins, 2001; Kalivas & McFarland, 2003; Robinson & Berridge, 2001). As such, drug-induced modifications in DA systems have been proposed to play a key role in maintaining motivated behaviors characteristic of addiction, in which the addict persists to compulsively seek out and administer the drug despite aversive consequences related to withdrawal or social and monetary repercussions (DSM IV, 1994; Hyman & Malenka, 2001; Ikemoto & Wise, 2004; Mameli & Luscher, 2011).

8 3 The objective of this communication is to first discuss evidence for drug-induced neuroadaptations within dopaminergic systems, and how such adaptations may contribute to addictive behaviors. This will be followed by a review of evidence for the behavioral consequences of drug-induced sensitization of DA transmission within these systems. The effects of repeated drug exposure on negative incentive motivational processes will then be discussed in relation to both neurochemical and behavioral drug-induced adaptations. This communication will then culminate with a discussion of results from an original study that was conducted to examine the behavioral effects of repeated cocaine exposure on approach and avoidance behaviors of rats tested in a conflict paradigm wherein positive and negative incentive motivations were simultaneously evoked. 1.1 Neurochemical and Behavioral Consequences of Drug Exposure DA neurons originate primarily in the VTA and substantia nigra pars compacta (SNc), and project to targeted areas of the striatum, prefrontal cortex (PFC), and amygdala (Lammel, Lim, & Malenka, 2013). DA neurons projecting from the VTA to the NAc in particular have been identified as a crucial substrate for the processing of reward-related information (Bromberg- Martin, Matsumoto, & Hikosaka, 2010; Lammel et al., 2013; Schultz, 1998). Under normal conditions, DA neurons in this pathway have been shown to fire bursts of action potentials in response to food rewards and reward-predicting stimuli (Schultz, 1998). This dopaminergic response has been posited to have evolved as a teaching signal that enables animals to learn to engage in behaviors that favor survival and reproduction, and to avoid behaviors that do not (Berridge, 2007; Cardinal, Parkinson, Hall, & Everitt, 2002; Everitt et al. 1999; Robinson and Berridge, 1993; Salamone & Correa, 2002).

9 4 Much evidence suggests that drugs of abuse elicit their reinforcing effects by inducing alterations within this dopaminergic system. Such alterations can be long-lasting and have been postulated to underlie critical components of the addiction process (Di Chiara, 1995; Di Ciano et al., 1995; Zapata, Chefer, Ator, Shippenberg, & Rocha, 2003). When administered acutely, cocaine induces a blockade of DA transporter proteins (DAT), resulting in a net increase of extracellular DA concentrations (Mameli & Luscher, 2011). Amphetamine induces a similar increase in extracellular DA through the induction of non-vesicular DA release (Seiden, Sabol, & Ricaurte, 1993). With repeated drug-exposure, the drug-induced increase of DA concentrations may trigger long-lasting activity-dependent synaptic modifications that evoke an augmentation in membrane excitability of DA neurons in the VTA (Luscher & Malenka, 2011; Mameli & Luscher, 2011). Evidently, electrophysiological recordings in brain slices have shown that repeated cocaine administrations evoke an increase in the ratio of VTA AMPA receptor (AMPAR)-mediated EPSCs to NMDA receptor (NMDAR)-mediated EPSCs (AMPAR/NMDAR ratio), which is indicative of cocaine-induced LTP in VTA DA neurons (Bellone & Luscher, 2006; Borgland, Malenka, & Bonci, 2004; Chen et al., 2008; Ungless, Whistler, Malenka, & Bonci, 2001). Consequently, it is suggested that VTA DA neurons become hyperexcitable, and therefore exhibit an augmented response to drug administrations and stimuli of motivational value. In rodents, sensitization of the brain s DA system can be verified by the observation of an augmented locomotor response to a single administration of the drug (Kalivas & Stewart, 1991). Following a regimen of repeated amphetamine treatment, a single dose of amphetamine has been

10 5 shown to induce an augmented release of extracellular DA in the NAc in parallel with enhanced locomotor activity relative to the amphetamine-induced response observed at the onset of treatment (Robinson, Jurson, Bennet, & Bentgen, 1988). This effect has also been observed after repeated cocaine administration (Pettit, Pan, Parsons, & Justice, 1990; Post & Weiss, 1988; Zapata et al., 2003). Sensitization of the dopaminergic system is also expressed behaviorally in the form of increased motivation to seek and administer drugs that induce a dopaminergic response. Cocaine and amphetamine sensitized rats have been shown to display an enhanced acquisition of CPP for locations that have been previously paired with drug availability (Lett, 1989; Seymour & Wagner, 2002). Cocaine and amphetamine sensitized rats have also been shown to display increased break-points at which they would stop responding on a lever under a progressive ratio schedule of drug self-administration, which is indicative of increased motivation to obtain drug reinforcement (Liu, Roberts, & Morgan, 2005; Mendrek, Blaha, & Phillips, 1998). Further, utilizing a runway paradigm for cocaine administration, Deroche and colleagues have shown that over successive trials, rats that have been pretreated with 29 cocaine self-administration sessions will more quickly traverse a runway towards a goal compartment for intravenous injections of a low dose of cocaine compared to rats that were pretreated with only 6 self-administration sessions (Deroche, Moal, & Piazza, 1999). In this paradigm, the time it takes to traverse the runway provides an index of the animal s motivation to seek the reward within the goal compartment, with faster run times indicating higher motivation (Deroche et al., 1999; Ettenberg, 2009).

11 6 Under conditions of sensitization, motivation to seek the drug of abuse can be further facilitated by exposure to drug-associated stimuli (Robinson & Berridge, 2001). The incentive sensitization theory posits that repeated pairings of a drug and a drug-associated stimulus can increase the incentive value of the stimulus itself. Thus, exposure to the stimulus may contribute to drugcraving and drug-seeking behaviors (Robinson & Berridge, 1993). Supporting this hypothesis, it has been observed that presentation of a tone stimulus that had been repeatedly paired with intravenous injections of amphetamine induced an efflux of DA in mesolimbic areas (Schiff, 1982). Likewise, presentation of a light stimulus that had been repeatedly paired with intravenous cocaine self-administration led to an increase in DA-related electrochemical signals in the NAc (Gratton & Wise, 1994). Further, cocaine sensitized animals exhibited elevated locomotor activity following an i.p. administration of a cocaine challenge dose in an environment previously paired with cocaine injections, when compared to sensitized animals that received the challenge dose in an environment not previously paired with cocaine administration (Stewart, de Wit, & Eikelboom, 1984). Together, these observations suggest that drug-associated stimuli and contexts may induce drug-seeking behaviors by evoking an enhanced dopaminergic response within mesolimbic areas. Exposure to drug-associated stimuli, as well as to drugassociated contexts, may also augment the effects of the drug itself in sensitized animals. 1.2 Cross-Sensitization for Natural Reinforcers Indeed, the enhanced motivated behaviors characteristic of drug-induced sensitization is not exclusive to drug-related goal-seeking. Rather, the enhanced drug-seeking behaviors discussed thus far may be attributable to the sensitization of basic motivational processes that underlie reward-seeking behaviors in general. Wyvell and Berridge have shown that amphetamine

12 7 sensitized rats display augmented cue-induced goal-seeking behavior for sucrose reinforcement (Wyvell & Berridge, 2001). In this experiment, rats were trained to lever-press for sucrose, and were separately conditioned to associate an auditory cue with the availability of sucrose reinforcement. Following an amphetamine sensitization regimen, the rats were tested for instrumental responding under conditions of extinction with intermittent presentations of the auditory conditioned stimulus. Sensitized rats displayed significantly greater instrumental responding in the presence of the conditioned stimulus compared to controls (Wyvell & Berridge, 2001). Klein and colleagues have also shown that cocaine-sensitized rats reacquire instrumental responding for sucrose reinforcement in the presence of a sucrose-predictive light stimulus more efficiently than control rats after an extended period of time following the initial conditioning phase (Klein, Gehrke, Green, Zentall, & Bardo, 2007). The ability for reward-associated stimuli to enhance goal-seeking behaviors for natural reinforcers in drug-sensitized animals may in part be attributed to enhanced dopaminergic transmission in substrates that underlie associative learning processes. For example, amphetamine-sensitized rats display elevated levels of extracellular DA in both the NAc and amygdala following an amphetamine challenge dose (Harmer, Hitchcott, Morutto, & Phillips (1997). VTA DA projections to the amygdala have been identified as a substrate important for the acquisition of stimulus-reward associations, suggesting that drug-induced sensitization of this system may facilitate associative learning processes that allow for reward-related cues to take control over motivated behaviors (Packard, Cahill, & McGaugh, 1994; Harmer et al., 1997; Hitchcott, Harmer, & Phillips, 1997). Further supporting this hypothesis, Harmer and Phillips demonstrated that amphetamine-sensitized rats displayed enhanced associative learning for a

13 8 light stimulus that was repeatedly paired with sucrose reinforcement (Harmer & Phillips, 1998). They also showed that the enhanced Pavlovian learning was associated with augmented DA efflux in the amygdala during the conditioning sessions (Harmer & Phillips, 1999). 1.3 Drug-Induced Alterations upon Motivation to Avoid Aversive Stimuli The behavioral research reviewed thus far provide evidence that repeated exposure to drugs such as amphetamine and cocaine can lead to an enhancement in goal-seeking behaviors, while also enabling reward-associated cues to gain greater control over motivational processes (Di Chiara, 1995; Harmer & Phillips, 1999; Lett, 1989; Robinson & Berridge, 2001). Although it is wellsupported that such augmented goal-seeking behaviors may be attributable to long-lasting druginduced neuroadaptations in dopaminergic systems, it is important to consider how such neuroadaptations may also influence motivation to avoid aversive stimuli (Bellone & Luscher, 2006; Borgland, et al., 2004; Chen et al., 2008; Ungless et al., 2001). This consideration is particularly important given that a major component of addiction pathology includes the persistence to pursue drug reinforcement despite the drug s association with aversive consequences (DSM IV, 1994). It is conceivable that drug-induced alterations in both appetitive and aversive motivations may function synergistically to maintain compulsive drug-seeking behaviors. Evidence supporting drug-induced alterations upon motivation to avoid aversive stimuli will now be reviewed. The opponent process theory of drug addiction posits that drugs of abuse possess both positive and aversive properties (Koob, Caine, Parsons, Markou, & Weiss, 1997). The positive properties include the rewarding effects of the drug, while the withdrawal effects are perceived as aversive.

14 9 Ettenberg and Geist have demonstrated these opposing properties of cocaine utilizing a runway paradigm, in which rats were trained to traverse an alley way towards a goal compartment for intravenous cocaine reinforcement (Ettenberg & Geist, 1991). Rats displayed shorter latencies in leaving the start compartment and initiating runway performance over subsequent trials; however, longer latencies to enter the goal compartment were also observed. Longer goal latencies were not a result of slower runway performance, but rather, it was observed that rats would repeatedly retreat from and re-approach the goal compartment before ultimately entering it. This observation led to the interpretation that the goal compartment had become associated with both appetitive and aversive qualities, and the rats were thereby faced with an approachavoidance conflict wherein they were motivated to both pursue and avoid cocaine administration (Ettenberg, 2004). Given the opponent properties of cocaine, it is suggested that impulsive drug-taking and addiction development may initiate from a process of negative reinforcement wherein the individual is repeatedly motivated to administer the drug in order to alleviate negative symptoms that result from withdrawal of the drug (Koob et al., 1997). After repeated experiences with escaping the aversive effects of withdrawal through re-administration of the drug, the user may then learn that some withdrawal symptoms can be avoided with further drug administration before the onset of the aversive effects. Drug-seeking may then become a form of active avoidance, wherein successful avoidance of some of the aversive effects associated with prolonged withdrawal can, in itself, become rewarding.

15 10 Indeed, recent research has indicated that the mesolimbic DA system is importantly implicated in avoidance behaviors (Dombrowski et al., 2013). Utilizing a microdialysis probe extending from the dorsoloateral striatum through the NAc core and shell, Dombrowski and colleagues have shown an increase of extracellular DA when rats successfully emitted a response that led to the avoidance of footshock administration in the presence of a footshock administration-predicting cue. The increase of striatal DA was evident during the early training trials and returned to baseline as training progressed (Dombrowski et al., 2013). This finding suggests that DA neurons projecting to the striatum were initially encoding a positive reward prediction error when the rats learned the avoidance response. Considering the prediction error hypothesis, this finding would imply that the outcome of their behavior was better than expected, and the avoidance response was therefore encoded as rewarding (Schultz, 1998). Evidently, when the successful avoidance of footshock became fully expected upon avoidance behavior, the increase of dopaminergic firing no longer occurred (Dombrowski et al., 2013). However, it is important to consider a caveat in that the increase in DA concentration observed in this study was not identified as specifically occurring in either the dorsal or ventral striatum. Therefore, the results should be interpreted with caution. Similar to the previously discussed study, Oleson and colleagues have published findings that implicate a role for reward prediction in active avoidance (Oleson, Gentry, Chioma, & Cheer, 2012). They demonstrated an increase of DA in the NAc when rats were presented with a cue predictive of footshock, and the increase was associated with the subsequent exhibition of a conditioned response leading to successful avoidance of footshock. There was a further increase of DA during a safety period following the avoidance response, suggesting that DA was

16 11 encoding the safety period as a reward consequential to successful avoidance. In this task, rats also had the option of performing a conditioned response to escape footshock if they initially failed to avoid it. It was found that escape behavior was associated with a parallel decrease of DA in the NAc. The researchers conducted a separate experiment in which rats were conditioned to associate a cue with impending inescapable footshock. Here, it was found that presentation of the cue induced a decrease of NAc DA, thus providing further evidence that the cue-induced increase of DA observed in the avoidance task was due to the anticipation of reward (i.e., successful avoidance) (Oleson et al., 2012). The discussed findings on active avoidance suggest that instrumental responses leading to the avoidance of aversive stimuli are learned because the response is initially encoded as a behavior that leads to a rewarding outcome. When the response is well-learned, stimuli predictive of the adverse consequence shift to being predictive of the rewarding outcome, which takes the form of successful avoidance. Given these implications, it can be hypothesized that drug-induced sensitization of the mesolimbic DA system should be associated with an augmented increase of NAc DA under the implied reward-related conditions of active avoidance. Therefore, animals exhibiting a sensitized DA response should more efficiently acquire and display avoidance behaviors in situations of impending aversive consequences, which could suggest enhanced motivation to avoid aversive stimuli. However, evidence to directly support this hypothesis is currently lacking in the literature. Taken within the framework of addictive behaviors, it is also important to consider that the first 48 hours following cocaine withdrawal is associated with elevated levels of corticotropin-

17 12 releasing factor (CRF) and increased display of behavioral anxiety (Erb, 2010). CRF neurons project to, and synapse with, neurons in the VTA (Tagliaferro & Morales, 2008). It has been shown that CRF release in the VTA induces activation of glutamate neurons in rats that have undergone repeated cocaine self-administration sessions, but not in cocaine-naïve rats. The CRFinduced glutamate release then triggers the firing of VTA-mesolimbic DA neurons (Wang et al., 2005). It is therefore conceivable that rats with a history of cocaine exposure may consequently express enhanced motivation to actively avoid further experiencing a state of heightened anxiety during the 48-hour period of presumed CRF-induced elevation of mesolimbic DA. Indeed, the avoidance response may take the form of drug-seeking behaviors. Although speculative, it is possible that once the active avoidance response (i.e., drug seeking) becomes well-learned through repeated trials of withdrawal, the elevation of CRF levels characteristic of the withdrawal period may then serve as a neurochemical cue that is predictive of successfully avoiding the persistence of the state of heightened anxiety. Although this interpretation provides a viable explanation as to why addicts may persist to express enhanced motivation to seek drugs despite aversive consequences, it is complicated by findings indicating that the first 48 hours of cocaine withdrawal are actually associated with decreased concentrations of NAc DA (Erb, 2010; Markou & Koob, 1992). Compulsive drug-seeking during periods of withdrawal may be further exacerbated by druginduced alterations within neural circuits involved in behavioral impulsivity (Dalley, Everitt, & Robbins, 2011). A recent study has shown that a regimen of either chronic cocaine systemic injections or self-administration effectively induces potentiation in presynaptic glutamatergic synapses in the PFC to NAc pathway, which has been postulated to be a key substrate underlying

18 13 impulsive behaviors (Dalley et al., 2011; Suska, Lee, Huang, Dong, & Schluter, 2013). Specifically, electrophysiological recordings of brain slices revealed an enhancement in release probability for synapses in the PFC to NAc pathway, which was evident during both short-term withdrawal (1d) and long-term withdrawal (45d) following either the systemic or selfadministration regimen. The potentiation of glutamatergic synapses in this pathway would imply a consequential enhancement of excitatory synaptic transmissions in the NAc. Taken together with the previously discussed findings, it is suggested that a repeated cocaine exposure-induced increase in impulsivity during periods of withdrawal may give rise to compulsive drug-seeking behaviors due to synergistic functioning with augmented motivation to avoid aversive physical and emotional states associated with withdrawal, as well as with enhanced incentive motivation for rewards. 1.4 The Present Study Thus far, it is well documented that repeated administration of cocaine induces synaptic alterations within cortical and striatal dopaminergic and glutamatergic circuits, which have been shown to elicit profound effects upon responding to stimuli of motivational value (Luscher & Malenka, 2011; Mameli & Luscher, 2011; Smith et al., 2013). Behaviorally, this is expressed as enhanced incentive motivation to obtain rewards, increased sensitivity to cues predictive of reward, increased impulsivity, and presumably enhanced motivation to avoid aversive states associated with withdrawal (Berridge, 2007; Dombrowski et al, 2013; Oleson et al., 2012; Suska et al., 2013). Such drug-induced augmentations are postulated to underlie compulsive drugseeking behaviors during periods of withdrawal. Given that addiction pathology is characterized as a disorder in which drug-seeking behaviors persist despite negative consequences associated

19 14 with using the drug, it is important to elucidate upon the mechanisms that contribute to both positive and negative incentive motivational processes. Although both processes have been thoroughly explored separately, studies examining behavioral and neurochemical responses under circumstances wherein both motivational processes are experienced simultaneously are currently lacking (Bromberg-Martin et al., 2010; Hikida et al., 2010; Lammel et al., 2013). Therefore, the purpose of the present study was to examine the effects of repeated cocaine exposure upon approach and avoidance behaviors, utilizing paradigms wherein the animal experiences conflicting motivational processing induced by the paired presentation of appetitive and aversive stimuli. Here, it was found that cocaine-experienced animals displayed attenuated learning for aversive associations, as well as enhanced approach and avoidance behaviors, depending upon the procedural conditions put forth in each experiment. 2 Methods The subjects were 46 male Long Evans rats (Charles River Laboratories) weighing between 323 and 598g at the time of drug treatment. Subjects were pair-housed in transparent plastic cages and maintained under a standard 12h light/dark cycle (lights on at 0700h). At the time of behavioral testing, subjects were kept on a food (laboratory chow, Purina) restricted diet to maintain 85% of baseline weight. Water was available ad libitum. All procedures were performed in accordance with the Canadian Council of Animal Care guidelines. 2.1 Cocaine Drug Administrations Habituation phase. Rats were individually placed in separate opaque plastic chambers in which locomotor activity was monitored over a 60min period by an overhead CCD camera and infrared

20 15 sensor connected to an Ethovision tracking system (Noldus Information Technology). Rats then received an intraperitoneal (i.p.) injection of saline (0.9%), and were immediately replaced into the activity chambers. Locomotor activity was monitored for an additional 60min. Pre-exposure phase. Rats were administered an i.p. injection of cocaine (n = 22) or saline (n = 24) daily over a period of seven consecutive days beginning 1d after the habituation phase. The first and last injections (15 mg/kg, i.p., in a volume of 1 mg/ml) were administered immediately following a 60min recording period in the activity chambers. Activity was then monitored for an additional 60min. Injections 2-6 were administered in a room separate to their housing and behavioral testing (30 mg/kg, i.p., in a volume of 1 mg/ml). No recordings took place on the days of injections 2-6. Following the final injection, all rats underwent a 10d washout period prior to beginning any behavioral testing. 2.2 Elevated Plus Maze At the end of the 10d washout period, all rats were tested for anxiety in an elevated plus maze (EPM) apparatus. The apparatus consisted of 4 arms arranged in the shape of a plus (43.18 cm length, cm width). Two arms enclosed by walls (24.77 cm height) emanating from the border edges of the arms. The entrances to these arms were positioned directly across from each other, and were accessible from the maze center. The other two arms were not enclosed by walls, and were also accessible from the maze center. The apparatus was elevated cm from the ground by a pedestal. Each rat was tested in a single trial, which began with the rat being placed in the center of the apparatus, which was placed in a well-lit room. The rat was allowed to explore the apparatus for 10min.

21 Stimuli of Motivational Value Sucrose Appetitive Stimulus. A solution consisting of 20% sugar and 80% distilled water was used as the appetitive stimulus. Footshock Aversive Stimulus. Scrambled footshocks were administered using a shock generator (Med Associates/ Lafayette Instrument Co.). Each administration of footshock consisted of a current between 0.25 ma and 0.3 ma lasting 0.5s. 2.4 Experiment 1 (cocaine N = 13, saline N = 13) Radial Arm Maze Apparatus Behavioral training and testing was conducted using a six-arm radial maze apparatus (Med Associates). The six arms converged at a hexagonal hub at which automated steel guillotine doors allowed access into each arm (45.7 cm length, 9 cm width, 16.5 cm height). The floor of each arm consisted of steel rods laid parallel across the width of the arm 0.5 cm apart. The steel rods were connected to a footshock generator (Med Associates). A cup receptacle for sucrose delivery was located at the end of each arm. The entire apparatus was covered with red cellophane to block out extra-maze stimuli. Med PC IV software (Med Associates) was used to control the maze apparatus. A ceiling-mounted camera positioned above the apparatus was used to monitor the test sessions (see Diagrams 1-2).

22 17 Discrete Cues During the training and test trials (described below), wooden bars (45 x 2.5 cm) covered with either duct-tape or cloth material was placed along the entire length of two maze arms. The textured bars were cues predictive of either sucrose or footshock administration during the training and testing sessions. The valence of the cues was determined for each rat following the habituation sessions (described below). An uncovered wooden bar was placed along the entire length of a third arm, signaling that the arm was neutral. All cues were attached along the bottom of the maze wall inside of their respective arms with Velcro. Procedure Habituation (day 1). Two habituation sessions were conducted on the first day of the experiment. No unconditioned stimuli (US) were administered during either habituation sessions. Only three of the six maze arms were utilized during both habituation sessions. For the first habituation session, no intra-maze cues were presented to distinguish between the arms. Individual rats began the session placed in the central hub of the radial maze with all guillotine doors closed. After a 1min period, three doors were raised allowing free exploration of three arms in addition to the central hub for a period of 5min. Upon expiration of the 5min, all three doors were lowered, and the rat was removed from the apparatus. For the second habituation session, the cues, as described above, were placed in three separate arms, and the same protocol utilized for the first habituation session was followed. The amount of time rats spent within each arm was recorded to determine the valence of each cue. The most preferred texture bar cue was paired with the aversive US during the training and testing sessions on an individual basis for

23 18 each rat. The uncovered wooden bar was always designated as the neutral cue. After completion of maze habituation, rats received a sample of sucrose solution in their home cages. Habituation (day 2). A third habituation session was conducted on day 2. Rats began the session by being placed into the central hub for a period of 1min. Upon expiration of this period, two doors were raised allowing access to the two corresponding arms. One arm contained the uncovered wooden bar, while the other contained a superposition of both duct tape and cloth textured bars. Rats were allowed to explore the two arms along with the central hub over a period of 5min. US were not administered during habituation. Upon expiration of the period, both doors were lowered, and the rat was removed from the apparatus. Training (days 2-10). Training sessions were conducted once per day over a period of 9 consecutive days, with the first session occurring immediately after the third habitation session on day 2. The appetitive, aversive, and neutral cues were placed in the appropriate arms prior to the start of each training session. A syringe for sucrose administration was connected to the cup receptacle via tubing for the arm containing the appetitive valence cue. The steel rod flooring in the arm containing the aversive valence cue was connected to the footshock generator. At the start of each session, a rat was placed in the center hub with no access to any of the arms. After a 30s period, the first door was raised. Upon entrance, the door was lowered to contain the rat in the arm for a period of 120s during which the respective US was administered. In the appetitive arm, the US was administered as an infusion of 2.0 ml of sucrose once randomly within four 15s intervals. Each 15s interval containing sucrose administration was followed by a single nonreinforced 15s interval. In the aversive arm, footshock lasting 0.5s was administered once

24 19 randomly within each 30s interval. There was no US presentation in the neutral arm. Upon expiration of the 120s period within each arm, the door was raised allowing reentry into the central hub. Upon entry of the central hub, the door was lowered, containing the rat in the central hub for 30s. The next door was then raised to allow entry into that arm. Once the rat completed the full duration of containment within each of the three arms, the rat was removed from the apparatus. For the first two training sessions, the appetitive arm was presented first, followed by the neutral arm, and then the aversive arm. For the remaining sessions, the order of entry into the arms was randomized. Valence was randomly assigned to different arms for each session to avoid conditioning to spatial orientation and unintended intra-maze cues. Testing (days 5, 8, 10). A single testing session was conducted on each of the designated days, and all testing sessions were followed by a single training session. Testing sessions followed the same protocol used for the second habituation session. Rats had free access to all three arms simultaneously with each containing one of the three cues. No US was administered. A ceiling mounted camera recorded each session for later behavioral scoring. Final testing (day 11). A single final test session was conducted on the 11 th day. The aversive cue was superimposed with the appetitive cue within one arm. Another arm contained the neutral cue. Rats were placed inside of the central hub at the start of the session with all doors closed. After a 1min period, two doors were raised, allowing access to the superimposed and neutral arms. The rats were allowed to explore both arms in addition to the central hub for a period of

25 20 5min. US was not administered. A ceiling mounted camera recorded each session for later behavioral scoring. Sucrose preference test. All rats underwent a 16h sucrose preference test once before the first habituation session and once again following the final conflict test. Each test was conducted at 18:00 to 10:00. At the start of this test, all rats were placed individually into separate cages identical to their home cages. Two identical bottles were inserted into each cage, with one containing a solution of 1% sucrose and the other containing water. The weight of the bottles were measured once before insertion at the start of the 16h period, and once again after removal at the end of the test. 2.5 Experiment 2a (cocaine N = 6, saline N = 7) Runway Apparatus Daily trials were conducted in a single enclosed runway apparatus (Lafayette Instrument Co.) divided into three compartments separated by steel guillotine doors. Identically-sized start and goal compartments (24.5 cm length, 11.4 cm width, 21.1 cm height) were located at opposite ends of an alley (98.8 cm length, 11.4 cm width, 21.1 cm height). The floor of the apparatus consisted of steel rods laid parallel across the width of the entire apparatus 1.3 cm apart. The steel rods located inside of the goal compartment were connected to a footshock generator (Lafayette Instrument Co.). The steel doors separating the three compartments were manually controlled to allow access to the alley and goal compartments. The entire apparatus was covered with red cellophane to minimize distraction by extra-maze stimuli. A ceiling-mounted infrared camera was positioned above the apparatus to recorded each trial (see Diagram 3).

26 21 Procedure Habituation (days 1-2). Single daily habituation sessions were conducted over a period of 2d. Rats began each habituation session inside of the start compartment where it remained for a period of 1min. Upon expiration of the 1min period, the steel guillotine door was raised allowing access to the alley. Rats were allowed to explore the alley along with the start compartment for a period of 3min. During habitation day 1, rats were removed from the apparatus upon cessation of the 3min period. On habituation day 2, the guillotine door blocking off the goal compartment was lifted following the 3min period. The goal compartment contained a cup filled with sucrose. Upon sampling the sucrose, rats were allowed to explore the entire apparatus for an additional 1min period before being removed. Behavioral testing phase 1 (days 3-8). Rats were trained to traverse the alley for sucrose reinforcement with 2 trials per day over a period of 6d. At the start of each trial, the guillotine door blocking the goal compartment was completely raised, and a cup containing sucrose solution was placed inside the goal compartment area. Rats were then placed in the start compartment for 1min. Upon expiration of the 1min period, the guillotine door was raised allowing access to the alley and goal compartment. Upon entering the goal compartment, the guillotine door was lowered, separating the goal compartment from the alley. Rats remained inside the goal compartment for 1min. Rats were then removed from the apparatus and placed in their home cage. The second trial commenced immediately after all rats completed the first trial.

27 22 Behavioral testing phase 2 (days 9-24). Footshock administrations were paired with sucrose inside the goal compartment for 2 trials per day over a period of 16d following phase 1 of behavioral testing. Trials followed the same protocol utilized for phase 1 of behavioral testing up until entry into the goal compartment. Upon entry into the goal compartment, the guillotine door was lowered confining the rat inside the compartment for 1min. During the 1min period, footshocks were administered once randomly within each 15s interval. Upon expiration of the 1min period, rats were removed from the apparatus and placed into their home cage. If a rat did not enter the goal compartment within 300s, the rat was removed from the apparatus and placed in to its home cage. The second trial commenced immediately after all rats completed the first trial. Devaluation trial (day 25). A single devaluation trial was conducted following the completion of behavioral testing phase 2. On this day, rats were individually placed into separate cages, where they remained for a duration of 1h without access to food or water. Following the 1h period, a bottle containing 20% sucrose solution was inserted into cage, and rats were allowed access to the solution for 1h. Immediately following the 1h period, rats were tested in the runway apparatus under the same protocol utilized for behavioral testing phase 1. Footshocks were not administered in the goal compartment, and only a single trial was conducted. Sucrose preference test. The same procedures described earlier were utilized for the sucrose preference test. However, rats in experiment 2a underwent only one 16h test session. The test occurred 1d following completion of the devaluation test.

28 Experiment 2b (cocaine N = 4, saline N = 4) Runway Apparatus Daily trials were conducted in a modified version of the runway apparatus utilized for experiment 2a. All aspects of the apparatus were exactly identical to the apparatus utilized for experiment 2a, except for the introduction of a pulley mechanism to operate the opening and closing of the guillotine door to the goal compartment. The pulley mechanism consisted of a rope suspended from a hook attached to the ceiling above the runway apparatus. One end of the rope was attached to the guillotine door of the goal compartment, while the other end was handled by the experimenter to control the movement of the guillotine door (see Diagram 4). Procedure All experimental procedures were followed exactly the way they were described for experiment 2a, with the exception of the control over the guillotine door. At the start of each trial, the guillotine door was raised only partially, exposing an opening that was just large enough for the rat to enter. Approximately half of the door remained inserted and visible to the rat throughout each trial. The door was immediately lowered upon entry of the goal compartment, confining the rat inside for a 1min period. All other procedures were conducted in accordance to the description proved for experiment 2a. Sucrose preference test. A 16h sucrose preference test was conducted following the devaluation session, in accordance with the description provided for experiment 2a.

29 24 Light-dark box. A test for anxiety utilizing a light-dark box apparatus was conducted 1d following the completion of the sucrose preference test. The apparatus consisted of two adjacent chambers divided by a wall containing a doorway (12.7 cm width, 12.7 cm height) allowing access to either chamber. Both chambers were of identical dimensions (30.48 cm length, cm width, cm height). The apparatus was raised cm from the ground by a table. One chamber was enclosed by opaque black walls, whereas the other was enclosed by transparent walls. A light bulb was inserted into the transparent chamber, and remained on throughout each test trial. Each trial was conducted in an unlit room. Each rat was tested in 1 trial, which began with the rat being place inside of the light chamber. The rat was then allowed to explore both chambers for 10min. 2.7 Locomotor Sensitization Test After completion of behavioral testing, rats remained in their home cages with ad libitum access to food and water for a period of 8-14d before undergoing a locomotor sensitization test. The sensitization test took place over a period of 2d. On the first, rats were habituated individually in opaque activity chambers for a period of 2h. Rats were then returned to their home cages. No recordings took place during this habituation session. On the second day, rats again underwent a 2h habituation session in the activity chambers, during which locomotor activity was recorded using an overhead CCD camera with infrared sensors connected to an Ethovision tracking system (Noldus Information Technology). Upon cessation of the 2h period, all rats received an i.p. injection of a low dose of cocaine (10mg/kg, i.p., in a volume of 1 mg/ml), immediately followed by an additional 2h recording period inside of the activity chambers. Rats were then returned to their home cages.

30 25 3 Results 3.1 Experiment 1 Locomotor sensitization. Cocaine-induced locomotor sensitization was determined with a comparison between the total distance travelled during the 2h recording period following administration of the cocaine challenge dose with the total distance travelled during the 2h habituation period preceding the challenge dose. This was accomplished by calculating distance travelled during the post-challenge period as a percentage of the distance travelled during the pre-challenge period, deriving a ratio representative of a cocaine-induced effect on locomotor activity. Independent-samples t-test was then conducted to compare the ratio between the two treatment conditions. This analysis revealed that rats with a history of repeated cocaine exposure exhibited a significantly greater locomotor response to the challenge dose than rats without a history of prior cocaine exposure (t(23) = 4.01, p <.001) (Figure 1). This result confirms that the repeated cocaine administration regimen was effective at inducing locomotor sensitization. Tests 1-3. For test 1, ANOVA revealed a significant main effect for valence on exploration time (F(2, 46) = 20.59, p <.001), indicating that the amount of time spent exploring each arm differed and was dependent upon the arm s emotional valence. An analysis of contrasts was conducted to compare the amount of time spent exploring the appetitive and aversive arms relative to time spent exploring the neutral arm. This analysis revealed that, overall, rats spent significantly more time exploring the appetitive arm than the neutral arm, (F(1, 23) = 26.37, p <.001, r =.73), indicating effective acquisition of cue-induced motivation to seek the sucrose reward. However, the amount of time spent in the aversive arm did not significantly differ from time spent in the

31 26 neutral arm, (F(1, 23) = 1.361, ns.). Moreover, a significant valence x treatment interaction was observed, (F(2, 46) = 3.21, p =.05). Post hoc (Bonferroni) analyses revealed that cocaine-naïve rats spent significantly more time exploring the appetitive arm compared to the neutral arm (p <.001), and significantly less time exploring the aversive arm compared to the neutral arm (p <.05). Cocaine-treated rats also spent significantly more time in the appetitive arm compared to the neutral arm (p <.01); however, the time spent exploring the aversive arm did not significantly differ from time spent in the neutral arm (Figure 2). Thus, cocaine-treated rats showed an impairment in aversive cue learning at the time of test 1, leaving appetitive cue learning intact. An analysis of the latency to enter each arm for test 1 revealed a violation of sphericity (χ 2 (2) = 17.81, p <.001). Therefore, degrees of freedom were corrected using the Greenhouse-Geisser estimate for sphericity (ε =.64). ANOVA revealed a significant main effect for valence on latency to enter the arms (F(1.29, 29.58) = 15.67, p <.001). The overall latency for rats to enter the aversive arm was significantly longer than their latency to enter the neutral arm (F(1, 23) = 15.53, p <.001, r =.63); however, the latency to enter the appetitive arm did not differ from the neutral arm. Further, an interaction effect with treatment was not found (F(1.29, 29.58) = 1.08, ns.). These results indicate that both cocaine-experienced and naïve rats were hesitant to enter the aversive arm, suggesting this behavior was guided by avoidance motivation. ANOVA for test 2 also revealed a significant main effect for valence on exploration time (F(2, 46) = 37.48, p <.001). Specifically, rats spent significantly more time exploring the appetitive arm than the neutral arm (F(1, 23) = 29.78, p <.001, r =.75), and significantly less time

32 27 exploring the aversive arm (F(1, 23) = 13.49, p <.001, r =.61) (Figure 3). No valence x treatment interaction was observed (F(2, 46) =.29, ns.). A separate ANOVA was then conducted on latency for entering each arm for test 2. Mauchly s test again indicated a violation of sphericity (χ 2 (2) = 24.34, p <.001), and the Greenhouse-Geisser estimate for sphericity was used to correct the degrees of freedom (ε =.60). This revealed a significant main effect for valence on latency (F(1.20, 27.56) = 14.89, p <.001). Similar to test 1, latency to enter the appetitive arm did not differ from the latency to enter the neutral arm (F(1, 23) = 1.67, ns.), while latency to enter the aversive arm was significantly higher (F(1, 23) = 13.70, p <.001, r =.62). An interaction with treatment was not observed (F(1.20, 27.56) =.35, ns.). These results indicate that by test 2, all rats in both treatment conditions effectively acquired the emotional valence associated with both the appetitive and aversive cues, in accordance with exploration times. ANOVA for exploration time in test 3 revealed a violation of sphericity (χ 2 (2) = 10.78, p <.01), therefore Greenhouse-Geisser was used to correct the degrees of freedom (ε =.72). A significant main effect for valence on exploration time was once again revealed (F(1.44, 33.16) = 24.00, p <.001), with time spent exploring the appetitive arm being significantly greater than time spent exploring the neutral arm (F(1, 23) = 20.74, p <.001, r =.69), and time spent exploring the aversive arm being significantly lower (F(1, 23) = 10.08, p <.01, r =.55) (Figure 4). An interaction was not observed (F(1.44, 33.16) =.63, ns.). ANOVA for latency also revealed a violation of sphericity (χ 2 (2) = 32.89, p <.001). Greenhouse-Geisser estimate for sphericity was used to correct for degrees of freedom (ε =.56). There was a significant main effect for valence on latency (F(1.13, 25.91) = 19.09, p <.001), with latency for entering the appetitive arm being not significantly different compared to latency to enter the neutral arm (F(1, 23) = 2.15, ns.), and