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THE EFFECT OF REPEATED EXPOSURE TO UNPREDICTABLE REWARD ON

DOPAMINE NEUROPLASTICITY

by

Sarah Mathewson

A thesis submitted in conformity with the requirements

for the degree of Master of Arts

Graduate Department of Psychology

University of Toronto

© Copyright by Sarah Mathewson 2009

Unpredictable Reward

The Effect of Repeated Exposure to Unpredictable Reward

on Dopamine Neuroplasticity

Master of Arts 2009

Sarah Mathewson

Department of Psychology, Program in Neuroscience, University of Toronto

Abstract

Drugs of abuse elicit dopamine release unconditionally, sensitizing the reward system to

drugs and drug-associated stimuli resulting in compulsive drug-seeking and drug-taking

behaviour. It has been discovered that these same dopamine neurons consistently respond to

natural rewards when the reward delivery is at maximum uncertainty (50%). Reward

uncertainty is a defining feature of gambling. Therefore, chronic increases in dopamine

release from gambling-like stimuli could lead to sensitization of the reward pathways and

contribute to gambling pathology. This study investigated the effects of repeated exposure to

different probabilities of sucrose reward (0, 25%, 50%, 75%, 100%) on sensitivity to an

amphetamine challenge (0.5 mg/kg) and development of sensitization after multiple

amphetamine doses (5 x 1.0/kg) in Sprague–Dawley and Lewis rats. No significant group

differences were found during the amphetamine challenge or amphetamine sensitization in

either strain. Opportunities for improvement in the experimental paradigm and for future

research are discussed.

ii


Acknowledgements

This study was supported by grants from Natural Science and Engineering Research Council.

The author would like to thank: Dr. Paul Fletcher, who served as thesis supervisor, for his

assistance in conducting the study and his helpful suggestions and insightful comments on

the final version; Dr. Suzanne Erb and Dr. Martin Zack for their input and feedback on the

experimental design as it evolved; and Christie Burton, Zoë Rizos and Judy Sinyard for their

technical guidance during the study.

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Table of Contents

1. Introduction 1

1.1 Objective 1

1.2 Dopamine and its Role in Addiction 3

1.3 Sensitization 5

1.4 Cross Sensitization 6

1.5 Comorbidity between Gambling and Drug Use 7

1.6 Gambling and Dopamine 8

1.7 Genetic Factors in Pathological Gambling 10

1.8 Lewis Inbred Strain 11

1.9 Purpose 13

1.10 Hypotheses 14

2. Methods 15

2.1 Animals 15

2.2 Training Apparatus 15

2.3 Experimental Procedures 16

2.4 Statistical Analyses 17

3. Results 20

3.1 Experiment 1: Sprague-Dawley Strain 20

3.1.1 Reward Probability Training 20

3.1.2 Locomotor Activity: Habituation 21

3.1.3 Locomotor Activity: Acute Amphetamine Challenge 22

3.1.4 Locomotor Activity: Amphetamine Sensitization 22

iv


3.1.5 Locomotor Activity: AMPH Challenge Retest 22

3.2 Experiment 2: Lewis Strain 24


3.2.2 Locomotor Activity: Habituation 25

3.2.3 Locomotor Activity: Acute Amphetamine Challenge 25

3.2.4 Locomotor Activity: Amphetamine Sensitization 26

3.2.5 Locomotor Activity: AMPH Challenge Retest 26

4. Discussion 28

4.1 Experiment 1: Sprague-Dawley Strain 29


4.1.2 Habituation 30

4.1.3 Locomotor Response to Amphetamine 31

4.2 Experiment 2: Lewis Strain 32


4.2.2 Habituation 34

4.2.3 Locomotor Response to Amphetamine 34

4.3 Strain Characteristics 35

4.4 Conclusion 36

5. References 43

6. Figure Captions 50

7. Figures 53

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vi

List of Figures

Figure 1: Experimental Design

Figure 2A: Sprague-Dawley Nose Pokes during Conditioned Stimulus Presentation

Figure 2B: Sprague-Dawley Nose Pokes Prior to Conditioned Stimulus Presentation

Figure 3: Sprague-Dawley Locomotor Response during First Habituation Session

Figure 4: Sprague-Dawley Locomotor Response across Three Habituation Sessions

Figure 5: Sprague-Dawley Locomotor Response to Saline and First Amphetamine Challenge

Figure 6: Sprague-Dawley Locomotor Response across Amphetamine Sensitization Regime

Figure 7: Sprague-Dawley Locomotor Response during Second Amphetamine Challenge

Figure 8: Sprague-Dawley Locomotor Response to First and Second Amphetamine

Challenge

Figure 9A: Lewis Nose Pokes during Conditioned Stimulus Presentation

Figure 9B: Lewis Nose Pokes Prior to Conditioned Stimulus Presentation

Figure 10: Lewis Locomotor Response during First Habituation Session

Figure 11: Lewis Locomotor Response across Three Habituation Sessions

Figure 12: Lewis Locomotor Response to Saline and First Amphetamine Challenge

Figure 13: Lewis Locomotor Response across Amphetamine Sensitization Regime

Figure 14: Lewis Locomotor Response during Second Amphetamine Challenge

Figure 15: Lewis Locomotor Response to First and Second Amphetamine Challenge

Unpredictable Reward 1


on Dopamine Neuroplasticity

1.1 Objective

Addiction has been defined as a compulsive pattern of drug-seeking and drug-taking

behaviour that occurs at the expense of most other activities and persists despite adverse

consequences (Robinson & Berridge, 2003). Addiction research traditionally focused solely on

drugs of abuse such as morphine, cocaine, amphetamine (AMPH) and alcohol. Recently both

researchers and clinicians have proposed that the term addiction be expanded to include a variety

of non-drug-related behaviours, such as pathological gambling (Martin & Petry, 2005; Petry

2006). Pathological gambling is a persistent and recurrent maladaptive gambling behaviour,

affecting between 0.5 to 1% of the adult population, that disrupts social, occupational, and

financial functioning (DSM-IV-TR, 2000; Statistics Canada, 2004). Although pathological

gambling is currently classified as an impulse disorder, research indicates that it shares many

diagnostic features with drug addiction, including compulsive reward seeking, tolerance (a need

to increase the wager or frequency of gambling), withdrawal (restlessness or irritability when

prevented from gambling), unsuccessful efforts to reduce or stop the behaviour, and forgoing

social, occupational, or recreational activities (in order to gamble) (DSM-IV-TR, 2000; Potenza,

2008).

According to the Incentive Sensitization Theory of Addiction (Robinson & Berridge, 1993)

repeated exposure to addictive drugs results in increased drug craving or “wanting” that is

independent from the pleasurable effects of the drug. It has been proposed that enduring

hypersensitivities (or sensitization) of the reward-related dopamine pathway in the brain, from

repeated drug administration, causes drugs to become compulsively and enduringly “wanted”


mediating the transition from casual drug use to drug addiction. If pathological gambling should

be characterized as a behavioural addiction, then gambling-like stimuli should act on and alter

this system in a manner similar to that seen with drugs of abuse.

Animal models can play an important role in unraveling the complex neurobiological

processes underlying pathological gambling. These models allow for the development and direct

testing of hypotheses about the neural mechanisms responsible for certain pathologies

(Szechtman & Eilam, 2005). For example, the notion that dopamine is implicated in the

rewarding properties of a variety of drugs of abuse was derived from studies that demonstrated

that disruption of the dopamine system attenuates drug-conditioned place preference and drug

reinforcement (Acquas, Carboni, Leone, & Chiara, 1989; Spyraki, Fibiger, & Phillips, 1983;

Yokel & Wise, 1976). Animal models can also be used to screen candidate medications and to

assist in understanding the pharmacodynamics of these drugs. For example, the drug

reinstatement procedure, an animal model of drug relapse, has played an important role in the

development of methadone and buprenorphine used in the treatment of opiate addiction (Epstein,

Preston, Stewart, & Shaham, 2006). It is likely that an animal model of the fundamental features

of gambling will be necessary for understanding the basic neurobiology of pathological gambling

and its possible relation to drug addiction. The purpose of the present study is to investigate

whether repeated exposure to gambling-like stimuli, characterized by unpredictable reward,

results in sensitization of the dopamine pathways, as assessed by an increased sensitivity to an

AMPH challenge and greater susceptibility to AMPH sensitization in two different strains of

rats. These findings may provide an animal model for the compulsive reward-seeking believed to

underlie pathological gambling in humans.


1.2 Mesolimbic Dopamine and its Role in Addiction

The mesolimbic dopamine pathway is one of the primary pathways involved in reward

(for review see Berridge, 2007). It originates in the ventral tegmental area (VTA) of the midbrain

and innervates several structures of the limbic system, including the nucleus accumbens (N.Acc.)

and prefrontal cortex. It has been established that all addictive drugs, regardless of their primary

neurochemical actions, either directly or indirectly increase neurotransmission of dopamine in

this pathway (Hyman, Malenka, & Nesler, 2006). For example, the psychostimulant drug AMPH

increases dopamine transmission by entering dopamine neurons and interacting with the

dopamine transporter (DAT) to reverse transport of dopamine out of the neuron into the synapse

(Azzaro & Rutledge, 1973; Besson, Cheramy, & Glowinski, 1969). Heroin, an opiate narcotic,

increases dopamine firing in the mesolimbic pathway indirectly by decreasing inhibitory

GABAergic neurotransmission, thus disinhibiting dopamine neurons in the VTA, which then

releases dopamine in the N.Acc. (Johnson, & North, 1992).

Despite the enormous research focus on dopamine and its known role in reward, debate

continues over the precise causal contribution made by mesolimbic dopamingeric neurons to

reward in general and to drug addiction in particular (Berridge, 2007). Dopamine activation in

response to drugs of abuse was originally thought to represent the hedonic (pleasurable) effects

of the drug; hence addiction resulted from the desire to obtain pleasure (Wise, 1982). This

hypothesis however has been shown to be unlikely since animals can display hedonic responses

to both natural rewards and drugs of abuse in the absence of dopamine. Both pharmacological

blocking of dopamine neurotransmission and lesions of dopamine terminals in the N.Acc. fail to

alter hedonic preferences for sucrose in rats (Peciña, Berridge, & Parker, 1997; Berridge &

Robinson, 1998). In addition, dopamine-deficient mice display a robust conditioned place


preference (an indirect measure of hedonic value) for morphine when given caffeine, which

stimulates locomotion in the absence of dopamine, suggesting that the hedonic experience of

morphine is independent from dopamine release (Hnasko, Sotak, & Palmiter, 2005).

Recent evidence in humans also indicates that dopamine may not mediate the subjective

pleasurable effects of drugs of abuse. Leyton et al., (2002) found that dopamine levels in the

mesolimbic pathway of healthy male volunteers, as measured by positron emission tomography

(PET), correlated significantly with drug “wanting” as oppose to drug “liking”. This finding has

been replicated in patients with Parkinson’s disease who show an addiction-like phenomenon

called dopamine dysregulation syndrome. These patients compulsively consume greater amounts

of their dopaminergic medication (L-DOPA) than prescribed, despite the external appearance of

being well-medicated and the disabling drug-induced effects of abnormal involuntary

movements. Using PET, Evans et al., (2006) found that these patients displayed enhanced

dopamine release in the mesolimbic pathway when challenged with an oral dose of L-DOPA

compared to other Parkinson’s patients. Importantly, the excessive dopamine release measured

by the PET correlated with self-reported compulsive drug “wanting” but not “liking”.

Experimental evidence indicating that dopamine affects “wanting” for drugs and that this is

dissociable from the drug’s subjective pleasurable effects has led Berridge and Robinson (1998)

to propose that dopamine transmission in the mesolimbic pathway mediates the assignment of

incentive salience or “wanting” for drugs and drug-related stimuli. The Incentive Sensitization

Theory of Addiction (Berridge & Robinson, 1993) posits that repeated intermittent drug-use

enhances mesolimbic dopamine release, resulting in neuroadaptations to this system, which

renders it hypersensitive or sensitized causing pathological incentive salience or “wanting”

motivation for drugs and drug-associated cues. They further argue that it is this sensitization of


the mesolimbic dopamine system that produces excessive drug craving triggering addictive

behaviour. This implies that addiction is caused primarily by neuroplastic changes in the

functioning of the dopamine system resulting from repeated experience with drugs.

1.3 Sensitization

Sensitization refers to the increased behavioural and neurochemical response to the effects of

a drug following its repeated intermittent administration. A common behavioural manifestation

of sensitization to many drugs of abuse is a progressive and enduring enhancement of their

psychomotor activating effects after repeated drug administration (Stewart & Badiani, 1993).

This behavioural sensitization has been extensively studied under experimental conditions in

animals. It has been well established that repeated injections of psychomotor stimulant drugs,

such as AMPH, result in increased locomotor activity in rats relative to the first exposure, when

challenged with the drug at a later time (Robinson & Becker, 1986; Anagnostaras & Robinson,

1996). Research has also revealed that sensitization is remarkably persistent. Psychomotor

sensitization in animals has been shown to endure for months to years after drug treatment is

discontinued (Paulson, Camp, & Robinson, 1991).

Recently, direct evidence for AMPH-induced sensitization has also been demonstrated in

humans (Strakowski & Sax, 1998; Boileau et al., 2006). For example, Strakowski and Sax

(1998) found that both eye-blink rate and motor activity in drug-naïve participants progressively

increased with each exposure when given three single low doses of AMPH at 48-hour intervals.

Similar behavioural findings were reported by Boileau et al., (2006), when they administered

three single doses of AMPH to healthy participants on alternating days. Consistent with

behavioural sensitization, participants showed an increased eye-blink response across exposures

and after a two-week latency period. This increased psychomotor response was still present with


AMPH re-exposure one year later indicating that sensitization after repeated AMPH

administrations in humans is also long-lasting.

Sensitization of psychomotor behaviours reflects reorganization and enhanced sensitivity

(sensitization) of the brain’s reward system (Robinson & Kolb, 1997; Lorrain, Arnold, & Vezina,

2000). The reward system includes the mesolimbic dopamine pathway, in addition to dopamine

projections to prefrontal cortex, and glutamate innervations from the prefrontal cortex, amygdala

and hippocampus to N.Acc. (Robinson & Berridge, 2003). Neural sensitization is shown by an

enhanced ability of a number of drugs to increase extracellular levels of dopamine in the N.Acc.

(Lorrain, Arnold & Vezina, 2000; Vezina, Lorrain, Arnold, Austin, & Suto, 2002), in addition to

a variety of molecular, morphological and neurochemical changes that extend beyond the

dopamine system (Nestler, 2001; Robinson & Kolb, 1997; Everitt & Wolf, 2002). For example,

repeated exposure to AMPH has been shown to decrease the sensitivity of metabotropic

glutamate receptors in the ventral tegmental area (Wolf, 1998) and to increase the length of

dendrites and the density of dendritic spines in the N.Acc. and prefrontal cortex (Robinson &

Kolb, 1997). The sensitization related structural changes to dendrites in the reward pathway are

thought to reflect changes in synaptic connectivity, and therefore may alter information

processing within this brain region (Robinson & Berridge, 2003).

1.4 Cross-sensitization

Both humans and animals sensitized to one substance often show cross-sensitization to a

different drug or substance (Stewart & Badiani, 1993). This phenomenon has been demonstrated

most often with several different classes of drugs of abuse. For example, chronic treatment with

either morphine or caffeine has been shown to enhance the locomotor response to AMPH

(Vezina, Giovino, Wise, & Stewart, 1989; Cauli, Pinna, Valentini, & Morelli, 2003). Cross-


sensitization can also occur between drugs of abuse and other non-drug rewarding events. For

example, a diet of intermittently high sugar consumption produces behavioral cross-sensitization

to a low dose of AMPH (Avena & Hoebel, 2003b), and animals sensitized to AMPH display

enhanced appetitive behaviour for sexual reward (Nocjar & Panksepp, 2002). Cross-sensitization

is thought to occur when different drugs or substances activate overlapping components of, or

the same, neural circuitry (Nestler, 2005). Therefore, cross-sensitization between a non-drug

manipulation and an addictive substance can be taken as evidence that the manipulation induced

similar neural adaptations as repeated drug administrations (Avena & Hoebel, 2003a).

1.5 Comorbidity between Gambling and Drug Use

Considerable epidemiological and clinical research has shown that pathological gambling

and substance use disorders often co-occur (for review see Petry, 2007). Research reports higher

rates of alcoholism and other substance use among problem gamblers than among the general

population. In the Canadian Community Health Survey, Statistics Canada (2002) reported that 15

percent of problem gamblers also suffered from alcohol dependence, compared to two percent of

non-gamblers. Data from a survey of 2,016 adults in Ontario also demonstrated a strong

association between alcohol use and gambling (Smart & Ferris, 1996). Using logistic regression

models the authors reported that alcohol dependence was a significant predictor of future

gambling problems. Gambling is also related to other drug use problems. From a sample of 580

individuals admitted to a Canadian residential addictions program, Toneatto & Brennan (2002)

found that the rate of pathological gambling was considerably higher for cannabis (24%),

cocaine (11.5%) and opiate (4.8%) abusers than the rate found in the general population. These

high rates of comorbidity between pathological gambling and drug addiction suggest that these


two psychiatric disorders may be linked by some shared neurobiology. It may be that both are

associated with alterations in some of the same neurotransmitter systems.

1.6 Dopamine and Gambling

Most drugs of abuse elicit dopamine release unconditionally; in time, this leads to

sensitization of dopamine pathways (Vezina, 2007). Indirect evidence in humans indicates that

dopamine may be implicated in gambling behaviours and may be altered in individuals who

suffer from pathological gambling. Plasma concentrations of dopamine, although a weak

indicator of neural dopamine activity, is elevated in healthy participants while playing Pachinko,

a Japanese gambling game, which is essentially a combination of pinball and slots (Shinohara, et

al., 1999). Neuroimaging studies of healthy volunteers indicate that cues associated with the

delivery of monetary reward result in activation of the ventral striatum, a region of the brain

containing the N.Acc. and innervated by dopamine neurons (Knutson, Fong, Adams, Varner, &

Hommer, 2001). Pathological gamblers, compared to healthy controls, show reduced activation

of the ventral striatum during a simulated gambling paradigm where the probability of reward is

at maximum uncertainty--that is probability of reward was 50% (Reuter et al., 2005). In the same

study, gambling severity among pathological gamblers was inversely correlated with responses

in this brain region. The finding of relatively diminished activation in the ventral striatum of

severe pathological gamblers parallels findings from studies of reward anticipation in individuals

with drug addiction. For example, reduced activation of the ventral striatum during anticipation

of monetary gain has been reported in individuals with alcohol and cocaine dependence (Wrase,

et al., 2007; Potenza, 2008). The findings from neuroimaging studies suggest that gambling-like

stimuli may alter reward systems in a manner similar to that seen with drugs of abuse. The

specific role of dopamine, however, cannot be fully established from these results, since other


neurotransmitters apart from dopamine, including glutamate and opioids, innervate these brain

regions. Furthermore, it is unclear whether activational differences in pathological gamblers,

compared to controls, existed prior to their involvement with gambling or whether these altered

responses resulted from gambling exposure (Zack, 2008).

Natural rewards, such as food, water, and sex, initially cause dopamine release. In contrast to

drugs of abuse, dopamine release induced by natural rewards diminishes with repeated exposure

(Avena, Rada, Hoebel, 2008). Recently, electrophysiological studies have shown that midbrain

dopamine neurons consistently respond to natural rewards if they are unexpected (Fiorillo,

Tobler, & Schultz, 2003; Fiorillo, Newsome & Schultz, 2008). It has been suggested that

dopamine neurons encode a ‘reward prediction error’: the difference between the probability of

an expected reward and the actual outcome (Waelti, Dickson, & Schultz, 2001). This information

is then subsequently used as a teaching signal in order to improve future predictions (Shizgal &

Arvanitogiannis, 2003). Thus, when an unpredicted (surprise) reward is first presented, it elicits

strong firing of dopaminergic neurons. With repeated presentation, cues (conditioned stimuli)

that reliably predict the occurrence of the reward become capable of activating these dopamine

neurons, while the reward itself, now redundant, elicits weaker activation. Fiorillo, et al., (2003;

2008) found that midbrain dopamine neuron firing after conditioned stimulus (CS) presentation

increased as a function of reward uncertainty. According to the authors, when a CS completely

predicts a particular outcome, probability=0.0 (no reward) or probability=1.0 (certain reward),

post-CS dopamine activation is negligible. In contrast, if a CS signals the maximum uncertainty,

probability = 0.5, when a reward is equally likely to be delivered or absent, post-CS dopamine

firing is maximal. At intermediate uncertainty, probability = 0.25 or probability = 0.75, post-CS

dopamine firing is moderate. These findings suggest that dopamine is repeatedly elevated during


gambling due to uncertain reward. This uncertainty-induced increase in dopamine can lead to

sensitization, which could explain the allure of gambling and the compulsive pattern of reward

seeking found in pathological gamblers.

1.7 Genetic Factors in Pathological Gambling

The results from Fiorillo et al. (2003, 2008) suggest a possible neurobiological mechanism,

dopamine sensitization, which could contribute to the development of pathological gambling.

However, these results do not explain the fact that the majority of Canadian adults participate in

some form of gambling and most do so without encountering significant problems (Canadian

Gaming Association, 2007). It is estimated that only 5% of the adult population exhibit

behaviours that would classify them as being either at-risk or problem gamblers (Statistics

Canada, 2007) and between 0.5 to 1% of adults go on to develop pathological gambling, the

most severe form of problem gambling (National Council of Welfare, 1996). A key question in

problem gambling research, therefore, is why do some individuals develop this persistent and

recurrent maladaptive behaviour while others remain unaffected.

Although environmental factors are involved in the etiology of pathological gambling

(Blaszczynski, & Nower, 2002), genetic factors are also thought to contribute to an individual’s

vulnerability to develop this disorder. Elevated rates of problem and pathological gambling have

been found in male monozygotic twins, compared to dizygotic twins, when their fathers were

problem or pathological gamblers (Eisen, et al., 1998). Biological investigations have suggested

that a shared genetic vulnerability could be a factor in explaining the similarities and high rates

of co-morbidity between substance use disorders and pathological gambling. One study of male

twins found that between 12% and 20% of the genetic variation in the risk for pathological

gambling was accounted for by the risk for alcohol dependence (Slutske, et al., 2000).


Ascertaining genetic vulnerabilities that contribute to the development of pathological gambling

in humans is difficult due to an inability to control environmental factors and to directly establish

the relative genetic contribution (Shah, Eisen, Xian & Potenza, 2005). However, the role genetic

factors play in this susceptibility can potentially be evaluated by examining neurobiological

differences between a heterogeneous outbred and a genetically-uniform inbred strain and relating

these differences to particular behaviours.

1.8 Lewis Inbred Strain

A common strategy used to examine genetic contributions to a particular disorder has been to

compared inbred Lewis (LEW) with Fisher 344 (F344) rats, since both strains are derived from

Sprague-Dawley’s (SD) yet they show marked differences in their behavioural and

neurochemical responses to stress and a variety of addictive substances (for review see Kosten &

Ambrosio, 2002). LEW rats acquire self-administration of opiates (Ambrosio, Goldberg, &

Elmer, 1995; Martin, et al.,1999), cocaine (Kosten, et al., 1997; Kosten Freeman, Kearns, Kohut,

& Riley, 2009), and alcohol (Suzuki, George, & Meisch, 1988 ) faster and display greater

conditioned place preference for morphine (Guitart, Beitner-Johnson, Marby, Kosten, & Nestler,

1992), cocaine (Kosten, Miserendino, Chi & Nesler, 1994) and nicotine (Horan, Smith, Gardner,

Lepore, & Ashby, 1997) compared to F344 rats. LEW rats display greater behavioural

sensitization to methamphetamine, exhibiting lower levels of activity initially that increase with

repeated drug administrations, (Camp, Browman, & Robinson, 1994), and to low and high doses

of cocaine (Kosten et al., 1994; Haile, Hiroi, Nestler, & Kosten, 2001). In addition, LEW rats

have a greater preference for naturally rewarding behaviours such as running. When given free

access to running wheels LEW rats will run excessively covering approximately 10 km per day

compared to F344 rats who run considerable less averaging 1.5km per day (Werme, Thoren,


Olson, & Brene 1999; Werme, et al., 2002). These studies have led some researchers to suggest

that the LEW rat may have a generalizable enhanced sensitivity to the rewarding effects of

addictive drugs and behaviours and may provide a model of genetic vulnerability for factors

related to drug abuse (Nestler, 1992; George, 1993; Werme, et al., 2002).

Like substance addictions, pathological gambling is thought to involve disturbances in

dopamine function (Reuter et al., 2005; Steeves, et al., 2009). Drug-naive LEW rats display

many features in the mesolimbic dopamine pathway that are similar to those of addicted SD rats

(Nestler, 1992). Tyrosine hydroxylase (TH), the rate-limiting enzyme for dopamine synthesis, is

lower in the nucleus accumbens and higher in the ventral tegmental area in LEW versus F344

rats (Guitart, et al., 1992; Haile et al., 2001). The level of dopamine transporter, a primary target

for AMPH and cocaine, in both the dorsal striatum and nucleus accumbens is lower in the LEW

versus F344 strain (Flores, Wood, Barbeau, Quirion, & Srivastava 1998; Gulley, Everett, &

Zahniser, 2007). LEW rats have lower dopamine metabolite levels in the nucleus accumbens;

however, they have only slightly lower extracellular dopamine levels compared to F344 rats

(Camp et al., 1994). Finally, LEW compared to F344 rats have lower levels of dopamine D2 and

D3 receptors in the striatum and nucleus accumbens (Flores et al., 1998), a direct site of action

for both AMPH and cocaine (Ferris, Tang, & Maxwell, 1972). These biochemical differences in

the mesolimbic dopamine system between LEW and F344 rats presumably reflect differences in

dopaminergic functioning that could account for their distinctive behavioural responses to drugs

of abuse.

Given the similarities and co-occurrence between pathological gambling and substance

abuse, the role of dopamine in gambling behaviour and the abnormalities in this neurochemical

system in pathological gamblers, and that LEW rats displays greater behavioural sensitization to


psychostimulants and alterations in the mesolimbic dopamine system, when compared to F344

rats, it seems plausible that the addiction-prone LEW rat might be especially sensitive to the

effects of repeated exposure to gambling-like stimuli.

1.9 Purpose

The previous sections have established that repeated exposure to drugs of abuse results in

sensitization of the mesolimbic dopamine pathway, which is thought to underlie drug addiction.

A common behaviour indicator of dopamine sensitization is increased locomotor activity in

response to a psychostimulant challenge. Electrophysiological results have shown that these

same dopamine neurons consistently respond when reward delivery is most uncertain (50%).

This reward schedule closely resembles that found in gambling. Therefore, repeated exposure to

unpredictable reward may result in intermittent increases in dopamine activation that could lead

to sensitization of the mesolimbic dopamine system and could partly explain compulsive reward-

seeking in pathological gamblers. In order to explore this possibility, the present study sought to

examine the effects of repeated exposure to different probabilities of reward delivery on acute

AMPH response and AMPH sensitization in both the outbred SD and inbred LEW strain. This

was achieved by first exposing separate groups of animals to a CS that differentially predicted

sucrose reward on 0, 25%, 50%, 75% and 100% of the trails. One week following the last

training session, animals were tested for their locomotor response to an acute low dose AMPH

challenge. They were then administered a sensitizing regimen of AMPH following by an AMPH

re-challenge.


1.10 Hypotheses

Experiment 1 and Experiment 2 were conducted sequentially, since it was thought that an

effect would be seen in the SD strain. Experiment 2, therefore, was designed based on the results

obtained from Experiment 1.

Experiment 1: Sprague-Dawley Strain

1) It is predicted that repeated exposure to unpredictable reward (50%) should induce

sensitization of the mesolimbic dopamine neurons by virtue of intermittently elevating dopamine

release. Further this should result in an increased sensitivity to the locomotor stimulant effects of

an AMPH challenge (cross-sensitization) and/or facilitate the development of sensitization after

multiple AMPH doses compared to intermediate (25% or 75%) or certain (0 or 100%) reward

probabilities.

Experiment 2: Lewis Strain

1) It is predicted that the vulnerable LEW strain will show sensitization of the

mesolimbic dopamine neurons after repeated exposure to unpredictable reward (50%), resulting

in an increased sensitivity to the locomotor stimulant effects of an AMPH challenge (cross-

sensitization) and/or facilitating the development of sensitization after multiple AMPH doses

compared to intermediate (25% or 75%) or certain (0 or 100%) reward probabilities.

2) It is hypothesized that the inbred LEW strain will display greater sensitivity to an

AMPH challenge and sensitization regime after reward exposure that the outbred SD strain.


Method

2.1 Animals

The experimental procedures followed the guidelines of the Canadian Council on Animal

Care and were approved by the Centre for Addiction and Mental Health Animal Care

Committee. Forty adult male Sprague-Dawley rats (300-350 g upon arrival; Charles River

Laboratories, St. Constant, QC, Canada) and 40 adult male Lewis rats (200-225 g upon arrival;

Charles River Laboratories, St. Constant, QC, Canada) were used. All animals were housed

individually in clear polycarbonate cages (20cm x 43cm x 22cm). The housing room was

maintained at approximately 22°C under a 12:12 light-dark cycle (lights on at 07:00), with

testing occurring during the light phase of the cycle (between 10:00 and 15:00). All rats were

drug-and-behaviourally naïve at the start of the experiment. Except for the one week

acclimatization period during which food and water was freely available, rats were maintained

on five pellets of laboratory chow per day with water freely available.

2.2 Training Apparatus

Training on the reward probability task was conducted in eight operant conditioning

boxes, measuring 33 x 31x 29 cm, (Med Associates, St. Albans, VT). A 5 cm square reinforcer

magazine was centered in the front wall 2.5 cm above the floor. The magazine contained an

infrared photo-detector at the entrance and a light mounted in the roof. A motor-driven dipper

arm could be raised to deliver 0.06 ml of liquid through a hole in the floor of the magazine. Each

operant box was illuminated by a houselight and was enclosed in a sound-attenuating chamber

equipped with a ventilation fan. The boxes were controlled by an IBM-compatible computer

running Med-PC for Windows.


2.3 Experimental Procedures

The effects of repeated unpredictable reward on sensitivity and sensitization to the

locomotor stimulant effect of AMPH in the 1) Sprague-Dawley and 2) Lewis strains were

measured in separate experiments; however the experimental procedures used in each

experiment were the same. A schematic of the experimental design is displayed in Figure 1.

Reward probability training. One day prior to training, rats were provided with free

access to a bottle containing 10% sucrose in the home cage in order to familiarize them with this

solution. In each experiment forty rats were randomly assigned to one of five experimental

conditions: probability of reward delivery: 0, 25%, 50%, 75%, 100% (n=8 per group). Each

session began with the illumination of the houselight, which remained illuminated throughout the

session. All experimental groups received 20 CS presentations (illumination of the magazine

light for 30 seconds). During the last 5 seconds of CS presentation, depending on the

experimental group, sucrose (unconditioned stimulus US) was available in the magazine on 0,

25%, 50%, 75% or 100% of the trials. The sucrose dipper was either raised for sucrose access or

not activated. Nose pokes into the magazine 30 seconds prior to CS presentation (preCS

responses) and during the 30 second CS presentation (CS responses) were measured. Each CS

presentation was separated by a variable interstimulus interval that averaged 90 seconds and

ranged between 30-180 seconds. Each training session lasted 40 minutes, and animals received

15 training sessions in total, five days per week for three weeks. This reward training paradigm

was adapted from previous work with Pavlovian conditioning (Fletcher, Tenn, Sinyard, Rizos, &

Kapur, 2007).

Acute response to low dose amphetamine. Nine days after the last reward training

session, locomotor responses to a threshold AMPH challenge was assessed in standard clear


Plexiglas housing cages (27 x 48 x 20 cm). A row of six infrared photocell emitters and detectors

was positioned along the long axis of the cage, 3 cm above the floor, capable of detecting

horizontal movements. Before any drug treatment was administered all rats were given a 90

minute session to habituate them to the test boxes for three consecutive days. The following day,

rats were placed in the boxes for 30 minutes and received one intraperitoneal (i.p.) vehicle

injection (0.9% saline, 1.0mg/kg) followed by 90 minutes in the box. On the AMPH test day, rats

were given 30 minutes to habituate to the test boxes, then received an i.p. injection of 0.5mg/kg

D-amphetamine sulfate (Sigma-RBI, Oakville, ON) and immediately returned to the boxes(Tenn,

Fletcher, & Kapur, 2003). A computer was used to detect and record the number of photocell

interruptions during the 90-minute post injection period. The amount of locomotion activity was

quantified as the number of beam breaks measured in 5 min intervals.

Sensitization. Three days after the AMPH challenge rats received one i.p. injection of

AMPH (1.0 mg/kg) per day on a Monday, Wednesday, Friday, Monday, Wednesday, for a total

of five exposures. After each AMPH injection rats were immediately tested for their locomotor

response (as described above). This AMPH treatment regime has been previously shown to

produce a long-lasting sensitized locomotor response to AMPH (Fletcher, Tenn, Rizos, Lovic, &

Kapur, 2005). One week after completion of the sensitization regimen, a retest of their locomotor

responses to an AMPH challenge (0.5mg/kg) was conducted. Rats were given 30 minutes to

habituate to the activity boxes, and then received an i.p. injection of 0.5mg/kg D-amphetamine

sulfate (Sigma-RBI, Oakville, ON) and immediately returned to the boxes. A computer was used

to detect and record the number of photocell interruptions during the 90-minute post injection

period. The amount of locomotion activity was quantified as the number of beam breaks

measured in 5 min intervals.


2.4 Statistical Analysis

The effect of repeated unpredictable reward on sensitivity and sensitization to the

locomotor stimulant effect of AMPH (AMPH) in SD and LEW rats was conducted in separate

experiments; therefore results from each experiment were analyzed separately. All statistical

analyses were conducted with SPSS (v.17 SPSS Inc., Chicago IL) with alpha level set at 0.05.

Reward probability training. Nose-poking into the magazine 30 seconds prior to and

during the CS presentation was analyzed by a three-way repeated measures analysis of variance

(ANOVA) with two between-subject factors (group-5 levels, period (preCS and CS)-2 levels)

and one within-subject factor (day-15 levels). Significant interactions were analyzed using

separate two-way repeated measures ANOVAs for the PreCS and CS period with one between-

subject factor (group -5 levels) and one within-subject factor (day-15 levels). Significant

interactions during each period were further analyzed using separate one-way ANOVAs for each

training session followed by post-hoc comparison using Tukey’s HSD test.

Locomotor activity: habituation. Novelty-induced locomotor activity during the first

exposure to the activity boxes was measured every five minutes for a 90-minute period and

analyzed by a two-way repeated measures ANOVA with one between-subject factor (group -5

levels) and one within-subject factor (5-min periods for 90 mins -18 levels). Habituation to the

activity boxes over the three sessions was assessed by a two-way repeated measures ANOVA

with one between-subject factor (group --5 levels) and one within-subject factor (sessions -3

levels).

Locomotor activity: acute AMPH challenge. A two-way repeated measures ANOVA with

one between-subject factor (group -5 levels) and one within-subject factor (5-min periods for 90

mins -18 levels) assessed locomotion over a 90-minute period post AMPH injection. To analyze


the locomotor response to AMPH treatment a two-way repeated measures ANOVA with one

between-subject factor (group -5 levels) and one within- subject factor (vehicle, AMPH—2

levels) assessed locomotion over the 90 minute period post vehicle and AMPH injection.

Locomotor activity: AMPH sensitization. Locomotor responses to 1.0mg/kg AMPH

across the five test sessions was analyzed with a two-way repeated measures ANOVA with one

between-subject factor (group -5 levels) and one within-subject factor (test session -5 levels).

Locomotor activity: AMPH challenge retest. A two-way repeated measures ANOVA

with one between-subject factor (group --5 levels) and one within-subject factor (5-min periods

for 90 mins --18 levels) assessed locomotion over a 90-minute period post injection. To analyze

sensitization, a two-way repeated measures ANOVA with one between-subject factor (group --5

levels) and one within-subject factor (drug challenge --2 levels) compared the initial 0.5mg/kg

AMPH challenge to the 0.5mg/kg AMPH retest post sensitization regime.


Results

3.1 Experiment 1


on Dopamine Neuroplasticity in Sprague-Dawley Rats

3.1.1 Reward Probability Training

Cumulative nose-poking during the 30-second PreCS period and cumulative nose-poking

during the 30-second CS period were analyzed for all groups across the 15 training sessions. The

results revealed a significant main effect of period (F(1,35)=34.25, p<0.001), indicating that all

animals performed significantly more nose pokes during the CS period compared to the PreCS

period. In addition, a significant main effect of group (F(1,35)=5.40, p<0.01), a significant

period x group interaction (F(4,35)=4.73, p<0.01), a significant day x period interaction

(F(14,56)=3.57, p<0.001) and a significant day x period x group interaction F(56,490)=1.87,

p<0.001) was found, thus indicating that the rate of nose-poke responses was influenced by

group as well as by session.

Figure 2 A and B show cumulative nose pokes during the PreCS and CS period for all

groups across training sessions. It can been seen from Figure 2A that after four days of reward

training, rats exposed to 100% probability of reward showed a significantly higher level of nose-

poking during the CS presentation compared to rats exposed to 0 or 25% probability of reward

(F(4,35) =4.34, p<0.01). This pattern of behaviour continued until the eighth day of reward

training, where rats exposed to 75% probability of reward displayed an increase in nose-poking

during the CS presentation compared to rats exposed to 0 or 25% probability of reward (F(4,39)

=4.49, p<0.01), reaching similar levels as rats exposed to100% reward. Rats exposed to 50%


probability of reward showed a stable midrange pattern of nose-poking behaviour during the CS

periods throughout the training.

During the PreCS period animals exposed to 75% or 100% probability of reward delivery

displayed a higher rate of nose-poking than animals exposed to 0 or 25% probability of reward

(Group F(4,39) =6.34, p<0.01); however, the amount of nose-poking for both the 75% and 100%

groups was significantly less than that displayed during the CS period (75% group F(1,7)=9.11,

p<0.05; 100% group F(1,7)=11.88, p<0.01). Across the 15 sessions there was an initial increase

in nose-poking behaviour during the PreCS period followed by a decrease and then plateau at a

stable low rate of responses (Day F(14,490) =2.24, p<0.01) (Fig. 2B).

3.1.2 Locomotor Activity: Habituation

A two-way repeated measures ANOVA of beam breaks during the initial habituation to

the activity boxes revealed a significant main effect of time (F(17, 595) =89.84, p<0.001),

reflecting a decline in locomotion during the session (Fig. 3). No significant main effect of group

(F(4, 35) =0.57, p=n.s) or significant time x group interaction (F(68, 595) =1.04, p=n.s) was

found, indicating that initial exposure to the activity boxes was not affected by reward

experience.

A two-way repeated measures ANOVA of locomotor response across the three

habituations sessions yielded a significant main effect of session (F(2, 70) =60.01, p<0.001),

indicating a decline in locomotion over the sessions (Fig. 4). No other significant effects (group

main effect (F(4,35) =0.62, p=n.s) and time x group interaction (F(8, 70) =0.38, p=n.s) were

found, indicating that habituation was not affected by reward experience.


3.1.3 Locomotor Activity: Acute AMPH Challenge

A two-way repeated measures ANOVA revealed a significant main effect of time (F(17,

595) =6.42, p<0.001), reflecting a reduction in locomotor activity over time. No significant main

effect of group (F(4,35) =0.62, p=n.s) or significant time x group interaction (F(68, 595) =0.86,

p=n.s) was found, indicating that locomotor response to an acute AMPH challenge was not

affected by reward experience.

Compared to the vehicle injection, all groups displayed a significant increase in

locomotor response to AMPH (Fig. 5). A two-way repeated measures ANOVA revealed a

significant main effect of treatment (F(1, 35) =213.44, p<0.001), but no other significant effects

(group main effect (F(4,35) =0.70, p=n.s) and treatment x group interaction (F(8, 70) =0.52,

p=n.s).

3.1.4 Locomotor Activity: AMPH Sensitization

A two-way repeated measures ANOVA of locomotor response to a 1 mg/kg dose of

AMPH over five exposures revealed a significant main effect of test session (F(4,140) =6.72,

p<0.001), indicating that locomotor activity increased in all groups over sessions (Fig. 6).

No significant main effect of group (F(4,35) =0.44, p=n.s) or significant test session by group

interaction (F(16,140) =1.57, p=n.s) was found, indicating that locomotor activity to a 1 mg/kg

dose of AMPH over five exposures was not affected by reward experience.

3.1.5 Locomotor activity: AMPH challenge retest.

Figure 7 shows the 90-minute time course of the locomotor response to the AMPH

challenge post sensitization. A two-way repeated measures ANOVA revealed a significant main

effect of time (F(17, 595) =17.07, p<0.001), reflecting a reduction in locomotor activity over

time. No significant main effect of groups (F(4,35) =0.99, p=n.s) or significant time x group


interaction (F(68, 595) =1.19, p=n.s) was found, indicating that locomotor response to the

AMPH challenge post sensitization was not affected by reward exposure.

To confirm that the AMPH treatment regime induced a sensitized state, the locomotor

data from the first challenge were compared to the second challenge. A two-way ANOVA

revealed a significant main effect of drug challenge (F(1,35) =76.26, p=0.00), but no significant

main effect of group (F(4,35) =0.84, p=n.s) or significant challenge x group interaction (F(4,35)

=0.87, p=n.s). These results indicate that a significant increase in locomotor response to the

0.5mg/kg AMPH challenge post sensitization regime occurred when compared to the initial

AMPH challenge; however, this response was not influence by reward experience (Fig. 8).


3.2 Experiment 2


on Dopamine Neuroplasticity in Lewis Rats


Cumulative nose-poking during the 30-second PreCS period and cumulative nose-poking

during the 30-second CS period were analyzed for all groups across the 15 training sessions. The

results revealed a significant main effect of period (F(1,35)=23.25, p<0.001), indicating that all

animals performed significantly more nose pokes during the CS period compared to the PreCS

period. In addition, a significant main effect of day (F(14,490)=3.17, p<0.001), a significant

main effect of group (F(4,35)=6.38, p<0.001), a significant day x group interaction

(F(56,490)=3.17, p<0.05), a significant period x group interaction(F(4,35)=4.58, p<0.01), a

significant day x period interaction (F(14,56)=2.89 p<0.001) and a significant day x period x

group interaction F(56,490)=1.59, p<0.001) was found, thus indicating that the rate of nose-poke

responses was influenced by group as well as by session.

Figure 9 A and B show cumulative nose pokes during the PreCS and CS period for all

groups across training sessions. It can been seen from Figure 9A that initially all groups except

for rats exposed to 100% probability of reward showed little nose-poking behaviour during the

CS period. After five days of training rats exposed to100% probability of reward showed a

significantly higher level of nose-poking during this period compared to all other groups

(F(4,39) =3.10, p<0.05). By the seventh day of training, rats exposed to 75% probability of

reward showed enhanced nose-poking behaviour during the CS period compared to the 0

probability of reward group (F(4,39) =6.35, p<0.01), while all other groups remained stable. This

pattern of behaviour continued until the thirteenth day of training, when rats exposed to 50%


probability of reward gradually increased nose-poking and rats exposed to 75% probability of

reward decreased this behaviour responding at levels similar to the 50% group.

Nose-poking behaviour during the PreCS period varied across days (Day F(14,490)

=3.08, p<0.01) (Fig. 9B). Animals exposed to 100% probability of reward performed more nose-

pokes during this period than groups exposed to 0, 25% or 50% probability of reward (Group

F(4,35) =7.24, p<0.01); however, the amount of nose-poking for the 100% group was

significantly less than that displayed during the CS period (F(1,9)=9.14 p<0.05).

3.2.2 Locomotor Activity: Habituation

A two-way repeated measures ANOVA of locomotor response during the initial

habituation to the activity boxes revealed a significant main effect of time (F(17,595) =85.13,

p<0.001), reflecting a decline in locomotion during the session (Fig. 10). No significant main

effect of group (F(4,35) =0.58, p=n.s) or significant time x group interaction (F(68,595) =0.85,

p=n.s) was found, indicating that initial exposure to the activity boxes was not affected by

reward experience.

A two-way repeated measures ANOVA of locomotor response across the three

habituations sessions yielded a significant main effect of session (F(2,70) =22.39, p<0.001),

indicating a decline in locomotion over the sessions (Fig. 11). No other significant effect (group

main effect (F(4,35) =0.41, p=n.s) and time x group interaction (F(8,70) =1.09, p=n.s) was

found, indicating that habituation was not affected by reward experience.

3.2.3 Locomotor Activity: Acute AMPH Challenge

A two-way repeated measures ANOVA revealed a significant main effect of time (F(17,

595) =13.24, p<0.001), reflecting a reduction in locomotor activity over time. No significant

main effect of group (F(4,35) =0.33, p=n.s) or significant time x group interaction (F(68, 595)


=0.94, p=n.s) was found, indicating that locomotor response to an acute AMPH challenge was

not affected by reward experience.

Compared to the vehicle injection, all groups displayed a significant increase in

locomotor response to AMPH (Fig. 12) A two-way repeated measures ANOVA revealed a

significant main effect of treatment (F(1, 35) =289.11, p<0.001), but no other significant effects

(group main effect (F(4,35) =0.87, p=n.s) and treatment x group interaction (F(8, 70) =0.33,

p=n.s).

3.2.4 Locomotor Activity: AMPH Sensitization

A two-way repeated measures ANOVA of locomotor response to a 1 mg/kg dose of

AMPH over five exposures revealed a significant main effect of test session (F(4,140) =7.10,

p<0.001), indicating an increase in locomotor activity in all groups over sessions (Fig. 13). No

significant main effect of group (F(4,35) =0.67, p=n.s) or significant test session by group

interaction (F(16,140) =0.66, p=n.s) was found, indicating that locomotor activity to a 1 mg/kg

dose of AMPH over five exposures was not affected by reward experience.

3.2.5 Locomotor Activity: AMPH Challenge Retest

Figure 14 shows the 90-minute time course of the locomotor response to the AMPH

challenge post sensitization. A two-way repeated measures ANOVA revealed a significant main

effect of time (F(17, 595)=21.99, p<0.001), reflecting a reduction in locomotor activity over

time. No significant main effect of group (F(4,35)=1.05, p=n.s) or significant time x group

interaction (F(68, 595)=0.76, p=n.s) was found, indicating that locomotor response to the AMPH

challenge post sensitization was not affected by reward experience.


To confirm that the AMPH treatment regime induced a sensitized state the locomotor

data from the first challenge was compared to the second challenge. A two-way ANOVA

revealed a significant main effect of drug challenge (F(1,35)=93.40, p=0.00), but no significant

main effect of group (F(4,35)=0.55, p=n.s) or significant challenge x group interaction

(F(4,35)=1.02, p=n.s). These results indicate that a significant increase in locomotor response to

the 0.5mg/kg AMPH challenge post sensitization regime occurred when compared to the initial

AMPH challenge; however this response was not influenced by reward experience (Fig. 15).


Discussion

Recurrent dopamine release induced by repeated exposure to drugs of abuse leads to

sensitization of the dopamine pathways. This process has been proposed to underlie compulsive

drug-seeking and drug-taking behaviour (Robinson & Berridge, 2000). Exposure to a CS for a

non-drug reward when the probability of reward delivery is at maximum uncertainty (50%) also

leads to high levels of midbrain dopamine activation (Fiorillo et al., 2003, 2008). Reward

unpredictability is a distinguishing feature of gambling, suggesting that repeated exposure to

gambling or gambling-like stimuli could lead to intermittent elevations in dopamine and thus to

sensitization of the dopamine pathways. This repeated uncertainty-induced increase in dopamine

could contribute to the compulsive pattern of reward-seeking behaviour seen in pathological

gamblers.

The present study sought to determine whether repeated exposure to unpredictable

reward (50%) induces sensitization of mesolimbic dopamine neurons in SD and LEW rats. This

was done by repeatedly pairing a CS (illumination of the magazine light) with differing

probabilities of sucrose reward (0, 25%, 50%, 75%, 100%) and then testing for evidence of

sensitization of mesolimbic dopamine neurons. Dopamine sensitization was assessed by

locomotor response to; an acute AMPH challenge, repeated AMPH administrations, and an

AMPH challenge retest. It was hypothesized that SD and LEW animals exposed to 50% reward

delivery would display increased sensitivity to the locomotor stimulant effects of the AMPH

challenge and enhanced development of sensitization after multiple AMPH doses compared to

animals exposed to more certain reward schedules. In addition, it was predicted that the

addiction-prone inbred LEW rats would display greater sensitivity to the AMPH challenge and

sensitization regime than the outbred SD rats. The results indicate that unpredictable reward


delivery did not the affect the acute response to AMPH or AMPH sensitization in either strain,

nor were any significant differences between strains in sensitivities to AMPH observed.

4.1 Experiment 1: Sprague-Dawley Strain


All animals, except those exposed to low probabilities (0% and 25%) of reward delivery,

consistently displayed increased nose-poking behaviour during the 30 second CS presentation

compared to 30 seconds prior to CS presentation (PreCS), indicating that for these animals the

CS had acquired motivational significance. It was expected that if the animals had learned

something about the structure of their probability schedule their behavioural responses to the CS,

as measured by nose-poking into the magazine, would be greater under conditions of higher

reward probability. By the 8th day of reward training a clear differentiation in nose-poking

behaviour during the CS presentation emerged, such that animals exposed to 75% or 100%

probability of reward displayed greater nose-poking behaviour than animals exposed to lower

reward probabilities, with the 50% group showing intermediate behavioural responses. This

behavioural distinction between reward probability groups remained consistent throughout the

rest of the reward training. Both the PreCS and CS behavioural distinction and the apparent

group differences in nose-poking behaviour during the CS presentation indicate that the differing

probabilities of reward delivery governed nose-poking behaviour and that the classical

conditioning regime (15 sessions) used was sufficient for probability learning in the SD rats.

4.1.2 Habituation

A number of studies have demonstrated that locomotor response to a novel environment

is predictive of sensitivity to acute AMPH and of the magnitude of sensitization (Piazza,

Deminiere, Le Moal, & Simon, 1989; Exner, & Clark, 1993; Hooks, Jones, Smith, Neill, &


Justice, 1991). Therefore, individual variations in novelty-induced locomotion could moderate

group differences to the reward probability treatment during the AMPH challenge and

sensitization regime. In addition, research indicates that locomotor response to a novel

environment is influenced by dopamine activation. Hooks and Kalivas (1995) found that

microinjections of fluphenazine, a dopamine receptor antagonist, into the N.Acc. prevented

novelty-induced locomotor activity in SD rats, but did not suppress locomotor activity in

previously habituated animals. Reduced novelty-induced locomotor activity was also found in

rats subcutaneously injected with another dopamine receptor antagonist, haloperidol, while an

effect was not seen in similarly treated habituated animals (Bardo, Bowling & Pierce, 1990).

These studies provide pharmacological evidence that locomotor response induced by exposure to

a novel environment is dependent on dopamine. If repeated exposure to differing probabilities of

reward delivery affects dopamine activation, then group differences in locomotor response

during the initial exposure to the activity boxes could reasonably be expected. Our results,

however, showed no group differences in locomotor response during the initial exposure to the

activity boxes or across the three habituation sessions. These results indicate that reward history

did not affect novelty-induced locomotion or the rate of habituation in SD rats.

4.1.3 Locomotor Response to Amphetamine

All reward probability groups responded similarly to the initial AMPH challenge, the

sensitization regime and the AMPH challenge retest. It has been established that locomotor

response to both acute and repeated intermittent AMPH administration is mediated by

mesolimbic dopamine activation (for review see Stewart & Badiani, 1993). Therefore, these

findings indicate that there is no evidence for mesolimbic dopamine alterations after repeated

exposure to unpredictable reward delivery in SD rats. It is important to note that comparison


between the initial and the final AMPH challenge did produce a robust sensitized locomotor

response indicating that the AMPH injection regime used in this experiment was adequate to

induce sensitization in this strain. Therefore, the lack of group differences during the final

AMPH challenge cannot be attributed to a lack of AMPH sensitization.

To the best of our knowledge, this is the first study to use behavioural measures to

examine the effects of maximum uncertainty of reward delivery (50%) on dopamine functioning

in rats. Our findings are in contrast to the electrophysiological results found in primates from

Fiorillo et al., (2003, 2008). This discrepancy, however, could have resulted from

methodological differences. Fiorillo et al., (2003, 2008) directly assessed activation of midbrain

dopamine neurons in primates, via single unit recordings, during a classical conditioning

procedure where unique visual stimuli were associated with differing probabilities of liquid

reward. In contrast, the present study indirectly inferred dopamine activation in rats under

differing conditions of reward probability from their locomotor response to AMPH. Although

this method has been shown to be a valid means of inferring mesolimbic dopamine sensitization

(Lorraine, Arnold, & Vezina, 2000), direct assessment of dopamine levels by microdialysis

might provide a more sensitive and direct measure of subtle group differences in dopamine

function.

Clinical research indicates that only certain susceptible individuals develop gambling

pathology (Potenza, Kosten, & Rounsaville, 2001; Blaszczynski, & Nower, 2002). One possible

reason for the lack of group differences found in the SD rats during the AMPH challenge and

subsequent sensitization in the first experiment could relate to the strain of rat used. Repeated

exposures to unpredictable reward did not increase sensitivity to the locomotor response to an

AMPH challenge or enhance sensitization in the SD rat; this suggests that perhaps dopamine


neuroadaptations due to reward uncertainty might only be induced in a vulnerable strain. The

LEW rat was selected as a plausible “vulnerable candidate” because of: alterations in its

mesolimbic dopamine functioning, its sensitivity to the rewarding effects of a variety of

addictive substances and behaviours and its enhanced behavioural sensitization to

psychostimulants.

4.2 Experiment 2: Lewis Strain

In the second experiment LEW rats underwent the same experimental procedure that had

been used with the SD rats.


By the 12th day of reward training, all animals, except those exposed to low probabilities

(0% and 25%) of reward delivery, displayed increased nose-poking behaviour during the 30

second CS presentation compared to 30 seconds prior to CS presentation (PreCS). These results

indicate that by the end of training the CS had acquired motivational significance for animals

exposed to 50%, 75% or 100% reward delivery. Unlike the SD rats, which showed a clear

divergence between groups in nose-poking during the CS presentation, a divergence of

behavioural responses for LEW rats during the CS presentation between all groups did not

emerge. Although the behavioural responses for LEW rats from the 100% group and

subsequently the 75% group were distinctly higher than the other groups, the 50% group failed to

differentiate from the lower reward probability groups until the second last day of training. The

lack of divergence in nose-poking behaviour during the CS period suggests that LEW rats

exposed to 50% probability of reward may have had difficulty learning the CS-US connection.

It is also possible, however, that the behavioural measure used to assess CS-US learning,

nose-poking into the magazine (which is one of a range of possible conditioned responses), did


not accurately reflect the learning of the CS-US contingency. Research has established that in

rats there are large individual differences in response to cues associated with rewards (Boakes,

1977; Davey & Cleland, 1982; Robinson & Flagell, 2009). Upon cue presentation some animals

will come to approach the cue and engage it, performing a “sign-tracking” conditioned response;

others will not approach the cue, but instead, approach the site of reward delivery, performing a

“goal-tracking” conditioned response (Boakes, 1977).

Visual observation of rats exposed to 50% reward delivery during the final training

session revealed that some animals displayed sign-tracking like behaviour upon CS presentation

(illumination of the magazine light) by nose-poking into the magazine, while others displayed

goal-tracking like behaviour by approaching the magazine upon CS presentation, and only nose-

poking into the magazine when the motor-driven dipper arm was raised in order to retrieve the

liquid sucrose. For these goal oriented animals relatively few nose pokes were performed during

the training session. Therefore, continuous video recording and subsequent scoring of all activity

in the operant boxes during the CS presentation might provide a more valid measure of all

conditioned behaviours. However, in both cases the CS produced a conditioned response directed

either at the CS or at the reward, which suggests that by the end of training the CS had acquired

some predictive value.

4.2.2 Habituation

The group similarities during the initial exposure to the activity boxes and across the

three habituation sessions indicated that reward history did not affect dopamine dependent

novelty-induced locomotor activity or the rate of habituation in the LEW rats.


4.2.3 Locomotor Response to Amphetamine

The lack of significant group differences in response to the initial AMPH challenge, the

sensitization regime and the AMPH challenge retest did not support our hypothesis that

unpredictable reward delivery would alter dopamine activation in LEW rats. Comparison of the

initial and the final AMPH challenge showed a robust sensitized locomotor response indicating

that the AMPH injection regime used in this experiment was adequate to induce sensitization in

this strain. Despite the lack of significant group differences during the sensitization regime and

final AMPH challenge, it is worth noting that by the fifth 1 mg/kg dose of AMPH, there appears

to be a trend where LEW rats exposed to 50% probability of reward displayed slighter greater,

although not significantly greater, locomotor responses than animals under the other probability

conditions (Fig. 13). This suggests that an enhanced dopaminergic tone may be required to reveal

the effects of differing reward probability schedules in this strain. Exposure to a low-dose

AMPH regime prior to reward training, in order to augment dopamine release, might amplify the

slight group differences observed here. In addition, AMPH treatment prior to reward training

might enhance the acquisition of the CS-US contingency. Harmer and Philips (1998)

demonstrated that repeated injections of AMPH (1 injection of 2mg/kg i.p. for 5 days) in rats two

weeks before moderate training (ten 1hr sessions of 13 CS-US trails) on a Pavlovian

conditioning task (light or tone paired with 10% sucrose solution) resulted in more rapid

acquisition of conditioned appetitive approach responses.

4.3 Strain Characteristics

Since the first and second experiments were not conducted simultaneously direct

comparison between strains is impossible; however, it is interesting to note certain distinguishing

strain characteristics. In the reward training portion of the study, SD rats appeared to require


fewer reward probability conditioning trials (8 sessions) than LEW rats to demonstrate learning

the CS-US probability schedule, as shown by the differentiation in nose-poke behaviour during

the CS period. LEW rats required considerably more reward probability trials (13 sessions) to

produce a moderate behavioural differentiation, and unlike the SD rats, a clear divergence of

behavioural responses to the CS between all groups never occurred. This raises the possibility

that LEW rats may possess a learning deficit relative to SD rats. Currently no comparative

research using LEW and SD strains on learning tasks exist; however, it has been found that LEW

rats more readily acquire self-administration of alcohol, opiates and psychostimulants (Kosten &

Ambrosio, 2002) and autoshaping behaviour compared to F344 rats (Kearns, Gomez-Serrano,

Weiss & Riley, 2006). Both of these conditioning tasks are more complicated than the simple

Pavlovian procedure used in the present study, suggesting that LEW rats do not possess any

general operant or appetitive learning difficulties relative to F344 rats. A possibility for future

research would be to examine LEW and SD strains on simple and complex learning tasks, in

order to elucidate the strain differences observed in this study.

In addition to differences in the rate of acquisition of the conditioned behaviour, these

strains exhibited differences in their initial locomotor response to the activity boxes. LEW rats

displayed a lower level of novelty-induced locomotion during the first 90 minute habituation

session (Mean beam breaks = 1041) compared to SD rats (Mean beam breaks = 2162).

Locomotor activity in an unfamiliar environment has been shown to be influence by dopamine

(Hooks & Kalivas, 1995; Bardo, Bowling & Pierce, 1990). Several studies have compared

locomotor response to a novel environment in inbred LEW and F344 strains; however, the

findings are inconsistent. Upon initial exposure to an experimental apparatus, some researchers

have reported greater locomotor activity in LEW rats (Camp et al., 1994; Stöhr, Wermeling,


Weiner, & Feldon, 1998; Rex, Sondern, Voigt, Franck, & Fink, 1996), while others have

reported reduced activity in comparison to F344 rats (Chaouloff, Kulikov, Sarrieau, Castanon, &

Mormède, 1995; Haile, et al., 2001). Despite these inconsistencies, as discussed previously,

considerable research has shown strain differences in mesolimbic dopamine functioning between

LEW and F344 rats (Kosten & Ambrosio, 2002). LEW rats have lower levels of tyrosine

hydroxylase (TH) in the N.Acc (Guitart, et al., 1992), dopamine transporter in dorsal striatum

and N.Acc. (Flores, et at., 1998; Gulley et al., 2007), dopamine metabolite in the N.Acc (Camp

et al., 1994) and dopamine D2 and D3 receptors in the striatum and N.Acc (Flores, et at., 1998).

Currently, few studies have examined strain differences in the dopamine system between LEW

and SD rats. However, one study by Minabe, Gardner, and Ashby (1998) found that LEW rats

have significantly fewer spontaneously active substantia nigra pars compacta and ventral

tegmental area dopamine neurons compared to both SD and F344 rats. The strain differences in

the dopamine system between LEW and F344 rats and the results from Minabe et al., (1998)

suggest that LEW rats may have a hypoactive midbrain dopamine system, which could explain

their reduced novelty-induced locomotion in the present study.

In addition to being dopamine dependent, locomotor activity in an unfamiliar

environment is also regarded as a behavioural response to stress (Dantzer & Mormède, 1983).

Exposure to stress activates the hypothalamic-pituitary-adrenal axis, which stimulates a cascade

of events including the release of adrenocorticotropic hormone (ACTH) from the anterior

pituitary gland leading to corticosteorne (CORT) release from the adrenal cortex (Nelson, 2005).

Data indicate that LEW rats exhibit a blunted HPA axis response to stress when compared to

F344 rats (Sternberg, et al., 1992; Dhabhar, McEwen, & Spencer 1993; Kosten & Ambrosio,

2002). Sternberg, et al., (1992) exposed LEW and F344 rats to an open field test, swim stress,


and restraint and found that LEW rats consistently exhibited lower levels of ACTH and CORT in

response to these stressors.

To date, relatively few studies have examined differential stress responses between SD

and LEW strains. Dhabhar, et al., (1993) reported that LEW rats fail to show a normal circadian

diurnal rise in CORT levels, which is expected at the beginning of their active period (evening),

and display impaired ACTH and CORT responses to one hour of restraint stress in comparison to

both F344 and SD rats. According to Cohen, Zohar, Gidron, Matar, Belkind et al., (2006) LEW

rats exhibit greater baseline startle responses and fearful behaviours in the elevated plus maze

and greater stress-induced increases in these behaviours following extreme stress (scent of the

urine of a prime predator) compared to SD rats. Although LEW rats display a reduced stress-like

response in the present study, inconsistencies in previous novelty-induced locomotor response

findings with F344 rats and limited research with the SD strain make drawing a conclusion

difficult. The findings from Dhabhar et al. (1993) and Cohen et al., (2006), however, do suggest

that differences between strains in the locomotor response during the first habituation observed

in the present study could be influenced by deficiencies in the HPA axis’ response to stress found

in the LEW rat.

Given that SD and LEW rats appear to differ on the rate of acquisition of the conditioned

nose-poking behaviour and on novelty-induced locomotor activity, it is surprising that the LEW

rats, a strain known for its high levels of drug intake (Kosten & Ambrosio, 2002) and sensitivity

to methamphetamine and cocaine sensitization when compared to F344 rats (Camp et al., 1994;

Kosten et al., 1994; Haile et al., 2001) did not show evidence of greater behavioural sensitization

to AMPH than SD rats. When comparing the locomotor responses from the initial AMPH

challenge to the AMPH challenge retest (post sensitization regime), LEW rats appear only


moderately more sensitive to repeated AMPH administration than SD rats (percent change 65 %

SD vs. 80% LEW, non significant). To the best of our knowledge, no research comparing

sensitivity to repeated AMPH exposure in SD and LEW rats has been published. Sicar and Kim

(1999), however, have reported that male LEW rats show an enhanced behavioural response to

multiple cocaine injections (15 mg/kg/day x 5) compared to F344 rats, with SD rats rank

intermediate in their behavioural sensitivity.

One possible explanation for the lack of substantial strain differences in response to

repeated AMPH administration seen in the present study could relate to the behavioural measure

of psychomotor activity used. In the study by Sicar and Kim (1999) the authors used a 12-point

behavioural ratings scale to assess all activity over a 60-minute post-injection period. A mean

behavioural score was then tabulated for each animal by adding the ratings from all post-

injection observation periods. The behavioural score for each animal at each testing was then

averaged to create a mean behavioural strain score. Various studies have reported that LEW and

F344 rats exhibit very different psychostimulant-induced locomotor behaviours, with LEW rats

displaying greater stereotypy than F344 rats (Camp et al., 1994; Numachi, Yoshida, Toda,

Matsuoka, & Sato, 2000). Stereotypic behaviour in rats involves repetitive activity (head

movements, gnawing, downward sniffing, and licking) not directed towards any specific goal,

that occurs in place (McKim, 2006). Some authors have criticized the reliance on cumulative

photobeam counts as a measure of psychostimulant-induced activity (Camp et al., 1994). When

comparing the behavioural response induced by repeated methamphetamine injections in LEW

and F344, Camp et al. 1994 found robust psychomotor activity differences between strains when

drug-induced behaviours were visually rated on a nine-point scale; however, these results were

less consistent when only photobeam counts were analyzed. Perhaps SD and LEW rats in the


present study displayed differing psychostimulant-induced behaviours, which are difficult to

interpret using cumulative photobeam counts. This might account for the similar increases in

locomotion from repeated AMPH administration seen in both strains. Videotaping each animal

following drug administration and subsequently rating drug-induced behaviours might provide a

more valid measure of stimulant-induced psychomotor activity and might reveal strain

differences.

4.4 Study Limitations

The present study had a number of limitations. First, the number of reward training

sessions (15) used in the present study may have not been sufficient to induce changes in the

mesolimbic dopamine system. Scott-Railton & Vezina (Society For Neuroscience poster, 2006)

examined the effects of chronic intermittent reinforcement compared to certain reinforcement on

AMPH sensitivity in rats by extensively training animals (55 training sessions) in an operant

conditioning paradigm to lever press for saccharin using escalating schedules of fixed or variable

ratio reinforcement. Two weeks post training all animals were given an AMPH challenge

(0.5mg/kg) followed by an AMPH sensitizing regimen. The authors found that animals trained

extensively on the variable-ratio reinforcement schedule displayed significantly greater

locomotor responses during the acute AMPH challenge and AMPH sensitizing regimen than rats

trained on the fixed-ratio schedule. Repeated exposure to intermittent reinforcement in the

variable ratio condition resulted in unpredictable reward delivery, and induced sensitization-like

neuroadaptations in the dopamine system, providing evidence that repeated exposure to

unpredictable reward alters dopamine functioning. This study’s experimental objective and

design were similar to the present study, which suggest that greater exposure to the reward

probability training may result in group differences in the SD rats and amplify the trend observed


in LEW rats. Second, dopamine activation was assessed indirectly by AMPH-induced locomotor

response. Although this method has been shown to be a valid means of inferring dopamine

sensitization (Lorraine, Arnold, & Vezina, 2000), future research might benefit from a direct

assessment of dopamine levels by using microdialysis. Third, although repeated exposure to

unpredictable reward is a distinguishing feature of gambling, the procedure used may have been

too simple to truly model the complicated phenomena of gambling. Gambling includes not only

uncertain reward, but also the risk of material loss. The loss of resources when a wager is

unsuccessful was a feature which was not included in this experimental design. Recently, Zeeb,

Robins and Winstanley (2009) have developed a novel rat gambling task which includes

differing probabilities of reward and loss of resources wagered. In this task rats choose among

four different options in order to earn as many sugar pellet rewards as possible during a 30

minute period. Each option is associated with different probabilities and amounts of reward as

well as different durations of punishing time-out periods, during which a reward can not be

obtained. Larger reward options are associated with a higher probability of long timeouts,

resulting in fewer rewards per session. In order to maximize reward earnings rats must learn to

avoid these risky options. These authors found that following repeated exposure to the gambling

task, acute administration of AMPH (1.0mg/kg or 1.5mg/kg, i.p.) impaired gambling

performance, by shifting preference towards the option associated with the smallest reward, and

the lowest frequency and duration of punishment. Despite the lack of significant group

differences in locomotor response to acute AMPH or AMPH sensitization in both strains in the

present study, the findings from Zeeb et al. (2009) give support to the hypothesis that there is a

critical relationship between dopamine and repeated exposure to gambling-like stimuli.


4.5 Conclusion

Over the last decade, gambling in Canada has grown rapidly and become an

increasingly popular form of recreation (Statistics Canada, 2007). New technologies, including

video lottery terminals, mega-lotteries and internet gambling, have resulted in wide accessibility.

As well, public perception of gambling has shifted from what was once viewed as disreputable to

an acceptable form of entertainment (Korn, 2000). Nonetheless, for susceptible individuals

gambling has the potential to develop into a serious problem affecting all aspects of their lives

(Centre for Addiction and Mental Health, 2004). As gambling becomes more accessible and

acceptable in Canada, the number of people affected by problem gambling will increase.

Currently, behavioural pharmacological research on the causal mechanisms of

pathological gambling is in its preliminary stages, largely due to limits in modeling the

fundamental features of gambling in animals. Repeated exposure to a CS for non-drug reward

consistently results in maximum midbrain dopamine release in primates when the probability of

reward is most uncertain (50%) (Fiorillo et al., 2003, 2008). This repeated increase in dopamine

release can lead to sensitization of the mesolimbic dopamine pathways and could contribute to

gambling pathology. In the present study, however, unpredictable reward delivery did not alter

dopamine activation as assessed by acute and sensitized locomotor response to AMPH in either

SD or LEW rats.


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Figure Captions

Figure 1. A schematic representation of the experimental design used for Experiment 1 and 2.

Figure 2A. Mean (± S.E.s) nose pokes during conditioned stimulus presentation (30 seconds) for

Sprague-Dawley rats (n=8 per group) exposed to 0, 25%, 50%, 75% or 100% sucrose reward

probability.

Figure 2B. Mean (± S.E.s) nose pokes 30 seconds prior to conditioned stimulus presentation for

Sprague-Dawley rats (n=8 per group) exposed to 0, 25%, 50%, 75% or 100% sucrose reward

probability.

Figure 3. Time-course (5-min bins) of beam breaks (mean ± S.E.s) during initial exposure to

locomotor boxes for Sprague-Dawley rats (n=8 per group) previously exposed to 0, 25%, 50%,

75% or 100% sucrose reward probability.

Figure 4. Mean beam breaks (± S.E.) across three 90 minute habituation session for Sprague-

Dawley rats (n=8 per group) previously exposed to 0, 25%, 50%, 75% or 100% sucrose reward

probability.

Figure 5. Mean beam breaks (± S.E.) after saline (1.0ml/kg, i.p.) and first amphetamine

challenge (0.5 mg/kg, i.p.) for Sprague-Dawley rats (n=8 per group) previously exposed to 0,

25%, 50%, 75% or 100% sucrose reward probability.

Figure 6. Mean beam breaks (± S.E.) over the course of 5 amphetamine injections (1.0mg/kg,

i.p.) for Sprague-Dawley rats (n=8 per group) previously exposed to 0, 25%, 50%, 75% or 100%

sucrose reward probability.

Figure 7. Time-course (5-min bins) of beam breaks (mean ± S.E.s) after second amphetamine

challenge (0.5 mg/kg, i.p.) for Sprague-Dawley rats (n=8 per group) previously exposed to 0,

25%, 50%, 75% or 100% sucrose reward probability.


Figure 8. Mean beam breaks (± S.E.) after the first and second amphetamine challenge for

Sprague-Dawley rats (n=8 per group) previously exposed to 0, 25%, 50%, 75% or 100% sucrose

reward probability.

Figure 9A. Mean (± S.E.s) nose pokes during conditioned stimulus presentation (30 seconds) for

Lewis rats (n=8 per group) exposed to 0, 25%, 50%, 75% or 100% sucrose reward probability.

Figure 9B. Mean (± S.E.s) nose pokes 30 seconds prior to conditioned stimulus presentation for

Lewis rats (n=8 per group) exposed to 0, 25%, 50%, 75% or 100% sucrose reward probability.

Figure 10. Time-course (5-min bins) of beam breaks (mean ± S.E.s) during initial exposure to

locomotor boxes for Lewis rats (n=8 per group) previously exposed to 0, 25%, 50%, 75% or

100% sucrose reward probability.

Figure 11. Mean beam breaks (± S.E.) across three 90 minute habituation session for Lewis rats

(n=8 per group) previously exposed to 0, 25%, 50%, 75% or 100% sucrose reward probability.

Figure 12. Mean beam breaks (± S.E.) after saline (1.0ml/kg, i.p.) and first amphetamine

challenge (0.5 mg/kg, i.p.) for Lewis rats (n=8 per group) previously exposed to 0, 25%, 50%,


Figure 13. Mean beam breaks (± S.E.) over the course of 5 amphetamine injections (1mg/kg,

i.p.) for Lewis rats (n=8 per group) previously exposed to 0, 25%, 50%, 75% or 100% sucrose

reward probability.

Figure 14. Time-course (5-min bins) of beam breaks (mean ± S.E.s) after second amphetamine

challenge (0.5 mg/kg, i.p.) for Lewis rats (n=8 per group) previously exposed to 0, 25%, 50%,



Figure 15 . Mean beam breaks (± S.E.) after the first and second amphetamine challenge for

Lewis rats (n=8 per group) previously exposed to 0, 25%, 50%, 75% or 100% sucrose reward

probability.


52

Experimental Design

Reward Probability Training Probability of reward delivery

0, 25%, 50%, 75%, 100% (n=8 per group) Day 1-21

Habituation to Locomotor Activity Boxes

Day 22-24

Vehicle Injection (1.0 ml/kg) Day 25

1st AMPH Challenge (0.5 mg/kg) Day 29

AMPH Sensitizing Regime (5 x 1.0 mg/kg)

Day 33-43

2nd AMPH Challenge (0.5mg/kg) Day 50


53

Training Sessions

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Mea

n N

ose

Poke

s

0

50

100

150

200025% 50% 75% 100%

A

Training Sessions

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Mea

n N

ose

Poke

s

0

50

100

150

200

025% 50% 75% 100%

B


Time (min)

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90

Mea

n B

eam

Bre

aks

0

100

200

300

400

500

600

025% 50% 75% 100%


Habituation Session

1 2 3

Mea

n B

eam

Bre

aks

0

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4000 0 25% 50% 75% 100%


Treatment

Vehicle AMPH (0.5 mg/kg)

Mea

n B

eam

Bre

aks

0

1000

2000

3000

4000

5000 0 25% 50% 75% 100%


Amphetamine Treament (1.0 mg/kg)

1 2 3 4 5

Mea

n B

eam

Bre

aks

0

2000

4000

6000

8000

10000

0 25% 50% 75% 100%


Time (min)

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90

Mea

n B

eam

Bre

aks

0

200

400

600

800

025% 50% 75% 100%


Amphetamine Challenge (0.5mg/kg)

1st 2nd

Mea

n B

eam

Bre

aks

0

2000

4000

6000

8000

10000

0 25% 50% 75% 100%


60

Training Session

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Mea

n N

ose

Pok

es

0

20

40

60

80

100

025% 50% 75% 100%

A

Training Session

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Mean

Nos

e P

oke

s

0

20

40

60

80

100

025% 50% 75% 100%

B


61

Time (min)

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90

Mea

n B

eam

Bre

aks

0

100

200

300

400

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600

025% 50% 75% 100%


Habituation

1 2 3

Mea

n B

eam

Bre

aks

0

1000

2000

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0 25% 50% 75% 100%


Treatment

Vehicle AMPH (0.5 mg/kg)

Mea

n B

eam

Bre

aks

0

1000

2000

3000

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5000

0 25% 50% 75% 100%


Amphetamine Treatment (1.0mg/kg)

1 2 3 4 5

Mea

n B

eam

Bre

aks

0

2000

4000

6000

8000

10000

0 25% 50% 75% 100%


Time (min)

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90

Mea

n B

eam

Bre

aks

0

200

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800

025% 50% 75% 100%


Amphetamine Challenge (0.5mg/kg)

1st 2nd

Mea

n B

eam

Bre

aks

0

2000

4000

6000

8000

100000 25% 50% 75% 100%

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