THE ROLE OF THE VENTRAL TEGMENTAL AREA AND NUCLEUS ACCUMBENS IN NICOTINE-INDUCED

ENHANCEMENT OF RESPONDING FOR CONDITIONED REINFORCEMENT

by

Rayane Tabbara

A thesis submitted in conformity with the requirements for the degree of Masters of Arts

Graduate Department of Psychology University of Toronto

© Copyright by Rayane Tabbara 2015

   

   

ii  

The role of the ventral tegmental area and nucleus accumbens in nicotine-induced enhancement of responding for conditioned

reinforcement

Rayane Tabbara

Masters of Arts

Department of Psychology University of Toronto

2015

Abstract Nicotine enhances the reinforcing properties of reward-paired cues but little is known

about neural mechanisms mediating this enhancement. We hypothesized that nicotine enhances

responding for conditioned reinforcement through nAChRs in the VTA but not the NAcc.

Thirsty male Long-Evans rats underwent Pavlovian conditioning sessions where a 5-sec CS was

paired with water. Next, responding for conditioned reinforcement was tested. The effects of

α4β2 nAChR antagonist DHβE infusion into the VTA or NAcc on nicotine-enhanced

responding for a CRf were assessed. The effects of nicotine infusion into the VTA or NAcc on

responding for a CRf were also examined. Results indicated that nicotine enhanced responding

for a CRf and this effect was attenuated following DHβE infusion into the VTA or NAcc.

Nicotine infused into the VTA, but not the NAcc, enhanced responding for a CRf. These data

suggest that nAChRs in the VTA primarily mediate the reinforcement enhancing actions of

nicotine.

   

   

iii  

Acknowledgments

This research project would not have been possible without the support of several

people. First and foremost, I would like to thank Dr. Paul Fletcher for allowing me to conduct

my master’s thesis project in his laboratory. Paul, your support, encouragement and guidance

have helped me tremendously throughout this year. Thank you for teaching me that research can

be flexible, that it is okay to make mistakes and, most importantly, that I can become an

independent researcher. I am looking forward for the next years of my PhD in your lab. A

special thank you to Zhaoxia Li who taught me all the surgical procedures required for this

thesis. Your patience and support have contributed to the success of these experiments. I would

also like to thank all members of the Fletcher lab for being a source of friendship and for their

encouragement. This research would not have been possible without support from Zoe Rizos

and Lori Dixon who guided me through the proper handling and care of laboratory animals.

Furthermore, I would like to express my appreciation to Dr. Suzanne Erb and Dr. Rutsuko Ito

for accepting to be part of my defense committee. I would also like to thank the Canadian

Institutes of Health Research (CIHR) for funding this research.

I would like to express my deepest gratitude to my parents. Mum and Dad, I know the

move to Toronto was hard for you to accept. However, you still supported me and you were

always there for me. Thank you for all the sacrifices that you have made and continue to make

for my education. I love you and I dedicate this thesis to you.

   

   

iv  

Table of Contents

1. Introduction 1 1.1. Acetylcholine receptors 1

1.1.1. Molecular structure of nicotinic receptors 2 1.1.2. Transition states of nicotinic receptors 3 1.1.3. Ligand binding sites on nicotinic receptors 3 1.1.4. Diversity of nicotinic receptors 4 1.1.5. Brain localization of α4β2 and α7 nicotinic receptors 5

1.2. The mesolimbic dopamine system and the reinforcing effects of drugs of abuse 5 1.2.1. The mesolimbic dopamine system and the reinforcing effects of 6 nicotine 1.2.2. The mesolimbic dopamine system and the distribution of nicotinic 6 receptors

1.3. The role of α4β2 and α7 nicotinic receptors in the ventral tegmental area and 8 nucleus accumbens in nicotine reinforcement

1.3.1. Nicotine-evoked stimulation of dopamine release in the nucleus 8 accumbens 1.3.2. Behavioural effects of nicotine 9

1.3.2.1. Nicotine-induced enhancement of locomotor activity 9 1.3.2.2. Nicotine self-administration 10 1.3.2.3. Nicotine-conditioned place preference 11

1.4. Conditioned responses to tobacco-related cues in humans 12 1.5. Brain activity responses to tobacco-related cues in humans 13 1.6. Reinforcement enhancing effects of nicotine in animal models 13

1.6.1. Reinforcement enhancing effects of nicotine and the mesolimbic 15 dopamine system

1.7. Aim of the present research 16 1.8. Specific hypotheses 17

2. Method 18 2.1. Subjects 18 2.2. Apparatus 18 2.3. Surgery 19 2.4. General procedure 20

2.4.1. Water deprivation 20 2.4.2. Pavlovian conditioning 20 2.4.3. Tests of responding for conditioned reinforcement 21

2.5. Experiment 1: effects of DHβE infusion into the ventral tegmental area on 21 nicotine-enhanced responding for conditioned reinforcement

2.6. Experiment 2: effects of DHβE infusion into the nucleus accumbens on 22 nicotine-enhanced responding for conditioned reinforcement

2.6.1. Experiment 2A: effects of DHβE infusion into an anterior site of the 22 nucleus accumbens on nicotine-enhanced responding for conditioned reinforcement

2.6.2. Experiment 2B: effects of DHβE infusion into a more posterior site 22

   

   

v  

of the nucleus accumbens on nicotine-enhanced responding for conditioned reinforcement

2.7. Experiment 3: effects of nicotine infusion into the ventral tegmental area on 23 responding for conditioned reinforcement

2.8. Experiment 4: effects of nicotine infusion into the nucleus accumbens on 23 responding for conditioned reinforcement

2.9. Drugs and microinfusion procedure 23 2.10. Histology 24 2.11. Statistical analyses 24

3. Results 26

3.1. Pavlovian conditioning 26 3.1.1. Latency to CS-elicited receptacle entry 26 3.1.2. Pre-CS and CS receptacle entries 26

3.2. Tests of responding for conditioned reinforcement 26 3.2.1. Experiment 1: effects of DHβE infusion into the ventral tegmental area 26 on nicotine-enhanced responding for conditioned reinforcement 3.2.2. Experiment 2: effects of DHβE infusion into the nucleus accumbens on 28 nicotine-enhanced responding for conditioned reinforcement

3.2.2.1. Experiment 2A: effects of DHβE infusion into an anterior 28 site of the nucleus accumbens on nicotine-enhanced responding for conditioned reinforcement 3.2.2.2. Experiment 2B: effects of DHβE infusion into a more 29 posterior site of the nucleus accumbens on nicotine-

enhanced responding for conditioned reinforcement 3.2.3. Experiment 3: effects of nicotine infusion into the ventral tegmental area 30 on responding for conditioned reinforcement 3.2.4. Experiment 4: effects of nicotine infusion into the nucleus accumbens on 31 responding for conditioned reinforcement

4. Discussion 32 4.1. Summary 32 4.2. Nicotine-induced enhancement of the reinforcing properties of reward-paired 32

cues 4.3. The role of the mesolimbic dopamine system in nicotine-enhanced responding 33

for conditioned reinforcement 4.4. The role of the nucleus accumbens in nicotine-enhanced responding for 34 conditioned reinforcement 4.5. The role of the ventral tegmental area in nicotine-enhanced responding for 37

conditioned reinforcement 4.6. Possible mechanisms 38 4.7. The role of α4β2 nicotinic receptors in nicotine-enhanced responding for 39

conditioned reinforcement 4.8. The role of the cholinergic system in the reinforcing properties of reward-paired 40

cues 4.9. Future directions 40 4.10. Summary and conclusion 42

   

   

vi  

5. References 44

6. Table captions 65

7. Tables 66

8. Figure Captions 69

9. Figures 73

   

   

vii  

List of Tables

Table 1. Distribution of binding sites for α4β2 and α7 nAChR subunits in the rat brain.

   

   

viii  

List of Figures

Figure 1. Mean latency to CS-elicited receptacle entry across 12 sessions of Pavlovian

conditioning.

Figure 2. Mean receptacle entries during periods of pre-CS or CS presentation across 12

sessions of Pavlovian conditioning.

Figure 3. Effects of DHβE infusion into the VTA on nicotine-enhanced responding for a CRf.

Figure 4. Location of injector tips within the VTA for experiment 1.

Figure 5. Effects of DHβE infusion outside of the VTA on nicotine-enhanced responding for a

CRf.

Figure 6. Location of injector tips outside of the VTA for experiment 1.

Figure 7. Effects of DHβE infusion into an anterior site of the NAcc on nicotine-enhanced

responding for a CRf.

Figure 8. Location of injector tips within the NAcc for experiment 2A.

Figure 9. Effects of DHβE infusion into a more posterior site of the NAcc on nicotine-enhanced

responding for a CRf.

Figure 10. Location of injector tips within the NAcc for experiment 2B.

Figure 11. Effects of nicotine infusion into the VTA on responding for a CRf.

Figure 12. Location of injector tips within the VTA for experiment 3.

Figure 13. Effects of nicotine infusion outside of the VTA on responding for a CRf.

Figure 14. Location of injector tips outside of the VTA for experiment 3.

Figure 15. Effects of nicotine infusion into the NAcc on responding for a CRf.

Figure 16. Location of injector tips within the NAcc for experiment 4.

   

  1

1. Introduction

Tobacco smoke, mostly consumed in the form of cigarettes (World Health Organization

[WHO], 2006), is associated with adverse health outcomes leading to an estimated yearly death

rate of 443 000 individuals in the USA (Centers for Disease Control and Prevention [CDC],

2008). Exposure to tobacco smoke leads to an increased risk and severity of lung disease,

cardiovascular disease, diabetes, cancer and other health issues (U. S. Department of Health and

Human Services [USDHHS], 2004). Nicotine, the most abundant alkaloid chemical in tobacco

leaf and the main psychoactive component in tobacco smoke, possesses highly addictive

properties, which contribute to the persistence in using tobacco products (USDHHS, 2010). A

national health interview survey conducted in the USA over a 10-year period by CDC (2011)

revealed that only 6.2% of Americans had successfully quit smoking within the past year of

taking the survey. One factor that promotes further tobacco smoking and contributes to nicotine

dependence is the reinforcement actions of nicotine. Experimental evidence suggests that

nicotine enhances the reinforcing efficacy of reinforcing stimuli in the environment, resulting in

greater reward-seeking behaviours (Caggiula et al., 2002; Chaudhri et al., 2006; Guy &

Fletcher, 2014a; Olausson, Jentsch, & Taylor, 2004a, 2004b). From a treatment perspective, a

comprehensive understanding of how nicotine impacts behaviour would be helpful in

developing therapeutic drugs that treat nicotine dependence. The experimental work described

in this thesis investigated neural mechanisms that mediate the enhancement effects produced by

nicotine on the reinforcing properties of reward-paired cues. Specifically, it examined the role of

the ventral tegmental area (VTA) and nucleus accumbens (NAcc) in mediating the

reinforcement-enhancing effects of nicotine.

1.1. Acetylcholine receptors

   

 

2

Acetylcholine receptors are activated by the neurotransmitter acetylcholine and can be

classified into metabotropic muscarinic receptors and ionotropic neuronal nicotinic receptors.

The metabotropic receptors are second messenger G-protein coupled receptors that are found at

the skeletal neuromuscular junction, whereas the neuronal receptors are ligand-gated ion

channels that are found throughout the peripheral and central nervous system (Albuquerque,

Pereira, Alkondon, & Rogers, 2009; Hogg, Raggenbass, & Bertrand, 2003). The neuronal

nicotinic acetylcholine receptors (nAChRs), which are activated by acetylcholine or by nAChR

agonists (Gotti, Zoli, & Clementi, 2006), have been heavily implicated in the reinforcing

properties of nicotine. Therefore, these receptors, rather than the metabotropic muscarinic

receptors, were the main focus of this thesis.

1.1.1. Molecular structure of nicotinic receptors

Neuronal nAChRs belong to the cys-loop family of ligand-gated ion channels that also

includes gamma aminobutyric acid (GABAA, GABAC), glycine and 5-hydroxytryptamine

receptors (5-HT3) (Changeux & Edelstein, 1998; Le Novère & Changeux, 1995). They are

preferentially located at presynaptic sites where they regulate neurotransmitter release, including

acetylcholine (Wilkie, Hutson, Stephens, Whiting, & Wonnacott, 1993), noradrenaline (Clarke

& Reuben, 1996; Snell & Johnson, 1989), dopamine (DA) (Grady, Marks, Wonnacott, &

Collins, 1992; Rapier, Lunt, & Wonnacott, 1990), glutamate (Gray, Rajan, Radcliffe, Yakehiro,

& Dani, 1996) and GABA (Alkondon, Pereira, Barbosa, & Albuquerque, 1997).

The purification of the nAChR protein from the nAChR-rich tissue of the electric organ

of Torpedo californica has allowed for the characterization of these receptors (Raftery,

Hunkapiller, Strader, & Hood, 1980). Five distinct types of subunits have been identified and

labeled as α, β, γ, δ and ε (Albuquerque et al., 2009). Muscarinic acetylcholine receptors are

composed of any of these five subunits, whereas neuronal nAChRs are composed of α and/or β

   

   

3

subunits (Hogg et al., 2003). All nAChRs form pentameric subunit combinations that span the

lipid bilayer to form a water-filled pore permeable to cations (Wonnacott, 2014). Each subunit

of this pentameric consists of 1) an extracellular N-terminal that participates in the formation of

the ligand-binding domain; 2) transmembrane regions that consist of four hydrophobic

membrane spanning segments (M1-M4), among which the M2 segment lines the ion channel; 3)

an intracellular loop between M3 and M4 that has been implicated in the transduction of agonist

binding into channel opening (Bouzat, 2012; Sine & Engel, 2006).

1.1.2. Transition states of nicotinic receptors

nAChR subunits can exist in four interconvertible conformational states, as reviewed by

Changeux and colleagues (1998). 1) At the resting state, the ion channel of nAChRs is closed

and the ligand-binding sites are unoccupied. 2) Upon agonists’ binding, the channel undergoes

an allosteric change from the resting state to the open state, which promotes an influx of Na+

and Ca2+ and an efflux of K+. In the active state, the nAChR binds agonists with low affinity. 3)

Despite the presence of agonists, the nAChR channel closes within sec to min and enters the

desensitized state. In this state, the nAChR is refractory to activation but displays higher affinity

for agonists relative to the open state. 4) The channel is then in the inactive state, which is also

referred to as the long-lasting desensitized state. The removal of agonists induces the channel to

return to the resting state.

1.1.3. Ligand binding sites on nicotinic receptors

Each nAChR carries binding sites for agonists, competitive and non-competitive

antagonists and allosteric modulators. Wonnacott (2014) describes the binding of these ligands

to their corresponding nAChR sites. For example, agonists, such as nicotine and cytisine, bind at

subunit interfaces in the extracellular domain, which leads to receptor activation. Competitive

antagonists, such as α-Bungarotoxin (α-Bgt), prevent access to agonists by binding at the

   

   

4

agonist binding sites. Upon antagonist binding, the channel remains closed and access to the

agonist binding sites is blocked. In contrast, non-competitive antagonists, such as

mecamylamine, bind at sites that are distinct from the agonist binding sites to prevent receptor

activation. Finally, allosteric modulators, such as desformylflustrabomine, are non-competitive

ligands that bind to distinct sites and positively or negatively influence nAChR function.

Although nAChR subunits have common structural features and similar patterns of activation

and conduction, they possess different physiological characteristics and exhibit distinct regional

expression.

1.1.4. Diversity of nicotinic receptors

Twelve genes coding for nAChR subunits that form functional receptors when expressed

in Xenopus laevis oocytes have been identified (Lindstrom, 1998). These genes are classified

into two subfamilies of nine alpha (α2-α10) and three beta (β2-β4) subunits (Hurst, Rollema, &

Bertrand, 2013; Millar & Gotti, 2009). Genes that code for subunits that include two adjacent

cysteines in the extracellular domain are termed α subunits, whereas genes that code for

subunits that lack the pair of adjacent cysteines are designated β subunits (Changeux &

Edelstein, 1998).

The mammalian brain expresses nine of the 12 nAChR subunit genes (α2-α7, β2-β4)

(Heinemann et al., 1991; Lindstrom, 1998). The remaining subunits (α8-α10) are not expressed

in the mammalian brain but can be found, for example, in rat cochlear hair cells or chick retina

(Elgoyhen, Johnson, Boulter, Vetter, & Heinemann, 1994; Keyser et al., 1993). The 12 nAChRs

can be divided into two classes based on their subunit composition. Heteromeric receptors are

comprised of α and β subunits and include two pairwise combinations of α2, α3, α4 or α6 with

β2 or β4 subunits (e.g. α4β2). The α5 and β3 subunits can also co-exist with other α and β

subunits (e.g. α4α5β2). In contrast, homomeric receptors are made up solely of α7, α8 or α9

   

   

5

subunits. These subunits can also combine with a second α subunit to form heteromeric

receptors (e.g. α7α8) (Alexander, Mathie, & Peters, 2008; Gotti & Clementi, 2004). The

remaining α10 subunit must co-assemble with an α9 subunit to form functional heteromeric

receptors (Elgoyhen et al., 2001; Vetter et al., 2007).

1.1.5. Brain localization of α4β2 and α7 nicotinic receptors

The mammalian brain possesses a heterogeneous distribution of α2-α7 and β2-β4

nAChR subunits. Among the various subunits, the heteromeric α4β2 are the most common in

the brain and the homomeric α7 are the next most common (Collins, Salminen, Marks,

Whiteaker, & Grady, 2009; Gotti et al., 2006; Sharma & Brody, 2009). These nAChR subunits

are widespread throughout the brain and their binding sites have been quantified, characterized

and localized using different radioactive nAChR agonists and antagonists (Clarke, Pert C, &

Pert A., 1984; Clarke, Schwartz, Paul, Pert C., & Pert,A., 1985; Hunt & Schmidt, 1978; Segal,

Dudai, & Amsterdam, 1978; Tribollet, Bertrand, Marguerat, & Raggenbass, 2004; Whiteaker et

al., 2000). Table 1 provides a comparison between the binding distribution of α4β2 and α7

subunits, which were identified using [3H]-nicotine and [125I]-α-Bgt, respectively. [3H]-nicotine

binding is detected in most brain regions, particularly in the thalamus, cortex and basal ganglia,

including the VTA and the NAcc, and at low levels in the hippocampus. [125I]-α-Bgt binding is

also detected throughout the brain, particularly in the hippocampus and cortex, and at low levels

in thalamic regions and the basal ganglia.

1.2. The mesolimbic dopamine system and the reinforcing effects of drugs of abuse

Drug addiction, also referred to as substance use disorder, is a chronically relapsing

disorder characterized by compulsive drug use, an inability to limit drug intake and persistence

in drug use despite harmful consequences (National Institute on Drug Abuse [NIDA]). Abused

drugs act as reinforcers that increase the probability of drug taking. Several major classes of

   

   

6

abused drugs exist and these include stimulants, such as amphetamine and nicotine, opiates,

such as morphine and heroin, cannabinoids, such as marijuana, and depressants, such as alcohol

(Pierce & Kumaresan, 2006). Although these classes of drugs of abuse have different primary

neurochemical actions, they produce their reinforcing effects through activation of the

mesolimbic system (Pierce & Kumaresan, 2006). This system consists of DAergic neurons

originating in the VTA and their axonal projections to the prefrontal cortex (PFC) and other

limbic structures, such as the NAcc. Overwhelming evidence indicates that increased DA

transmission within the mesolimbic pathway contributes, at least partially, to the reinforcing

properties of drugs of abuse (Koob, 1992; Wise, 2004; Wise & Bozarth, 1987).

1.2.1. The mesolimbic dopamine system and the reinforcing effects of nicotine

The mesolimbic DA system has been extensively implicated in the reinforcing effects of

nicotine (Balfour, 2004; Corrigall, Franklin, Coen, & Clarke, 1992; Nisell, Nomikos, &

Svensson, 1994a, 1994; Pierce & Kumaresan, 2006). For example, DA levels in the NAcc are

elevated following systemic nicotine administration (Benwell, Balfour, & Lucchi, 1993;

Damsma, Day, & Fibiger, 1989; Nisell et al., 1994b) or local infusion of nicotine into the VTA

(Nisell et al., 1994a, 1994b; Ferrari, Le Novère, Picciotto, Changeux, & Zoli, 2002) or NAcc

(Ferrari et al., 2002; Mifsud, Hernandez, & Hoebel, 1989; Nisell et al., 1994a, 1994b).

Furthermore, depletion of DA through intra-NAcc infusion of the selective catecholaminergic

neurotoxin 6-hydroxydopamine (6-OHDA) reduces nicotine self-administration (Corrigall et al.,

1992). Antagonism of D1 or D2 DA receptors also attenuates nicotine self-administration

(Corrigall & Coen, 1991). Together, these findings indicate that DA transmission within the

mesolimbic DA system plays a role in nicotine reinforcement.

1.2.2. The mesolimbic dopamine system and the distribution of nicotinic receptors

   

   

7

Nicotine crosses the blood-brain barrier and binds to nAChRs expressed on several

neurons in the mesolimbic DA system (Clarke & Pert A., 1985; Deutch, Holliday, Roth, Chun,

& Hawrot, 1987; Imperato, Mulas, & Di Chiara, 1986). In particular, nicotine binds to nAChRs

present on VTA neurons resulting in a sustained increase in DA release in the NAcc, which

contributes to the development of nicotine dependence (Balfour, 2004; Pidoplichko et al., 2004).

Within the VTA, nAChRs are found on DAergic cell bodies expressing α4α5β2, α4α5α6β2 and

α7 subunits and GABAergic cell bodies expressing α4α5β2 and α7 subunits (Changeux, 2010;

Klink, de Kerchove d’Exaerde, Zoli, & Changeux, 2001; Wonnacott, Sidhpura, & Balfour,

2005). These DAergic neurons receive two main types of excitatory inputs and one main type of

inhibitory inputs (Changeux, 2010). First, the DAergic and GABAergic neurons in the VTA

receive cholinergic innervation from the pedunculopontine tegmental nucleus (PPTg) and the

adjacent laterodorsal tegmental nucleus (LDTg) expressing α4β2 subunits at presynaptic

terminals (Bolam, Francis, & Henderson, 1991; Changeux, 2010; Wonnacott et al., 2005).

Second, the DAergic and GABAergic neurons in the VTA receive glutamatergic innervation

from the PFC and PPTg with α7 subunits expressed on presynaptic terminals (Changeux, 2010;

Jones & Wonnacott, 2004; Mansvelder, Keath, & McGehee, 2002; Picciotto, 2003; Wonnacott

et al., 2005). Third, in addition to the inhibitory GABAergic interneurons located within the

VTA, this area receives GABAergic innervation expressing α4β2 subunits at presynaptic

terminals (Changeux, 2010; Lu et al., 1998; Mansvelder et al., 2002; Walaas & Fonnum, 1980).

Furthermore, DAergic neurons within the VTA send axonal projections to the NAcc expressing

nAChRs with α4β2, α4α6β2β3, α4α5β2 and α6β2β3 subunits at presynaptic terminals

(Changeux, 2010; Luetje, 2004; Picciotto, 2003; Wonnacott et al., 2005; Zoli et al., 2002).

nAChRs in the NAcc are also found on glutamatergic and GABAergic presynaptic terminals

expressing α4β2 subunits (Lu et al., 1998; Pierce & Kumaresan, 2006; Wonnacott et al., 2005).

   

   

8

Presynaptic α7 nAChRs have also been inferred to reside on glutamatergic afferents projecting

to the NAcc (Wonnacott et al., 2005; Wonnacott, Kaiser, Mogg, Soliakov, & Jones, 2000).

1.3. The role of α4β2 and α7 nicotinic receptors in the ventral tegmental area and nucleus

accumbens in nicotine reinforcement

Enhanced DA transmission within the NAcc contributes to the reinforcing properties of

nicotine (Balfour, 2004; Pidoplichko et al., 2004). This increase in accumbal DA release is

produced following infusion of nicotine into the VTA or NAcc (Ferrari et al., 2002), suggesting

that these two regions could modulate nicotine reinforcement. However, an overwhelming body

of evidence has shown that nicotine-induced DA release following stimulation of nAChRs in the

VTA, rather than the NAcc, contributes to a greater extent to nicotine reinforcement (Nisell et

al., 1994a, 1994b; Nisell, Marcus, Nomikos, & Svensson, 1997). The following paragraphs will

review the role of nAChRs in the VTA and NAcc in nicotine reinforcement, with a main

emphasis on the high affinity α4β2 and α7 subunits.

1.3.1. Nicotine-evoked stimulation of dopamine release in the nucleus accumbens

Systemic nicotine administration or direct continuous nicotine infusion into the VTA or

NAcc produces a dose-dependent increase in extracellular DA concentration in the NAcc

(Imperato et al., 1986; Mifsud et al., 1989; Nisell et al., 1997). However, direct continuous intra-

VTA nicotine infusion produces a longer lasting increase in accumbal DA release compared to

intra-NAcc nicotine infusion (Nisell et al., 1994a). In addition, intra-VTA infusion of the non-

selective nicotinic antagonist mecamylamine blocks the nicotine-induced accumbal increase in

DA release following systemic nicotine administration, whereas intra-NAcc mecamylamine

infusion does not (Nisell et al., 1994b). Looking at the type of nAChRs involved in the nicotine-

induced increase in accumbal DA levels, this increase is abolished in mice lacking α4 (Marubio

et al., 2003) or β2 subunits (Picciotto et al., 1998). This increase is still observed in mice lacking

   

   

9

α7 subunits; however, it is sustained for a longer period of time relative to wild type mice that

do not lack these subunits (Besson et al., 2012). Overall, these results suggest that nAChRs in

the VTA contribute to a greater extent to the nicotine-induced accumbal DA increase, compared

to nAChRs in the NAcc.

1.3.2. Behavioural effects of nicotine

The actions of nicotine to increase accumbal DA release play a role in the behavioural

effects of nicotine. These behavioural effects can be examined in laboratory rodents while

assessing locomotor activity, self-administration behaviour and conditioned place preference

(CPP).

1.3.2.1. Nicotine-induced enhancement of locomotor activity

Systemic nicotine administration enhances locomotor activity and this effect results from

nicotine-induced activation of the mesolimbic DA system (Clarke & Kumar, 1983a). Indeed,

depletion of accumbal DA through 6-OHDA lesions abolishes the locomotor stimulant actions

of nicotine (Clarke, Fu, Jakubovic, & Fibiger, 1988). The NAcc was later shown to not directly

mediate the locomotor stimulant actions of nicotine given that intra-NAcc nicotine infusion

failed to potentiate locomotor activity (Reavill & Stolerman, 1990; Welzl, Bättig, & Berz,

1990). However, intra-VTA infusion of nicotine or the nicotinic agonist cytisine potentiated

locomotor activity, an effect that was blocked by systemic mecamylamine administration

(Museo & Wise, 1990a, 1990b; Reavill & Stolerman, 1990). Furthermore, the locomotor

stimulant actions of nicotine are mediated by α4β2 but not α7 nAChRs. For example, injection

of the α4β2 nicotinic agonist ligand SIB 1765F increases locomotor activity, whereas injection

of the α7 nicotinic agonist ligand AR-R 17779 fails to increase activity (Grottick et al., 2000).

Moreover, the competitive α4β2 nAChR antagonist Dihydro-β-Erythroidine (DHβE) reduces the

nicotine-induced enhancement of locomotor activity, whereas the competitive α7 nAChR

   

   

10

antagonist methyllycaconitine (MLA) does not. These findings first suggest that nicotine

enhances locomotor activity by acting on nAChRs in the VTA, rather than those in the NAcc.

These findings also indicate a role for α4β2, but not α7, nAChRs in underlying the locomotor

stimulant actions of nicotine.

1.3.2.2. Nicotine self-administration

The role of the VTA in nicotine reinforcement is further suggested by intravenous drug

self-administration studies where the number of infusions received for a self-administered drug

provides a measure of its reinforcing properties (Koob & Le Moal, 2006; Panlilio & Goldberg,

2007). The mesolimbic DA system was first implicated in nicotine self-administration following

the finding that infusion of 6-OHDA into the NAcc reduces this behaviour (Corrigall et al.,

1992). The NAcc was later shown to not directly mediate nicotine self-administration given that

intra-NAcc DHβE infusion had no impact on this behaviour (Corrigall, Coen, & Adamson,

1994). However, intra-VTA DHβE infusion significantly reduced nicotine self-administration

(Corrigall et al., 1994). Further evidence implicating the VTA in the reinforcing effects of

nicotine came from findings that rats self-administer nicotine into the VTA (Ikemoto, Qin, &

Liu, 2006). This behaviour is also blocked by co-administration of mecamylamine into the VTA

(Ikemoto et al., 2006). Looking at the type of nAChR subunits mediating nicotine self-

administration, Grottick and colleagues (2000) demonstrated that systemic pretreatment with the

α4β2 nAChR antagonist DHβE significantly reduced nicotine self-administration. However,

pretreatment with the α7 nAChR antagonist MLA did not affect the number of self-administered

nicotine infusions. These data are consistent with other reports demonstrating reduced

propensity for nicotine self-administration in β2-knockout mice (Epping-Jordan, Picciotto,

Changeux, & Pich, 1999; Léna & Changeux, 1999). Overall, these results suggest that self-

   

   

11

administered nicotine interacts with the mesolimbic DA system through stimulation of α4β2

nAChRs in the VTA.

1.3.2.3. Nicotine-conditioned place preference

The CPP procedure has been used to assess the rewarding properties of a variety of drugs

of abuse, including those pertaining to nicotine (Koob & Le Moal, 2006; Fudala & Iwamoto,

1986; Fudala, Teoh, & Iwamoto, 1985; Shoaib, Stolerman, & Kumar, 1994). In this procedure,

subjects are repeatedly exposed to two different environments, one of which is paired in time

and space with the pharmacological effects of a drug, such as nicotine. At test, subjects are

given access to both environments and the amount of time spent in each environment is

recorded. Animals exhibit a CPP if they spend more time in the environment where they had

received a rewarding drug injection compared to the environment where they had received a

saline injection. In contrast, animals exhibit a conditioned place aversion if they spend more

time in the saline-paired environment compared to the drug-paired environment. The preference

for the drug-paired environment over the saline-paired environment provides a measure of the

rewarding value of the drug (Koob & Le Moal, 2006; Prus, James, & Rosecrans, 2009).

Several lines of evidence implicate the mesolimbic DA system in the rewarding

properties of nicotine as assessed using the CPP procedure (Laviolette & van der Kooy, 2003a;

Spina, Fenu, Longoni, Rivas, & Di Chiara, 2006). Indeed, disruption of mesolimbic DA

transmission following administration of DA antagonists inhibits the acquisition of nicotine-

CPP (Spina et al., 2006). These rewarding effects of nicotine are modulated through the VTA

given that infusion of nicotine (Tan, Bishop, Lauzon, Sun, & Laviolette, 2009) or the nicotinic

agonist cytisine (Museo & Wise, 1994) into this region results in CPP to the nicotine-paired

environment. In addition, administration of DHβE along with nicotine into the VTA inhibits the

acquisition of nicotine-CPP (Laviolette & van der Kooy, 2003b). To our knowledge, no study

   

   

12

has investigated the effects of administering a nAChR agonist or antagonist into the NAcc on

nicotine-induced CPP. In order to examine the possible role of α4β2 and α7 nAChRs in the

rewarding effects of nicotine, Laviolette and van der Kooy (2003b) co-infused DHβE or MLA

with nicotine into the VTA. While DHβE blocked CPP produced by intra-VTA nicotine

infusion, MLA switched the motivational valence of nicotine from rewarding to aversive.

Overall, these findings first indicate that the VTA is a critical neural substrate for the mediation

of nicotine’s rewarding properties. These findings also suggest a functional dissociation

between α4β2 and α7 nAChRs in mediating the rewarding value of nicotine.

1.4. Conditioned responses to tobacco-related cues in humans

The associative processes that occur and develop with repeated tobacco use play an

important role in maintaining smoking (Caggiula et al., 2002; Chaudhri et al., 2006;

Chiamulera, 2005; Niaura et al., 1988). For instance, environmental stimuli that have been

previously paired with tobacco use, such as the sight of an ashtray, can evoke subjective and

physiological responses indicative of craving, which may in turn elicit a strong urge to smoke

(Carter & Tiffany, 1999; Payne, Schare, Levis, & Colletti, 1991; Rose & Levin, 1991). A link

between environmental stimuli associated with tobacco use and craving has already been

established (Lazev, Herzog, & Brandon, 1999; Lee et al., 2004; Mucha, Geier, & Pauli, 1999;

Payne et al., 1991; Rose & Levin, 1991). For example, Lazev and colleagues (1999) exposed

tobacco users to two sets of discrete stimuli. One set consisting of blue light, flute music and a

citrus scent was designated as the conditioned stimulus (CS+), whereas a second set consisting

of red light, string music and a cinnamon scent was designated as the CS-. Participants were

asked to smoke a cigarette that was available to them in the experimental room upon exposure

to the CS+. However, they were asked to refrain from smoking upon exposure to the CS-. For

each CS+ or CS- trial, reactivity to each set of discrete cues was measured by a self-report of

   

   

13

urge to smoke and pulse rate. Results indicated that participants’ self-report of urge to smoke

and pulse rate increased across CS+ trials. Importantly, these responses were higher upon

exposure to the CS+ compared to the CS-. These findings suggest that initially neutral

environmental stimuli, by virtue of being repeatedly paired with the pharmacological effects of

nicotine, can become CSs that elicit physiological conditioned responses.

1.5. Brain activity responses to tobacco-related cues in humans

Tobacco-associated cues not only elicit craving and drive continued tobacco use, but

also induce discrete patterns of brain activation in tobacco users (Brody, 2006; Due, Huettel,

Hall, & Rubin, 2002). For example, in one study conducted by Due and colleagues (2002),

nicotine-deprived smokers and nonsmokers were placed into a functional magnetic resonance

imaging scanner and were exposed to smoking-related images and neutral nonsmoking images.

Nicotine-deprived smokers consisted of nicotine-dependent smokers that abstained from

smoking for 10 hr prior to the scanning session. Results indicated that areas of the mesolimbic

DA system, including the VTA and posterior amygdala, and areas implicated in visuospatial

attention, including the PFC and parietal cortex, were activated following exposure to smoking-

related images. However, these areas were not activated following exposure to neutral

nonsmoking images. Importantly, these regions showed greater activation in nicotine-deprived

smokers than in nonsmokers. These results suggest that tobacco-related images can elicit

different patterns of brain activation in smokers compared to nonsmokers.

1.6. Reinforcement enhancing effects of nicotine in animal models

Evidence from studies using tobacco smokers has shown that environmental stimuli

associated with tobacco use can motivate tobacco smoking and contribute to nicotine

dependence. Studies using laboratory animals have also demonstrated that nicotine can, non-

associatively, enhance responding that is maintained solely by presentations of an

   

   

14

environmental cue previously paired with reward (Guy & Fletcher, 2013, 2014a, 2014b;

Mackintosh, 1974; Olausson et al., 2004a, 2004b; Palmatier et al., 2007; Quick, Olausson,

Addy, & Taylor, 2014). This enhanced responding suggests that nicotine potentiates the

conditioned reinforcing properties of reward-paired cues.

The reinforcement enhancing actions of nicotine can be examined using a test of

responding for conditioned reinforcement (Mackintosh, 1974). In an initial Pavlovian

conditioning phase of this test, animals are repeatedly exposed to pairings of a CS, such as a

tone, with the delivery of an unconditioned stimulus (US), such as sucrose. In the next phase,

subjects are allowed to make an operant response, such as a lever press, to obtain the CS. For

example, two novel levers are introduced into the conditioning chamber and responding on the

lever termed the CR lever results in presentations of the same CS used during Pavlovian

conditioning but no US is delivered, whereas responding on the lever termed the NCR lever has

no programmed consequences (Mackintosh, 1974). The rate of responding on the CR lever

compared to the NCR lever provides a measure of the conditioned reinforcing properties of the

CS (Mackintosh, 1974; Williams, 1994).

Olausson and colleagues (2004a) were the first to investigate the effects of nicotine on

responding for conditioned reinforcement. In their study, subjects received several Pavlovian

conditioning sessions where a light-tone CS was paired with water delivery. Rats were then

divided into two groups prior to being tested for responding for conditioned reinforcement. One

group received a systemic saline injection, whereas another group received a systemic nicotine

injection. Results indicated that responding on the CR lever was greater than responding on the

NCR lever for saline- and nicotine pre-treated rats, suggesting that the CS acquired conditioned

reinforcing properties. Responding on the CR lever was greater for nicotine than saline pre-

treated rats, suggesting that nicotine potentiated responding for conditioned reinforcement.

   

   

15

Among the different nAChRs, nicotine binds to α4β2 subunits to produce its

reinforcement enhancing effects. For example, in one study by Brunzell and colleagues (2006),

wild type and β2-knockout mice were first exposed to either nicotine or saccharin in their

drinking water for several days. Following exposure to these drinking solutions, all mice

received paired presentations of a light-tone CS with water delivery. Next, animals received

tests of responding for conditioned reinforcement. Results indicated that responding in the CR

nose-poke port was greater than responding on the NCR nose poke-port for all mice, suggesting

that the CS acquired conditioned reinforcing properties. Responding in the CR nose-poke port

was also greater for nicotine- than saccharine-exposed wild type mice, suggesting that prior

nicotine exposure enhanced responding for a conditioned reinforcer (CRf). Importantly,

nicotine-exposed β2-knockout mice had similar levels of responding on the CR nose poke hole

compared to saccharin-exposed β2-knockout mice, indicating that β2-subunit-containing

nAChRs are necessary for nicotine-induced enhancement of responding for a CRf. Guy and

Fletcher (2013) replicated these findings with the α4β2 nAChR antagonist DHβE. In their study,

systemic administration of DHβE, but not the α7 nAChR antagonist MLA, blocked nicotine-

enhanced responding for a CRf. Taken together, these data first suggest that a CS can reinforce

behaviour on its own after repeated association with the delivery of a reward. These data also

indicate that nicotine can potentiate the conditioned reinforcing properties of this reward-paired

CS by stimulating α4β2 nAChRs.

1.6.1. Reinforcement enhancing effects of nicotine and the mesolimbic dopamine system

The ability of nicotine to enhance responding for conditioned reinforcement is dependent

upon the mesolimbic DA system. For example, in one study by Guy and Fletcher (2014b), rats

were given Pavlovian conditioning sessions where a light-tone CS was paired with water

delivery. All rats received a systemic injection of nicotine prior to these sessions. Following

   

   

16

training, animals were separated into two groups before being tested for responding for

conditioned reinforcement. One group received a systemic injection of the D1 receptor

antagonist SCH 23390, whereas the other group received a systemic injection of the D2 receptor

antagonist eticlopride. All rats received an additional nicotine injection following administration

of the DA receptor antagonists. Results indicated that SCH 23390 and eticlopride blocked the

effects of nicotine to enhance responding for a CRf, suggesting a role for DA in the

reinforcement enhancing effects of nicotine.

As mentioned previously, the mesolimbic DA system plays a role in the reinforcing

effects of nicotine. Stimulation of nAChRs directly in the VTA, but not the NAcc, is the primary

mechanism underlying nicotine-induced increase in accumbal DA release and nicotine effects

on locomotor activity, self-administration behaviour and CPP. Despite the established role of

DA in the reinforcement enhancement effects of nicotine (Guy & Fletcher, 2014b), no study has

investigated whether stimulation of nAChRs directly in the VTA, rather than the NAcc,

contributes to these effects.

1.7. Aim of the present research

Experiments in this thesis investigated neural mechanisms mediating nicotine-induced

enhancement of responding for conditioned reinforcement. Specifically, they examined whether

nicotine enhances responding for a CRf by binding to nAChRs located in the VTA and/or NAcc

using two complementary approaches. One approach consisted of attempting to attenuate

nicotine-induced enhancement of responding for a CRf by infusing DHβE directly into the VTA

or NAcc. A second approach consisted of infusing various doses of nicotine directly into the

VTA or NAcc in an attempt to replicate the effects of systemic nicotine administration on

responding for a CRf. The nAChR antagonist DHβE, and not MLA, was chosen because α4β2,

but not α7, nAChRs have been primarily implicated in the behavioural effects of nicotine.

   

   

17

1.8. Specific hypotheses

Experiments in this thesis were conducted to test the hypothesis that nicotine binds to

α4β2 nAChRs in the VTA, and not the NAcc, to enhance responding for a CRf. Four predictions

were made based on this hypothesis:

1) DHβE infused into the VTA should attenuate nicotine-enhanced responding for a CRf.

2) DHβE infused into the NAcc should not attenuate nicotine-enhanced responding for a CRf.

3) Nicotine infused into the VTA should dose-dependently enhance responding for a CRf.

4) Nicotine infused into the NAcc should not dose-dependently enhance responding for a CRf.

   

  18

2. Method

2.1. Subjects

Male Long-Evans rats (Charles River, QC) weighing 250-275 g upon arrival were

housed in a temperature (22 ± 1 °C) and humidity-controlled (~ 50-60%) colony room on a 12

hr light/dark cycle (lights on at 8:00 a.m.). All procedures were conducted during the light

phase. Rats were initially housed in pairs and then single-housed prior to surgery. Food and

water were available ad libitum, except as described below. All procedures were in accordance

with guidelines from the Canadian Council on Animal Care and approved by the Centre for

Addiction and Mental Health Animal Care Committee.

2.2. Apparatus

Behavioural procedures were conducted in operant conditioning chambers (Med

Associates, St. Albans, VT) that were located in a different room from the animal colony. Each

chamber was contained within a ventilated, sound-attenuating cubicle and comprised of a clear

polycarbonate ceiling, front and back wall and stainless steel sidewalls. The floors were made of

metal bars that extended from rear to front. The right wall featured a recessed water receptacle

positioned 3 cm from the floor of the chamber and through which water was delivered by a

solenoid operated dispenser (Med Associates, St. Albans, VT). The right wall also contained

two retractable levers, each positioned 6.5 cm on either side of the receptacle. Located above

each lever was a red stimulus light. The upper left wall featured a white house light and a

Sonalert tone generator (2.9 kHz, 80-85 dB, Med Associates, St. Albans, VT). Entries into the

water receptacle were detected by interruptions of an infrared beam (Med Associates, St.

Albans, VT) located across the entrance of the receptacle. Water delivery and stimulus

presentations were controlled by a PC computer and Med PC IV software (Med Associates, St.

Albans, VT), which also recorded receptacle entries and responses on each lever.

   

   

19

2.3. Surgery

Rats were anaesthetized with isoflurane in an induction chamber and placed in a

stereotaxic frame. Anesthesia was maintained via a mask mounted on the incision bar (1-3%

isoflurane, 1 l/min O2). The animal's head was cleaned with 70% EtOH followed by betadine

and tear gel was applied onto the rat's eyes. The local anesthetic Marcaine (0.125 %, 0.1 ml) was

injected subcutaneously (s.c.) at the area where the incision was to be made. Rats then received

injections of the analgesic agent Anafen (5 mg/kg; s.c.), the antibiotic Derapen (0.1 ml; s.c.) and

saline (3 ml; s.c.). Next, an incision was made through the skin using a sterile scalpel to expose

the skull. Three holes were drilled for skull screws. Two small holes were drilled at the

appropriate stereotaxic coordinates corresponding to the VTA or NAcc. Bilateral stainless-steel

guide cannulae (22 gauge; Plastics One, QC) were then implanted aimed at the VTA or NAcc.

The following coordinates were used relative to bregma (mm): for the VTA with cannulae

positioned at a 10° angle and at 1 mm above the intended injection site, anteroposterior (AP)

- 5.0, lateral (L) ± 2.3 and from the dural surface, ventral (V) - 8.0; for the NAcc with cannulae

positioned 2 mm above the intended injection site, AP + 1.7, L ± 1.5 and V - 5.0. After

histological examination, cannulae placements were later found at a more anterior site of the

NAcc than intended. In order to determine that the neuroanatomical specificity of the NAcc did

not influence behaviour, additional experiments were conducted where cannulae were implanted

at a more posterior site of the NAcc using the following coordinates: AP + 1.28, L ± 1.5 and V

- 5.2. The coordinates were taken from the atlas of Paxinos and Watson (2006). Cannulae were

anchored to the skull with jeweller’s screws and dental cement. Plastic stylets were placed into

the cannulae to prevent occlusion. Rats were monitored following surgery until conscious and

were then placed back into their home-cage. In addition to dry food, wet-mash food was

available for 24 to 48 hr. Next, rats were given a minimum of seven days to recover from

   

   

20

surgery during which they had unrestricted access to food and water. Throughout this period,

rats were monitored and weighed daily.

2.4. General procedure

2.4.1. Water deprivation

Following recovery from surgery, rats were acclimated to a water deprivation regimen

for three days, after which water was provided for 2 hr per day for the remainder of the

experiment. This acclimation period consisted of restricting access to water for 12 hr on the first

day, 18 hr on the second day and 22 hr on the third day. During behavioural training, water was

provided 30 min after each conditioning session. At test, water was available immediately after

the session until approximately 22 hr prior to the next test. At times where training and testing

did not occur, rats had unrestricted access to food and water.

2.4.2. Pavlovian conditioning

Rats were first acclimated to a saline injection in the colony room. To allow tolerance to

develop to the locomotor suppressant effects of nicotine (Clarke & Kumar, 1983b; Matta et al.,

2007), rats received two nicotine injections (0.4 mg/kg; s.c.) in the colony room, each

administered on a separate day. Rats also received a nicotine injection (0.4 mg/kg; s.c.)

approximately 5 min prior to each conditioning session. Nicotine administration during

Pavlovian conditioning sessions is necessary to produce a nicotine-induced enhancement of

responding for conditioned reinforcement (Guy & Fletcher, 2013). The 0.4 mg/kg dose of

nicotine was chosen because it enhances responding to a reward-paired CS during Pavlovian

conditioning and potentiates responding for conditioned reinforcement (Guy & Fletcher, 2013,

2014a; Olausson et al., 2003, 2004a, 2004b). Pavlovian conditioning was conducted in 12

sessions. Each ~ 30-min session consisted of 30 trials in which a compound CS was presented

following a random time 60 sec schedule of reinforcement. The CS consisted of a 5-sec period

   

   

21

of the house light off and both light stimuli on. During the last 0.5 sec of this 5-sec period, a

tone stimulus was presented following which 0.05 ml of water (US) was delivered into the fluid

receptacle. The receptacle was checked at the end of each session to ensure that all water (1.5

ml) had been consumed.

2.4.3. Tests of responding for conditioned reinforcement

Following the last session of Pavlovian conditioning, rats were placed in their assigned

conditioning chambers with the CR and NCR levers present. This session was conducted to

decrease the levers’ novelty effects on responding during subsequent tests and to ensure that rats

had sampled the CR lever prior to drug testing. Presses on the CR lever resulted in presentation

of the CS without the US according to a random ratio 2 schedule, in which each CR lever press

had a ~ 0.5 probability of delivering the CS. Presses on the NCR lever had no programmed

consequences. This session terminated after 10 CR lever responses and the position of the CR

lever (left/right) was counterbalanced based on data from Pavlovian conditioning. This

counterbalancing was made to ensure that the position of the lever did not influence behaviour.

Data for counterbalancing purposes included latency to make a receptacle entry upon CS

presentation and proportion of receptacle entry made during CS presentation, averaged across

the last two sessions of Pavlovian conditioning. Next, the capacity of the CS to support

responding for a CRf was assessed. Test sessions were conducted following the same design as

described above, except that they lasted for 40 min.

2.5. Experiment 1: effects of DHβE infusion into the ventral tegmental area on nicotine-

enhanced responding for conditioned reinforcement

Sixteen rats with cannulae aimed at the VTA were used. All rats underwent behavioural

training and testing as described above with one exception. Prior to each test of responding for

conditioned reinforcement, rats first received an intracranial infusion followed by a s.c. injection

   

   

22

according to the following conditions: in the SAL/SAL condition, rats received a saline infusion

followed by a saline injection; in the SAL/NIC condition, they received a saline infusion

followed by a nicotine injection; in the DHβE/SAL condition, they received a DHβE infusion

followed by a saline injection; in the DHβE/NIC condition, they received a DHβE infusion

followed by a nicotine injection. Using a within-subject design, each rat was tested under these

four conditions with 72 hr intervening between tests to allow for drug-washout. Treatment order

was counterbalanced across all subjects using a Latin square design. At test, nicotine was

injected at a dose of 0.2 mg/kg. DHβE was administered at a dose of 10 nmol because this dose

blocks CPP produced by intra-VTA nicotine (Laviolette & van der Kooy, 2003b).

2.6. Experiment 2: effects of DHβE infusion into the nucleus accumbens on nicotine-

enhanced responding for conditioned reinforcement

2.6.1. Experiment 2A: effects of DHβE infusion into an anterior site of the nucleus

accumbens on nicotine-enhanced responding for conditioned reinforcement

Fourteen rats with cannulae aimed at an anterior site of the NAcc were used following

the coordinates mentioned above: AP + 1.7, L ± 1.5 and V - 5.0. This experiment followed the

same procedure as in experiment 1, except saline or DHβE was infused into the NAcc rather

than the VTA.

2.6.2. Experiment 2B: effects of DHβE infusion into a more posterior site of the nucleus

accumbens on nicotine-enhanced responding for conditioned reinforcement

Thirteen rats with cannulae aimed at a more posterior site of the NAcc were used

following the coordinates mentioned above: AP + 1.28, L ± 1.5 and V - 5.2. This experiment

also followed the same procedure as in experiment 1, except saline or DHβE was infused into

the NAcc rather than the VTA.

   

   

23

2.7. Experiment 3: effects of nicotine infusion into the ventral tegmental area on

responding for conditioned reinforcement

Fourteen rats with cannulae aimed at the VTA were used. Rats underwent behavioural

training and testing as described above. Using a within-subject design, each rat was tested under

four conditions with 72 hr intervening between tests to allow for drug-washout. The four

conditions were saline, 8, 16 and 32 nmol nicotine. Treatment order was counterbalanced across

all subjects using a Latin square design. These doses were selected to investigate a dose-

response relationship for nicotine-induced enhancement of responding for a CRf.

Following the last microinfusion test, one additional test was conducted where nicotine

was injected (0.2 mg/kg; s.c.) 5 min prior to testing. This test was conducted to compare the

effects of intracranial nicotine infusion with the effects of systemic nicotine injection on

responding for a CRf.

2.8. Experiment 4: effects of nicotine infusion into the nucleus accumbens on responding

for conditioned reinforcement

Fifteen rats with cannulae aimed at the NAcc were used. This experiment followed the

same procedure as in experiment 3, except saline or nicotine (8, 16, and 32 nmol) were infused

into the NAcc rather than the VTA. Cannulae were implanted using the coordinates chosen for

experiment 2B: AP + 1.28, L ± 1.5 and V - 5.2.

2.9. Drugs and microinfusion procedure

Nicotine hydrogen tartrate salt (Sigma-Aldrich, St. Louis, MO) and DHβE (Tocris

Bioscience, Ellisville, MO) were dissolved in sterile 0.9% saline solution and adjusted to a pH

of 7.0-7.2 using NaOH. All nicotine doses were expressed as the amount of free-base and were

administered s.c. at a volume of 1 ml/kg for experiments 1 and 2. For experiment 3 and 4,

nicotine was infused intracranially. Bilateral intracranial microinfusions of DHβE, saline or

   

   

24

nicotine were administered at 0.5 µl volume per site and were performed over 1 min. Injectors

were left in place for an additional min to ensure diffusion away from the injector tip.

Immediately after infusion, rats were placed in their assigned conditioning chamber. One day

prior to testing, an appropriate injector relative to the targeted brain region was inserted into the

guide cannulae for 1 min and no substance was diffused. This habituation session was

performed to cause a brain tissue lesion at a time when no testing occurred and to reduce the

stress of receiving a drug infusion on subsequent tests.

2.10. Histology

After completion of the experiments, animals were sacrificed and brains were removed

and fixed in 4% formaldehyde in 0.1 M phosphate buffered saline for a minimum of 72 hr. After

post-fixation, brains were transferred to a 30% sucrose solution for a minimum of 48 to 72 hr.

Next, brains were frozen, sliced into 40-µm sections on a freezing microtome and stained with

cresyl violet. The sections were examined under a light microscope to verify cannulae

placements according to the anatomical boundaries for these structures determined by Paxinos

and Watson (2006). Data from rats with cannulae implanted outside of the VTA were used as

placement controls to verify that the behavioural effects of DHβE or nicotine were not due to

diffusion of these drugs to nearby brain regions.

2.11. Statistical analyses

Dependent measures for Pavlovian conditioning included latency to make a receptacle

entry upon CS presentation, pre-CS receptacle entries and CS receptacle entries. Pre-CS

receptacle entries represent the mean of receptacle entries that occurred during the 5-sec prior to

each CS, whereas CS receptacle entries represent the mean of receptacle entries that occurred

during the 5-sec CS. Data from latency to make a receptacle entry were analyzed using repeated

measures analysis of variance (ANOVA) across the within-subject factor of session (1-12). Data

   

   

25

from pre-CS and CS receptacle entries were analyzed across the within-subject factors of

session (1-12) and period (pre-CS, CS).

Dependent measures for tests of responding for conditioned reinforcement included the

number of responses on the CR and NCR levers. These data were analyzed using repeated

measures ANOVA across the within-subject factor of lever (CR, NCR) and the between-subject

factors of infusion (saline, DHβE) and injection (saline, nicotine) for experiment 1 and 2. For

experiments 3 and 4, data were analyzed across the within-subject factor of lever (CR, NCR)

and the between-subject factor of infusion (saline, nicotine 8, nicotine 16, nicotine 32).

In all cases, significant interactions from the omnibus ANOVA were pursued using

targeted ANOVA and Post hoc pairwise comparisons using the Tukey’s honest significant

difference (HSD) test. Paired samples t-tests were also conducted where appropriate. Data were

screened to ensure that they met the assumptions of sphericity and equality of variance using

Mauchly’s test for sphericity and Levene’s test of equality of error variances, respectively.

Analyses were conducted using SPSS version 15.0 and Statistica version 7.0 with a significance

level of α = .05.

   

  26

3. Results

3.1. Pavlovian conditioning

Data from Pavlovian conditioning was similar across experiments 1-4. Therefore,

analyses from data on latency to CS-elicited receptacle entry, pre-CS receptacle entries and CS

receptacle entries were only reported from experiment 1 for illustrative purposes.

3.1.1. Latency to CS-elicited receptacle entry

Figure 1 shows that rats rapidly learned the predictive properties of the CS and were

faster to make a receptacle entry upon CS presentation starting session 2. Analysis of latency to

CS-elicited receptacle entry revealed a main effect of session, F(11, 165) = 12.12, p < .001,

confirming that latency decreased across session.

3.1.2. Pre-CS and CS receptacle entries

Figure 2 shows that rats demonstrated a conditioned approach response to the site of

water delivery only upon CS presentation. Results of the two-way Session (1-12) x Period

(preCS, CS) ANOVA revealed main effects of period, F(11, 165) = 23.69, p < .001, session,

F(11, 165) = 3.35, p < .001, and a Session x Period interaction, F(11, 165) = 8.61, p < .001. Post

hoc Tukey’s HSD tests confirmed that rats made more receptacle entries during the CS period,

relative to the pre-CS period in sessions 3-12 (ps < .035).

3.2. Tests of responding for conditioned reinforcement

3.2.1. Experiment 1: effects of DHβE infusion into the ventral tegmental area on nicotine-

enhanced responding for conditioned reinforcement

Figure 3 shows that nicotine enhanced responding for conditioned reinforcement and

that DHβE infused into the VTA attenuated this enhancement. The distribution of injection sites

in the VTA is presented in Figure 4. The number of CR and NCR lever responses for rats that

were implanted with cannulae into the VTA (n = 12) were analyzed using a three-way Lever

   

   

27

(CR, NCR) x Infusion (saline, DHβE) x Injection (saline, nicotine) ANOVA. There was a main

effect of lever, F(1, 11) = 21.74, p < .001, confirming greater CR lever responses than NCR

lever responses. ANOVA revealed a main effect of infusion, F(1, 11) = 8.88, p = .013,

indicating higher levels of responding following saline infusion than DHβE infusion. ANOVA

also revealed a main effect of injection, F(1, 11) = 14.68, p = .003, indicating higher levels of

responding following nicotine injection than saline injection. There was also a significant Lever

x Infusion x Injection interaction, F(1, 11) = 5.09, p = .045. Further decomposition of the 3-way

interaction indicated that nicotine preferentially enhanced responding on the CR lever, Lever x

Injection, F(1, 11) = 22.70, p = .001. Moreover, DHβE preferentially decreased responding on

the CR lever, Lever x Infusion, F(1, 11) = 10.56, p = .008, and reduced the ability of nicotine to

enhance responding, Infusion x Injection, F(1, 11) = 14.64, p = .003. Post hoc Tukey’s HSD

tests revealed that nicotine potentiated responding for a CRf, relative to saline (p < .001). In

addition, DHβE infusion into the VTA attenuated nicotine-induced enhancement of responding

for a CRf, relative to saline infusion (p = .020). Importantly, DHβE infusion into the VTA did

not alter responding on the CR or NCR lever, relative to saline infusion (p = .837).

Figure 5 shows that nicotine enhanced responding for conditioned reinforcement but that

DHβE infused outside the boundaries of the VTA did not affect this response. The distribution

of injection sites outside of the VTA is presented in Figure 6. Data on CR and NCR lever

responses for rats that were implanted with cannulae outside the VTA (n = 4) were analyzed

using a three-way Lever (CR, NCR) x Infusion (saline, DHβE) x Injection (saline, nicotine)

ANOVA. There was a main effect of lever, F(1, 3) = 26.97, p = .014, confirming greater

responding on the CR lever than the NCR lever. A main effect of injection was also found, F(1,

3) = 11.03, p = .045, indicating that responding on the CR lever alone was higher following

   

   

28

nicotine than saline. No other main effects or interactions were found (Fs(1, 3) < 4.23, ps >

.132).

3.2.2. Experiment 2: effects of DHβE infusion into the nucleus accumbens on nicotine-

enhanced responding for conditioned reinforcement

3.2.2.1. Experiment 2A: effects of DHβE infusion into an anterior site of the nucleus

accumbens on nicotine-enhanced responding for conditioned reinforcement

Figure 7 shows that nicotine enhanced responding for conditioned reinforcement and

that DHβE infused into an anterior site of the NAcc attenuated this enhancement. The

distribution of injection sites in an anterior site of the NAcc is presented in Figure 8. The

number of CR and NCR lever responses for rats that were implanted with cannulae into the

NAcc (n = 14) were analyzed using a three-way Lever (CR, NCR) x Infusion (saline, DHβE) x

Injection (saline, nicotine) ANOVA. There was a main effect of lever, F(1, 13) = 98.93, p <

.001, confirming greater CR lever responses than NCR lever responses. ANOVA revealed a

main effect of infusion, F(1, 13) = 5.39, p = .037, indicating higher levels of responding

following saline infusion than DHβE infusion. ANOVA also revealed a main effect of injection,

F(1, 13) = 39.11, p < .001, indicating higher levels of responding following nicotine injection

than saline injection. There was also a significant Lever x Infusion x Injection interaction, F(1,

13) = 6.14, p = .028. Further decomposition of the 3-way interaction indicated that nicotine

preferentially enhanced responding on the CR lever, Lever x Injection, F(1, 13) = 36.88, p <

.001. Moreover, DHβE preferentially decreased responding on the CR lever, Lever x Infusion,

F(1, 13) = 5.33, p = .038, and reduced the ability of nicotine to enhance overall responding,

Infusion x Injection, F(1, 13) = 5.99, p = .029. Post hoc Tukey’s HSD tests revealed that

nicotine potentiated responding for a CRf, relative to saline (p < .001). In addition, DHβE

infusion into the NAcc attenuated nicotine-induced enhancement of responding for a CRf,

   

   

29

relative to saline infusion (p < .001). Importantly, DHβE infusion into the NAcc did not result in

a different pattern of responding on the CR or NCR lever, relative to saline infusion (p = 1.00).

3.2.2.2. Experiment 2B: effects of DHβE infusion into a more posterior site of the nucleus

accumbens on nicotine-enhanced responding for conditioned reinforcement

Figure 9 shows that nicotine enhanced responding for conditioned reinforcement and

DHβE infused into a more posterior site of the NAcc attenuated this enhancement. The

distribution of injection sites in a more posterior site of the NAcc is presented in Figure 10. The

number of CR and NCR lever responses for rats that were implanted with cannulae within the

NAcc (n = 13) were analyzed using a three-way Lever (CR, NCR) x Infusion (saline, DHβE) x

Injection (saline, nicotine) ANOVA. There was a main effect of lever, F(1, 12) = 50.58, p <

.001, confirming greater CR lever responses than NCR lever responses. There was a main effect

of infusion, F(1, 12) = 11.60, p = .005, and a main effect of injection, F(1, 12) = 58.28, p < .001,

indicating that responding was higher following saline infusion than DHβE infusion and higher

following nicotine injection than saline injection. A significant Lever x Infusion x Injection

interaction was also found, F(1, 12) = 7.20, p = .020. Further decomposition of the 3-way

interaction indicated that nicotine preferentially enhanced responding on the CR lever, Lever x

Injection, F(1, 12) = 26.45, p < .001. Moreover, DHβE preferentially decreased responding on

the CR lever, Lever x Infusion, F(1, 12) = 5.53, p = .037, and reduced the ability of nicotine to

enhance overall responding, Infusion x Injection, F(1, 12) = 7.24, p = .020. Post hoc Tukey’s

HSD tests revealed that nicotine potentiated responding for a CRf, relative to saline (p < .001).

In addition, DHβE infusion into the NAcc attenuated nicotine-induced enhancement of

responding for a CRf, relative to saline infusion (p < .001). Importantly, DHβE infusion into the

NAcc did not result in a different pattern of responding on the CR or NCR lever, relative to

saline infusion (p = 1.00).

   

   

30

3.2.3. Experiment 3: effects of nicotine infusion into the ventral tegmental area on

responding for conditioned reinforcement

Figure 11 (left panel) shows that nicotine infused into the VTA dose-dependently

enhanced responding for conditioned reinforcement. The distribution of injection sites in the

VTA is presented in Figure 12. The number of CR and NCR lever responses for rats that were

implanted with cannulae into the VTA (n = 11) were analyzed using a two-way Lever (CR,

NCR) x Infusion (saline, nicotine doses) ANOVA. There was a main effect of lever, F(1, 10) =

43.79, p < .001, confirming greater responding on the CR lever than the NCR lever. A main

effect of infusion, F(1, 10) = 5.06, p = .015, and a significant Lever x Infusion interaction, F(1,

10) = 3.45, p = .049, were also found. Post hoc Tukey’s HSD tests revealed that responding on

the CR lever was greater than the NCR lever for each infusion condition, ps < .001. In addition,

nicotine infused at the 32 nmol dose resulted in greater responding on the CR lever alone

compared to infusion of saline (p < .001), 8 nmol nicotine dose (p < .001) and 16 nmol nicotine

dose (p = .005).

Following the last microinfusion test, rats were injected with nicotine (0.2 mg/kg; s.c.) and

then received an additional test of responding for conditioned reinforcement. Follow-up paired

sample t-tests were conducted to examine responding following nicotine injection with

responding following infusion of each nicotine dose (8, 16 and 32 nmol) into the VTA. As seen

in Figure 11 (right panel), responding on the CR lever alone was greater following nicotine

injection, compared to responding following nicotine infusion at a dose of 8 nmol, t(10) = 4.90,

p = .001, or 16 nmol, t(10) = 3.00, p = .013. However, responding on the CR lever was not

different following nicotine injection, relative to responding following nicotine infusion at a

dose of 32 nmol, t(10) = 2.00, p = .074.

   

   

31

Figure 13 shows that nicotine infused outside the boundaries of the VTA had no effect on

responding. The distribution of injection sites outside the boundaries of the VTA is presented in

Figure 14. The number of CR and NCR lever responses for rats that were implanted with

cannulae outside the VTA (n = 3) were analyzed using a two-way Lever (CR, NCR) x Infusion

(saline, nicotine doses) ANOVA. There were no main effects of lever and infusion, and no

Lever x Infusion interaction (Fs(1, 2) < 1.91, ps > .296).

3.2.4. Experiment 4: effects of nicotine infusion into the nucleus accumbens on responding

for conditioned reinforcement

Figure 15 (left panel) shows that nicotine infused into the NAcc failed to enhance

responding for conditioned reinforcement. The distribution of injection sites in the NAcc is

presented in Figure 16. The number of CR and NCR lever responses for rats that were implanted

with cannulae into the NAcc (n = 15) were analyzed using a two-way Lever (CR, NCR) x

Infusion (saline, nicotine doses) ANOVA. There was a main effect of lever, F(1, 14) = 30.84, p

< .001, confirming greater CR lever responses than NCR lever responses. No main effect of

infusion and no Lever x Infusion interaction were found (Fs(1, 14) < 1.86, ps > .185).

Following the last microinfusion test, rats were injected with nicotine (0.2 mg/kg; s.c.)

and then received an additional test of responding for conditioned reinforcement. Follow-up

paired sample t-tests were conducted to examine responding following nicotine injection with

responding following infusion of each nicotine dose (8 nmol, 16 nmol and 32 nmol) into the

NAcc. As seen in Figure 15 (right panel), responding on the CR lever was greater following

nicotine injection, compared to responding following nicotine infusion of 8 nmol, t(14) = 6.72, p

< .001, 16 nmol, t(14) = 5.97, p < .001, or 32 nmol, t(14) = 4.05, p = .001.

   

  32

4. Discussion

4.1. Summary

In the present research, paired presentations of a reward-predictive CS elicited conditioned

approach behaviour to the site of water delivery. This CS also acquired conditioned reinforcing

properties as shown by higher responding on a lever that resulted in its presentation (CR lever)

compared to a lever that did not (NCR lever). Nicotine administered systemically enhanced

responding for conditioned reinforcement and this enhancement was attenuated following

infusion of the α4β2 nAChR antagonist DHβE into the VTA or NAcc. Additionally, infusion of

nicotine into the VTA, but not the NAcc, dose dependently enhanced responding for a CRf.

These findings provide evidence that stimulation of α4β2 nAChRs in the VTA primarily

contributes to the reinforcement enhancing effects of nicotine. These findings also highlight the

role of the mesolimbic DA system in nicotine-induced reinforcement.

4.2. Nicotine-induced enhancement of the reinforcing properties of reward-paired cues

The test of responding for conditioned reinforcement measures the ability of a CS to

maintain responding where the reward-paired CS, rather than the primary reward (water), acts as

the reinforcer (Mackintosh, 1974). Therefore, this test is a behavioural method for examining

drug effects on the reinforcing properties of reward-paired CSs. In the present research, nicotine

administered systemically or into the VTA enhanced responding for conditioned reinforcement.

This enhancement was not due to a general increase in activity given that nicotine selectively

potentiated responding on the CR lever without affecting responding on the NCR lever, relative

to saline conditions. These data are consistent with previous findings demonstrating an

enhancement effect of nicotine on the reinforcing properties of reward-paired CSs. For instance,

in self-administration studies, environmental cues paired with nicotine promote the acquisition

of intravenous nicotine self-administration (Caggiula et al., 2001), and presentation of such cues

   

   

33

reinstates extinguished nicotine-seeking behaviour (Caggiula et al., 2001; LeSage, Burroughs,

Dufek, Keyler, & Pentel, 2004; Shaham, Adamson, Grocki, & Corrigall, 1997). In place

conditioning studies, nicotine administered systemically or intracranially results in a preference

for the nicotine-paired environment compared to the saline-paired environment (Fudala et al.,

1985; Laviolette & van der Kooy, 2003a, 2003b; Risinger & Oakes, 1995; Shoaib et al., 1994).

Furthermore, in conditioned reinforcement studies, nicotine administered systemically

potentiates responding for CSs previously associated with reward (Olausson et al., 2004a,

2004b, Palmatier et al., 2007; Guy & Fletcher, 2013; 2014a).

4.3. The role of the mesolimbic dopamine system in nicotine-enhanced responding for

conditioned reinforcement

The finding that nicotine infused into the VTA enhanced responding for conditioned

reinforcement suggests a role for DA in mediating this enhancement. This finding is consistent

with the substantial role for DA in the reinforcement actions of nicotine. For example,

manipulations that reduce DAergic activity, such as selective lesions of the mesolimbic DA

system, attenuate enhanced locomotor activity following systemic nicotine injection (Clarke et

al., 1988), decrease nicotine self-administration (Corrigall et al., 1992) and reduce nicotine-

induced CPP (Sellings, Baharnouri, McQuade, & Clarker, 2008). Systemic administration of

DA receptor antagonists, which also reduces mesolimbic DA signaling, decreases nicotine self-

administration (Corrigall et al., 1992; Corrigall & Coen, 1991), attenuates reinstatement of

nicotine seeking behaviour caused by presentation of nicotine-paired cues (Liu et al., 2010) and

blocks nicotine-induced enhancement of responding for conditioned reinforcement (Guy &

Fletcher, 2014b). Together, the present research and these previous reports suggest that the

reinforcement actions of nicotine are mediated by mesolimbic DAergic activity.

   

   

34

The role of DA in mediating the reinforcement enhancing effects of nicotine is

consistent with the role of this neurotransmitter in the reinforcement enhancing effects of other

psychomotor stimulant drugs. One example of a psychomotor stimulant drug that produces

reinforcement enhancing effects is amphetamine. Indeed, administration of amphetamine either

systemically (Ranaldi & Beninger, 1993; Robbins, Watson, Gaskin, & Ennis, 1983) or into the

NAcc (Chu & Kelley, 1992; Kelley & Delfs, 1991; Fletcher, Korth, Sabijan, & DeSousa, 1998;

Taylor & Robbins, 1984, 1986) enhances responding for a CRf. Furthermore, 6-OHDA-induced

depletion of DA from the NAcc attenuates the enhancement effects of amphetamine on

responding for a CRf (Taylor & Robbins, 1986). Antagonism of DA receptors also effectively

blocks amphetamine-induced responding (Chu & Kelley, 1992; Ranaldi & Beninger, 1993). The

findings of the present research indicate that nicotine, like amphetamine, enhances responding

for conditioned reinforcement by targeting the mesolimbic DA system.

4.4. The role of the nucleus accumbens in nicotine-enhanced responding for conditioned

reinforcement

Although nicotine and amphetamine act on the mesolimbic DA system to enhance the

reinforcing properties of reward-paired stimuli, these drugs may act on different regions within

the DA system to produce this enhancement. As mentioned above, direct infusion of

amphetamine into the NAcc enhances responding for conditioned reinforcement (Chu & Kelley,

1992; Kelley & Delfs, 1991; Fletcher et al., 1998; Taylor & Robbins, 1984, 1986). However, in

the present research, direct infusion of nicotine into the NAcc failed to enhance responding for a

CRf. These findings suggest that the NAcc plays an important role in directly mediating the

reinforcement-enhancing effects of amphetamine but not those of nicotine.

One explanation for the inability of nicotine infusion into the NAcc to enhance

responding for a CRf is that the reward-paired CS did not function as a CRf. In this case,

   

   

35

responding on the CR lever would have been lower or no different than responding on the NCR

lever. This explanation is unlikely given that responding on the CR lever was greater than

responding on the NCR lever following intra-NAcc saline or nicotine infusion. Systemic

administration of nicotine also selectively enhanced responding on the CR lever compared to

responding following intra-NAcc nicotine infusion. These results suggest that a CS previously

paired with water acquired conditioned reinforcing properties. These results also indicate that

systemic nicotine administration enhanced the reinforcing properties of the water-paired CS.

Therefore, the inability of intra-NAcc nicotine infusion to enhance responding for a CRf is

likely due to a lack of a local action of nicotine on nAChRs within this region.

DHβE infusion into the NAcc attenuated the nicotine-enhanced responding for a CRf.

This finding suggests that stimulation of α4β2 nAChRs in the NAcc is necessary for nicotine

reinforcement. However, this finding is incongruent with another finding where nicotine-

induced stimulation of nAChRs in the NAcc failed to enhance responding for a CRf. One

explanation for the discrepancy between these findings is that the doses of nicotine infused into

the NAcc were not sufficient to produce any enhancement effects. However, these doses

enhanced responding for a CRf following their infusion into the VTA. Furthermore, the

behavioural effects of intra-VTA nicotine infusion at the 32 nmol dose were not different from

the effects of systemic nicotine administration. Therefore, the nicotine doses chosen in the

present research would have been sufficient to produce an enhancement on responding for a

CRf if the NAcc directly mediated this enhancement.

Another explanation for the discrepancy between these findings is that DHβE infusion

into the NAcc may have produced motor impairments. These impairments could then account

for the reduction in nicotine-enhanced responding for conditioned reinforcement. If DHβE

resulted in motor impairments, then the pattern of responding on the CR and NCR levers would

   

   

36

have been lower in saline-treated rats that received a DHβE infusion, relative to saline-treated

rats that received a saline infusion. However, saline-treated rats that received a DHβE infusion

had a similar pattern of responding, compared to saline-treated rats that received a saline

infusion. Therefore, the effects of intra-NAcc DHβE infusion on the reinforcement enhancing

effects of nicotine were not due to a DHβE-induced decrease in motor behaviour.

The finding that DHβE infused into the NAcc attenuated nicotine-enhanced responding

for conditioned reinforcement is inconsistent with previous reports. For instance,

mecamylamine infused into the NAcc fails to diminish nicotine-induced enhancement of

accumbal DA release (Nisell et al., 1994b). DHβE infused into the NAcc also fails to reduce

nicotine-self administration behaviour (Corrigall et al., 1994). One explanation for the

discrepancy between these findings is that DHβE interacts with other classes of

neurotransmitters in the NAcc to attenuate the reinforcement enhancing effects of nicotine. For

instance, DHβE is an antagonist of 5HT3 receptors (Eiselé et al., 1993), which are located

presynaptically on DA terminals in the NAcc (Chen, Praag, & Gardner, 1991). The 5HT3

receptors have been implicated in some aspects of nicotine reinforcement. For example,

administration of a 5HT3 receptor antagonist inhibits nicotine-induced DA release (Carboni,

Acquas, Frau, & Di Chiara, 1989) and blocks nicotine-CPP (Carboni, Acquas, Leone, Perezzani,

& Di Chiara, 1988). However, the possibility of DHβE interacting with 5HT3 receptors to

attenuate the reinforcement enhancing effects of nicotine is unlikely given that DHβE is not as

potent at 5HT3 receptors as at nAChRs (Eiselé et al., 1993). Furthermore, the role of 5HT3

receptors in nicotine reinforcement is inconclusive given that administration of a 5HT3 receptor

antagonist fails to block nicotine-induced locomotor activity (Arnold et al., 1995) and fails to

reduce nicotine self-administration (Corrigall & Coen, 1994). Although the possibility of DHβE

interacting with other classes of neurotransmitters in the NAcc to inhibit the reinforcement

   

   

37

enhancing effects of nicotine cannot be ruled out, it is unlikely that DHβE does so via 5HT3

receptors.

4.5. The role of the ventral tegmental area in nicotine-enhanced responding for

conditioned reinforcement

The finding that nicotine infusion into the VTA enhanced responding for a CRf suggests

a local action of nicotine on nAChRs within this area. This result is consistent with previous

reports implicating the VTA in nicotine reinforcement. For example, studies using microdialysis

have shown that systemic nicotine administration increases extracellular accumbal DA levels

(Damsma et al., 1989; Imperato et al., 1986; Nisell et al., 1994b). This nicotine-induced

enhancement is blocked by mecamylamine infusion into the VTA (Nisell et al., 1994b).

Behavioural studies have also demonstrated that intra-VTA nicotine infusion enhances

locomotor activity (Reavill & Stolerman, 1990; Welzl et al., 1990). Furthermore, DHβE

infusion into the VTA blocks nicotine self-administration (Corrigall et al., 1994). Rats also

administer nicotine directly into the VTA (Farquhar, Latimer, & Winn, 2012; Ikemoto et al.,

2006) and display CPP for environments previously paired with intra-VTA nicotine infusion

(Laviolette & van der Kooy, 2003a, 2003b). Together, the present research and previous reports

suggest that nicotine reinforcement is mediated through stimulation of nAChRs in the VTA.

Due to the lipophilic nature of nicotine, this drug can diffuse away from the injection

sites in the brain. One possible explanation for the enhanced responding following intra-VTA

nicotine infusion can be due to activation of neuronal populations outside of the VTA. This

explanation is unlikely given that injections outside the boundaries of the VTA, as confirmed by

histological examination of injection sites, failed to dose-dependently enhance responding for a

CRf. Therefore, it appears that nicotine enhances the reinforcing properties of reward-paired

cues by activating specific neuronal elements within the VTA.

   

   

38

4.6. Possible mechanisms

The following mechanisms may account for the findings of the present research.

Nicotine binds to nAChRs expressed on DAergic cell bodies in the VTA, which results in

enhanced DA release in the NAcc. This enhanced accumbal DA release mediates nicotine-

enhanced responding for conditioned reinforcement following intra-VTA nicotine infusion.

Blockade of α4β2 nAChRs present on these DAergic cell bodies following DHβE infusion into

the VTA inhibits nicotine-induced DA release in the NAcc. This inhibition of accumbal DA

release results in diminished responding for conditioned reinforcement following systemic

nicotine administration. Furthermore, DAergic neurons within the VTA send axonal projections

to the NAcc expressing nAChRs at presynaptic terminals. Blockade of α4β2 nAChRs present at

these DAergic terminals following DHβE infusion into the NAcc may inhibit the nicotine-

induced accumbal DA release. This inhibition of DA release may then result in diminished

responding for conditioned reinforcement following systemic nicotine administration. However,

stimulation of nAChRs located at DAergic terminals following intra-NAcc nicotine infusion

does not result in enhanced responding for conditioned reinforcement. Although nicotine

infused into the VTA or NAcc increases extracellular accumbal DA levels to a comparable

extent (Imperato et al., 1986; Ferrari et al., 2002; Nisell et al., 1994a; Marshall, Redfern, &

Wonnacott, 1997), the increase in accumbal DA levels following intra-NAcc nicotine infusion

may not be sufficient to enhance responding for conditioned reinforcement. For instance, intra-

VTA nicotine infusion produces a longer lasting increase in accumbal DA release compared to

intra-NAcc nicotine infusion (Ferrari et al., 2002; Nisell et al., 1994a). This longer lasting

increase in accumbal DA levels following nicotine infusion into the VTA, but not the NAcc,

may account for the enhanced responding for a CRf following intra-VTA, but not intra-NAcc,

nicotine infusion. Another possibility is that intra-NAcc nicotine infusion activates other classes

   

   

39

of neurotransmitters with opposite action than those of DA on the reinforcement enhancing

effects of nicotine.

4.7. The role of α4β2 nicotinic receptors in nicotine-enhanced responding for conditioned

reinforcement

Infusion of the α4β2 nAChR antagonist DHβE into the VTA or NAcc attenuated

nicotine-enhanced responding for a CRf. This finding suggests a role for α4β2 nAChRs in

nicotine reinforcement. However, DHβE is selective to nAChR subunits other than α4β2. For

instance, DHβE can bind to α3β4 nAChRs (Harvey & Luetje, 1996; Harvey, Maddox, & Luetje,

1996) and, at high concentrations, bind to α7 nAChRs (Klink et al., 2001). These results may

suggest that blockade of α4β2, α3β4 and α7 nAChRs following DHβE infusion resulted in a

reduction in the reinforcement enhancing effects of nicotine. This suggestion is unlikely

considering that DHβE is 10-50 fold less potent at α3β4 and α7 than at α4β2 nAChR subunits

(Chavez-Noriega, Crona, & Washburn, 1997; Harvey & Luetje, 1996). Furthermore, DHβE

displays 100-fold greater selectivity for α4β2 relative to α7 nAChR subunits (Chavez-Noriega et

al., 1997). Although the present thesis did not provide data on DHβE binding levels to nAChR

subunits, it is believed that DHβE reduced nicotine-enhanced responding for a CRf by blocking

α4β2 nAChRs.

The finding that blockade of α4β2 nAChRs reduced the reinforcement enhancing effects

of nicotine is in agreement with previous reports. For instance, DHβE blocks nicotine-induced

enhancement of locomotor activity (Damaj, Welch, & Martin, 1995; Stolerman, Chandler,

Garcha, & Newton, 1997; Grottick et al., 2000), nicotine-induced responding for a visual

stimulus (Liu, Palmatier, Caggiula, Donny, & Sved, 2007), nicotine-enhanced efficacy of

rewarding brain stimulation (Kenny & Markou, 2006), nicotine self-administration (Corrigall et

al., 1994; Grottick et al., 2000; Watkins, Epping-Jordan, Koob, & Markou, 1999), nicotine-CPP

   

   

40

(Laviolette & van der Kooy, 2003b; Walters, Brown, Changeux, Martin, & Damaj, 2006) and

nicotine-induced responding for conditioned reinforcement (Guy & Fletcher, 2013).

Additionally, genetic deletion of the β2-containing nAChRs in mice attenuates nicotine-elicited

striatal DA release (Picciotto et al., 1998), nicotine self-administration (Picciotto et al., 1998)

and nicotine-enhanced responding for a CRf (Brunzell et al., 2006). Together, these data suggest

that α4β2 nAChRs mediate nicotine reinforcement.

4.8. The role of the cholinergic system in the reinforcing properties of reward-paired cues

In the present research, a dose of DHβE that blocked α4β2 nAChRs and attenuated the

effects of nicotine to enhance responding did not, by itself, reduce responding for conditioned

reinforcement. For instance, rats that received a DHβE infusion followed by a nicotine injection

responded less for a CRf, compared to rats that received a saline infusion followed by a nicotine

injection. However, rats that received a DHβE infusion followed by a saline injection responded

similarly for a CRf, compared to rats that received a saline infusion followed by a saline

injection. Together, these findings suggest a role for the cholinergic system in the reinforcement

enhancing effects of nicotine, but not in the reinforcing properties of reward-paired cues.

4.9. Future directions

Nicotine produces its psychoactive effects, at least partially, by stimulating nAChRs

located on DAergic neurons in the VTA that project to the terminal fields of the NAcc (Clarke

et al., 1988; Corrigall et al., 1994; Nisell et al., 1994a). Another region that receives DAergic

projections from the VTA is the PFC (Carr & Sesack, 2000; Deutch et al., 1987), an area

heavily involved in the control of executive function and attentional performance (Killcross &

Coutureau, 2003; Muir, Everitt, & Robbins, 1994; Passetti, Chudasama, & Robbins, 2002; Rossi

et al., 2012).

   

   

41

Nicotine administered systemically (Nisell, Nomikos, Hertel, Panagis, & Svensson,

1996) or infused into the PFC (Marshall et al., 1997) enhances mesocortical DA release.

Nicotine produces these effects by activating nAChRs located within the PFC. For example, the

α4β2 nAChR antagonist DHβE blocks DA release in the PFC following administration of

nicotine or the β2-selective agonist 5-lodo-A-85380 (Livingstone et al., 2009). Furthermore, the

α7 antagonist α-Bgt blocks DA release in the PFC following administration of the α7-selective

agonist choline (Livingstone et al., 2009). These findings suggest that nicotine enhances DA

release through stimulation of α4β2 and α7 nAChRs in the PFC.

Compelling evidence suggests that cholinergic innervation of the PFC is involved in

attentional processes (Bloem, Poorthuis, & Mansvelder, 2014). During attentional tasks,

acetylcholine levels in the medial PFC (mPFC) increase (Dalley et al., 2001; Kozak, Bruno, &

Sarter, 2006). Furthermore, stimulation of cholinergic projections to the mPFC enhances

attentional performance (St. Peters, Demeter, Lustig, Bruno, & Sarter, 2011), whereas lesions of

these cholinergic inputs to the mPFC reduce attentional performance (Croxson, Kyriazis, &

Baxter, 2011; Muir et al., 1994, 1996). nAChRs are one type of cholinergic receptors that play a

role in attentional processes. Drugs that stimulate these receptors within the PFC improve

cognitive function, including attentional performance, in both humans and laboratory animals

(Levin & Simon, 1998; Livingstone & Wonnacott, 2009; Mansvelder, van Aerde, Couey, &

Brussaard, 2006; Rezvani & Levin, 2001; Stolerman, Mirza, & Shoaib, 1995; Wallace &

Bertrand, 2013). These findings demonstrate that nicotine enhances attentional performance and

imply a role for nicotine-induced stimulation of accumbal DA release in the enhancement

effects of nicotine on attentional performance.

Responding for conditioned reinforcement involves responding for sensory reinforcers

that were previously paired with reward. The ability of nicotine to enhance this response is

   

   

42

mediated by stimulation of nAChRs in the VTA that results in enhanced accumbal DA release.

It is currently not known whether nicotine enhances the reinforcing properties of these sensory

reward-paired cues by acting on nAChRs in the PFC, which increases accumbal DA release and

results in enhanced attentional performance. Therefore, future studies should investigate

whether nicotine enhances responding for a CRf via a prefrontal attentional mechanism.

4.10. Summary and conclusion

The present experiments investigated the role of the VTA and NAcc in the enhancement

effects of nicotine on the reinforcing properties of reward-paired cues. Data indicated that

infusion of the α4β2 nAChR DHβE into the VTA attenuated nicotine-induced enhancement of

responding for a CRf. Moreover, infusion of nicotine into the VTA dose-dependently enhanced

responding for a CRf, with the 32 nmol dose of nicotine producing similar enhancement effects

compared to those observed following systemic nicotine administration. These findings suggest

that stimulation of nAChRs in the VTA is the primary mechanism underlying the reinforcement

actions of nicotine. Additionally, data indicated that infusion of the α4β2 nAChR DHβE into the

NAcc attenuated nicotine-induced enhancement of responding for a CRf. However, infusion of

nicotine into the NAcc failed to enhance responding for a CRf. These findings suggest that

direct stimulation of nAChRs on DAergic terminals in the NAcc does not mediate the

reinforcement actions of nicotine. However, enhanced DA release from these accumbal DAergic

terminals following stimulation of nAChRs in the VTA, which is produced by systemic

administration of nicotine, seems to be necessary for nicotine-induced reinforcement.

Nevertheless, the findings of the present research provide additional evidence for the role of the

mesolimbic DA system in the augmented reinforcement processes following nicotine exposure.

Future investigation of neurobiological mechanisms mediating nicotine reinforcement can be

beneficial for current and ex-tobacco smokers. For example, such investigation may provide

   

   

43

novel pharmacological interventions that ameliorate craving and relapse induced by exposure to

CSs associated with tobacco consumption.

   

  44

5. References

Albuquerque, E. X., Pereira, E. F. R., Alkondon, M., & Rogers, S. W. (2009). Mammalian

nicotinic acetylcholine receptors: from structure to function. Physiological Reviews,

89(1), 73–120.

Alexander, S. P. H., Mathie, A., & Peters, J. A. (2008). Guide to receptors and channels

(GRAC), 3rd edition. British Journal of Pharmacology, 153(Suppl. 2), S1–S209.

Alkondon, M., Pereira, E. F. R., Barbosa, C. T. F., & Albuquerque, E. X. (1997). Neuronal

nicotinic acetylcholine receptor activation modulates gamma-aminobutyric acid release

from CA1 neurons of rat hippocampal slices. The Journal of Pharmacology and

Experimental Therapeutics, 283(3), 1396–1411.

Arnold, B., Allison, K., Ivanová, S., Paetsch, P. R., Paslawski, T., & Greenshaw, A. J. (1995).

5HT3 receptor antagonists do not block nicotine induced hyperactivity in rats.

Psychopharmacology, 119(2), 213–221.

Balfour, D. J. K. (2004). The neurobiology of tobacco dependence: A preclinical perspective on

the role of the dopamine projections to the nucleus. Nicotine & Tobacco Research, 6(6),

899–912.

Benwell, M. E., Balfour, D. J. K., & Lucchi, H. M. (1993). Influence of tetrodotoxin and

calcium on changes in extracellular dopamine levels evoked by systemic nicotine.

Psychopharmacology, 112(4), 467–474.

Besson, M., David, V., Baudonnat, M., Cazala, P., Guilloux, J. P., Reperant, C., … Granon, S.

(2012). Alpha7-nicotinic receptors modulate nicotine-induced reinforcement and

extracellular dopamine outflow in the mesolimbic system in mice. Psychopharmacology,

220(1), 1–14.

Bloem, B., Poorthuis, R. B., & Mansvelder, H. D. (2014). Cholinergic modulation of the medial

   

   

45

prefrontal cortex: the role of nicotinic receptors in attention and regulation of neuronal

activity. Frontiers in Neural Circuits, 8(17), 1-16.

Bolam, J. P., Francis, C. M., & Henderson, Z. (1991). Cholinergic input to dopaminergic

neurons in the substantia nigra: a double immunocytochemical study. Neuroscience, 41,

483–494.

Bouzat, C. (2012). New insights into the structural bases of activation of Cys-loop receptors.

Journal of Physiology Paris, 106, 23–33.

Brody, A. L. (2006). Functional brain imaging of tobacco use and dependence. Journal of

Psychiatric Research, 40(5), 404–418.

Brunzell, D. H., Chang, J. R., Schneider, B., Olausson, P., Taylor, J. R., & Picciotto, M. R.

(2006). β2-Subunit-containing nicotinic acetylcholine receptors are involved in nicotine-

induced increases in conditioned reinforcement but not progressive ratio responding for

food in C57BL/6 mice. Psychopharmacology, 184, 328–338.

Caggiula, A. R., Donny, E. C., Chaudhri, N., Perkins, K. A., Evans-Martin, F. F., & Sved, A. F.

(2002). Importance of nonpharmacological factors in nicotine self-administration.

Physiology & Behavior, 77, 683–687.

Caggiula, A. R., Donny, E. C., White, A. R., Chaudhri, N., Booth, S., Gharib, M. A., … Sved,

A. F. (2001). Cue dependency of nicotine self-administration and smoking.

Pharmacology Biochemistry & Behavior, 70(4), 515–530.

Carboni, E., Acquas, E., Frau, R., & Di Chiara, G. D. (1989a). Differential inhibitory effects of

a 5-HT3 antagonist on drug-induced stimulation of dopamine release. European Journal

of Pharmacology, 164(3), 515–519.

Carboni, E., Acquas, E., Leone, P., Perezzani, L., & Di Chiara, G. (1988). 5-HT3 receptor

   

   

46

antagonists block morphine- and nicotine-induced place-preference conditioning.

European Journal of Pharmacology, 151(1), 159–160.

Carr, D. B., & Sesack, S. R. (2000). Projections from the rat prefrontal cortex to the ventral

tegmental area: target specificity in the synaptic associations with mesoaccumbens and

mesocortical neurons. The Journal of Neuroscience, 20(10), 3864-3873.

Carter, B. L., & Tiffany, S. T. (1999). Meta-analysis of cue-reactivity in addiction research.

Addiction, 94(3), 327–340.

Centers for Disease Control and Prevention (2008). Smoking-attributable mortality, years of

potential life lost, and productivity losses -- United States, 2000-2004. Morbidity and

Mortality Weekly Report, 57(45), 1226-1228.

Centers for Disease Control and Prevention (2011). Quitting smoking among adults – United

States, 2001-2010. Morbidity and Mortality Weekly Report, 60(44), 1513-1519.

Changeux, J. P. (2010). Nicotine addiction and nicotinic receptors: lessons from genetically

modified mice. Nature Reviews Neuroscience, 11(6), 389–401.

Changeux, J. P., Bertrand, D., Corringer, P. J., Dehaene, S., Edelstein, S., Léna, C., … Zoli, M.

(1998). Brain nicotinic receptors: structure and regulation, role in learning and

reinforcement. Brain Research Reviews, 26, 198–216.

Changeux, J. P., & Edelstein, S. J. (1998). Allosteric receptors after 30 years. Neuron, 21(5),

959–980.

Chaudhri, N., Caggiula, A. R., Donny, E. C., Palmatier, M. I., Liu, X., & Sved, A. F. (2006).

Complex interactions between nicotine and nonpharmacological stimuli reveal multiple

roles for nicotine in reinforcement. Psychopharmacology, 184, 353–366.

Chavez-Noriega, L. E., Crona, J. H., & Washburn, M. S. (1997). Pharmacological

   

   

47

characterization of recombinant human neuronal nicotinic acetylcholine receptors

hα2β2, hα2β4, hα3β2, hα3β4, hα4β2, hα4β4 and hα7 expressed in Xenopus Oocytes.

The Journal of Pharmacology and Experimental Therapeutics, 280, 346-356.

Chen, J., van Praag, H. M., & Gardner, E. L. (1991). Activation of 5-HT3 receptor by 1-

phenylbiguanide increases dopamine release in the rat nucleus accumbens. Brain

Research, 543(2), 354–357.

Chiamulera, C. (2005). Cue reactivity in nicotine and tobacco dependence: a “multiple-action”

model of nicotine as a primary reinforcement and as an enhancer of the effects of

smoking-associated stimuli. Brain Research Reviews, 48(1), 74–97.

Chu, B., & Kelley, A. E. (1992). Potentiation of reward-related responding by psychostimulant

infusion into nucleus accumbens: Role of dopamine receptor subtypes. Psychobiology,

20(2), 153–162.

Clarke, P. B. S., & Kumar, R. (1983a). Characterization of the locomotor stimulant action of

nicotine in tolerant rats. British Journal of Pharmacology, 80(3), 587–594.

Clarke, P. B. S., & Kumar, R. (1983b). The effects of nicotine on locomotor activity in non-

tolerant and tolerant rats. British Journal of Pharmacology, 78, 329–337.

Clarke, P. B. S., Fu, D. S., Jakubovic, A., & Fibiger, H. C. (1988). Evidence that mesolimbic

dopaminergic activation underlies the locomotor stimulant action of nicotine in rats. The

Journal of Pharmacology and Experimental Therapeutics, 246(2), 701–708.

Clarke, P. B. S., & Pert, A. (1985). Autoradiographic evidence for nicotine receptors on

nigrostriatal and mesolimbic dopaminergic neurons. Brain Research, 348(2), 355–358.

Clarke, P. B. S., Pert, C. B., & Pert, A. (1984). Autoradiographic distribution of nicotine

receptors in rat brain. Brain Research, 323(2), 390–395.

Clarke, P. B., & Reuben, M. (1996). Release of [3H]-noradrenaline from rat hippocampal

   

   

48

synaptosomes by nicotine: mediation by different nicotinic receptor subtypes from

striatal [3H]-dopamine release. British Journal of Pharmacology, 117(4), 595–606.

Clarke, P. B. S., Schwartz, R. D., Paul, S. M., Pert, C. B., & Pert, A. (1985). Nicotinic binding

in rat brain: autoradiographic comparison of [3H]acetylcholine, [3H]nicotine, and [125I]-

alpha-bungarotoxin. The Journal of Neuroscience, 5(5), 1307–1315.

Collins, A., Salminen, O., Marks, M., Whiteaker, P., & Grady, S. (2009). The road to discovery

of neuronal nicotinic cholinergic receptor subtypes. In J. E. Henningfield, E. D. London,

& S. Pogun (Eds.), Nicotine Psychopharmacology (Vol. 192, pp. 85-112): Springer

Berlin Heidelberg.

Corrigall, W. A., & Coen, K. M. (1991). Selective dopamine antagonists reduce nicotine self-

administration. Psychopharmacology, 104(2), 171–176.

Corrigall, W. A., & Coen, K. M. (1994). Nicotine self-administration and locomotor activity are

not modified by the 5-HT3 antagonists ICS 205-930 and MDL 72222. Pharmacology

Biochemistry & Behavior, 49(1), 67–71.

Corrigall, W. A., Coen, K. M., & Adamson, K. L. (1994). Self-administered nicotine activates

the mesolimbic dopamine system through the ventral tegmental area. Brain Research,

653, 278–284.

Corrigall, W. A., Franklin, K. B., Coen, K. M., & Clarke, P. B. S. (1992). The mesolimbic

dopaminergic system is implicated in the reinforcing effects of nicotine.

Psychopharmacology, 107, 285–289.

Croxson, P. L., Kyriazis, D. A., & Baxter, M. G. (2011). Cholinergic modulation of a specific

memory function of prefrontal cortex. Nature Neuroscience, 14(12), 1510–1512.

Dalley, J. W., McGaughy, J., O'Connell, M. T., Cardinal, R. N., Levita, L., & Robbins, T. W.

   

   

49

(2001). Distinct changes in cortical acetylcholine and noradrenaline efflux during

contingent and noncontingent performance of a visual attentional task. The Journal of

Neuroscience, 21(13), 4908–4914.

Damaj, M. I., Welch, S. P., & Martin, B. R. (1995). In vivo pharmacological effects of dihydro-

β-erythroidine, a nicotinic antagonist, in mice. Psychopharmacology, 117(1), 67-73.

Damsma, G., Day, J., & Fibiger, H. C. (1989). Lack of tolerance to nicotine-induced dopamine

release in the nucleus accumbens. European Journal of Pharmacology, 168(3), 363-368.

Deutch, A. Y., Holliday, J., Roth, R. H., Chun, L. L. Y., & Hawrot, E. (1987).

Immunohistochemical localization of a neuronal nicotinic acetylcholine receptor in

mammalian brain. Proceedings of the National Academy of Sciences USA, 84, 8697-

8701.

Due, D. L., Huettel, S. A., Hall, W. G., & Rubin, D. C. (2002). Activation in mesolimbic and

visuospatial neural circuits elicited by smoking cues: evidence from functional magnetic

resonance imaging. The American Journal of Psychiatry, 159(6), 954–960.

Eiselé, J. L., Bertrand, S., Galzi, J. L., Devillers-Thiéry, A., Changeux, J. P., & Bertrand, D.

(1993). Chimaeric nicotinic-serotonergic receptor combines distinct ligand binding and

channel specificities. Nature, 366(6454), 479–483.

Elgoyhen, A. B., Johnson, D. S., Boulter, J., Vetter, D. E., & Heinemann, S. (1994). α9: An

acetylcholine receptor with novel pharmacological properties expressed in rat cochlear

hair cells. Cell, 79(4), 705–715.

Elgoyhen, A. B., Vetter, D. E., Katz, E., Rothlin, C. V., Heinemann, S. F., & Boulter, J. (2001).

α10: A determinant of nicotinic cholinergic receptor function in mammalian vestibular

and cochlear mechanosensory hair cells. Proceedings of the National Academy of

Sciences of the United States of America, 98(6), 3501–3506.

   

   

50

Epping-Jordan, M. P., Picciotto, M. R., Changeux, J. P., & Pich, E. M. (1999). Assessment of

nicotinic acetylcholine receptor subunit contributions to nicotine self-administration in

mutant mice. Psychopharmacology, 147(1), 25–26.

Farquhar, M. J., Latimer, M. P., & Winn, P. (2012). Nicotine self-administered directly into the

VTA by rats is weakly reinforcing but has strong reinforcement enhancing properties.

Psychopharmacology, 220(1), 43–54.

Ferrari, R., Le Novère, N., Picciotto, M. R., Changeux, J. P., & Zoli, M. (2002). Acute and long-

term changes in the mesolimbic dopamine pathway after systemic or local single

nicotine injections. The European Journal of Neuroscience, 15(11), 1810–1818.

Fletcher, P. J., Korth, K. M., Sabijan, M. S., & DeSousa, N. J. (1998). Injections of D-

amphetamine into the ventral pallidum increase locomotor activity and responding for

conditioned reward: a comparison with injections into the nucleus accumbens. Brain

Research, 805, 29-40.

Fudala, P. J., & Iwamoto, E. T. (1986). Further studies on nicotine-induced conditioned place

preference in the rat. Pharmacology Biochemistry & Behavior, 25(5), 1041–1049.

Fudala, P. J., Teoh, K. W., & Iwamoto, E. T. (1985). Pharmacologic characterization of

nicotine-induced conditioned place preference. Pharmacology Biochemistry & Behavior,

22(2), 237–241.

Gotti, C., & Clementi, F. (2004). Neuronal nicotinic receptors: from structure to pathology.

Progress in Neurobiology, 74(6), 363-396.

Gotti, C., Zoli, M., & Clementi, F. (2006). Brain nicotinic acetylcholine receptors: native

subtypes and their relevance. Trends in Pharmacological Sciences, 27(9), 482-491.

Grady, S., Marks, M. J., Wonnacott, S., & Collins, A. C. (1992). Characterization of nicotinic

   

   

51

receptor-mediated [3H]dopamine release from synaptosomes prepared from mouse

striatum. Journal of Neurochemistry, 59(3), 848–856.

Gray, R., Rajan, A. S., Radcliffe, K. A., Yakehiro, M., & Dani, J. A. (1996). Hippocampal

synaptic transmission enhanced by low concentrations of nicotine. Nature, 383(6602),

713–716.

Grottick, A. J., Trube, G., Corrigall, W. A., Huwyler, J., Malherbe, P., Wyler, R., & Higgins, G.

A. (2000). Evidence that nicotinic α7 receptors are not involved in the hyperlocomotor

and rewarding effects of nicotine. The Journal of Pharmacology and Experimental

Therapeutics, 294(3), 1112–1119.

Guy, E. G., & Fletcher, P. J. (2013). Nicotine-induced enhancement of responding for

conditioned reinforcement in rats: role of prior nicotine exposure and α4β2 nicotinic

receptors. Psychopharmacology, 225(2), 429–440.

Guy, E. G., & Fletcher, P. J. (2014a). The effects of nicotine exposure during Pavlovian

conditioning in rats on several measures of incentive motivation for a conditioned

stimulus paired with water. Psychopharmacology, 231(11), 2261–2271.

Guy, E. G., & Fletcher, P. J. (2014b). Responding for a conditioned reinforcer, and its

enhancement by nicotine, is blocked by dopamine receptor antagonists and a 5-HT2C

receptor agonist but not by a 5-HT2A receptor antagonist. Pharmacology Biochemistry &

Behavior, 125, 40–47.

Harvey, S. C., & Luetje, C. W. (1996). Determinants of competitive antagonist sensitivity on

neuronal nicotinic receptor β subunits. The Journal of Neuroscience, 16(12), 3798-3806.

Harvey, S. C., Maddox, F. N., & Luetje, C. W. (1996). Multiple determinants of Dihydro-β-

Erythroidine sensitivity on rat neuronal nicotinic receptor α subunits. Journal of

Neurochemistry, 67, 1953-1959.

   

   

52

Heinemann, S., Boulter, J., Connolly, J., Deneris, E., Duvoisin, R., Hartley, M., … Patrick, J.

(1991). The nicotinic receptor genes. Clinical Neuropharmacology, 14, S45–S61.

Hogg, R. C., Raggenbass, M., & Bertrand, D. (2003). Nicotinic acetylcholine receptors: from

structure to brain function. Reviews of Physiology, Biochemistry and Pharmacology,

147, 1–46.

Hunt, S., & Schmidt, J. (1978). Some observations on the binding patterns of α-bungarotoxin in

the central nervous system of the rat. Brain Research, 157(2), 213–232.

Hurst, R., Rollema, H., & Bertrand, D. (2013). Nicotinic acetylcholine receptors: From basic

science to therapeutics. Pharmacology and Therapeutics, 137(1), 22–54.

Ikemoto, S., Qin, M., & Liu, Z. H. (2006). Primary reinforcing effects of nicotine are triggered

from multiple regions both inside and outside the ventral tegmental area. The Journal of

Neuroscience, 26(3), 723–730.

Imperato, A., Mulas, A., & Di Chiara, G. (1986). Nicotine preferentially stimulates dopamine

release in the limbic system of freely moving rats. European Journal of Pharmacology,

132, 337–338.

Jones, I. W., & Wonnacott, S. (2004). Precise localization of α7 nicotinic acetylcholine

receptors on glutamatergic axon terminals in the rat ventral tegmental area. The Journal

of Neuroscience, 24(50), 11244–11252.

Kelley, A. E., & Delfs, J. M. (1991). Dopamine and conditioned reinforcement. I. Differential

effects of amphetamine microinjections into striatal subregions. Psychopharmacology,

103(2), 187–196.

Kenny, P. J., & Markou, A. (2006). Nicotine self-administration acutely activates brain reward

systems and induces a long-lasting increase in reward sensitivity.

Neuropsychopharmacology, 31(6), 1203–1211.

   

   

53

Keyser, K. T., Britto, L. R. G., Schoepfer, R., Whiting, P., Cooper, J., Conroy, W., …

Lindstrom, J. (1993). Three subtypes of α-bungarotoxin-sensitive nicotinic acetylcholine

receptors are expressed in chick retina. The Journal of Neuroscience, 13(2), 442–454.

Killcross, S., & Coutureau, E. (2003). Coordination of actions and habits in the medial

prefrontal cortex of rats. Cerebral Cortex, 13(4), 400–408.

Klink, R., de Kerchove d'Exaerde, A., Zoli, M., & Changeux, J. P. (2001). Molecular and

physiological diversity of nicotinic acetylcholine receptors in the midbrain dopaminergic

nuclei. The Journal of Neuroscience, 21(5), 1452–1463.

Koob, G. F. (1992). Drugs of abuse: anatomy, pharmacology and function of reward pathways.

Trends in Pharmacological Sciences, 13(5), 177–184.

Koob, G. F., & Le Moal, M. (2006). Neurobiology of addiction. London, England: Academic

Press.

Kozak, R., Bruno, J. P., & Sarter, M. (2006). Augmented prefrontal acetylcholine release during

challenged attentional performance. Cerebral Cortex, 16(1), 9–17.

Laviolette, S. R., & van der Kooy, D. (2003a). Blockade of mesolimbic dopamine transmission

dramatically increases sensitivity to the rewarding effects of nicotine in the ventral

tegmental area. Molecular Psychiatry, 8(1), 50–59.

Laviolette, S. R., & van der Kooy, D. (2003b). The motivational valence of nicotine in the rat

ventral tegmental area is switched from rewarding to aversive following blockade of the

α7-subunit-containing nicotinic acetylcholine receptor. Psychopharmacology, 166, 306–

313.

Lazev, A. B., Herzog, T. A., & Brandon, T. H. (1999). Classical Conditioning of Environmental

Cues to Cigarette Smoking. Experimental and Clinical Psychopharmacology, 7(1), 56–

63.

   

   

54

Le Novère, N., & Changeux, J. P. (1995). Molecular evolution of the nicotinic acetylcholine

receptor: An example of multigene family in excitable cells. Journal of Molecular

Evolution, 40(2), 155–172.

Lee, J., Lim, Y., Graham, S. J., Kim, G., Wiederhold, B. K., Wiederhold, M. D., … Kim, S. I.

(2004). Nicotine craving and cue exposure therapy by using virtual environments.

CyberPsychology & Behavior, 7(6), 705–713.

Léna, C., & Changeux, J. P. (1999). The role of β2-subunit-containing nicotinic acetylcholine

receptors in the brain explored with a mutant mouse. Annals of the New York Academy

of Sciences, 868, 611–616.

LeSage, M. G., Burroughs, D., Dufek, M., Keyler, D. E., & Pentel, P. R. (2004). Reinstatement

of nicotine self-administration in rats by presentation of nicotine-paired stimuli, but not

nicotine priming. Pharmacology Biochemistry & Behavior, 79(3), 507–513.

Levin, E. D., & Simon, B. B. (1998). Nicotinic acetylcholine involvement in cognitive function

in animals. Psychopharmacology, 138(3-4), 217–230.

Lindstrom, J. (1998). Purification and cloning of nicotinic acetylcholine receptors. In S. P.

Arneric, & J. D. Brioni (Eds.), Neuronal nicotinic receptors: pharmacology and

therapeutic opportunities (pp. 3-23). New York, NY: Wiley.

Liu, X., Jernigen, C., Gharib, M., Booth, S., Caggiula, A. R., & Sved, A. F. (2010). Effects of

dopamine antagonists on drug cue-induced reinstatement of nicotine-seeking behavior in

rats. Behavioural Pharmacology, 21(2), 153–160.

Liu, X., Palmatier, M. I., Caggiula, A. R., Donny, E. C., & Sved, A. F. (2007). Reinforcement

enhancing effect of nicotine and its attenuation by nicotinic antagonists in rats.

Psychopharmacology, 194(4), 463–473.

Livingstone, P. D., Srinivasan, J., Kew, J. N. C., Dawson, L. A., Gotti, C., Moretti, M., …

   

   

55

Wonnacott, S. (2009). α7 and non-α7 nicotinic acetylcholine receptors modulate

dopamine release in vitro and in vivo in the rat prefrontal cortex. The European Journal

of Neuroscience, 29(3), 539–550.

Livingstone, P. D., & Wonnacott, S. (2009). Nicotinic acetylcholine receptors and the ascending

dopamine pathways. Biochemical Pharmacology, 78(7), 744-755.

Mackintosh, N. J. (1974). The Psychology of Animal Learning. New York, NY: Academic

Press.

Lu, Y., Grady, S., Marks, M. J., Picciotto, M., Changeux, J. P., & Collins, A. C. (1998).

Pharmacological characterization of nicotinic receptor-stimulated GABA release from

mouse brain synaptosomes. The Journal of Pharmacology and Experimental

Therapeutics, 287(2), 648–657.

Luetje, C. W. (2004). Getting past the asterisk: the subunit composition of presynaptic nicotinic

receptors that modulate striatal dopamine release. Molecular Pharmacology, 65(6),

1333–1335.

Mansvelder, H. D., Keath, J. R., & McGehee, D. S. (2002). Synaptic mechanisms underlie

nicotine-induced excitability of brain reward areas. Neuron, 33(6), 905–919.

Mansvelder, H. D., van Aerde, K. I., Couey, J. J., & Brussaard, A. B. (2006). Nicotinic

modulation of neuronal networks: from receptors to cognition. Psychopharmacology,

184, 292-305.

Marshall, D. L., Redfern, P. H., & Wonnacott, S. (1997). Presynaptic nicotinic modulation of

dopamine release in the three ascending pathways studied by in vivo microdialysis:

comparison of naive and chronic nicotine-treated rats. Journal of Neurochemistry, 68(4),

1511–1519.

Marubio, L. M., Gardier, A. M., Durier, S., David, D., Klink, R., Arroyo-Jimenez, M. M., …

   

   

56

Changeux, J. P. (2003). Effects of nicotine in the dopaminergic system of mice lacking

the alpha4 subunit of neuronal nicotinic acetylcholine receptors. The European Journal

of Neuroscience, 17(7), 1329–1337.

Matta, S. G., Balfour, D. J., Benowitz, N. L., Boyd, R. T., Buccafusco, J. J., Caggiula, A. R., . . .

Zirger, J. M. (2007). Guidelines on nicotine dose selection for in vivo research.

Psychopharmacology, 190(3), 269-319.

Mifsud, J. C., Hernandez, L., & Hoebel, B. G. (1989). Nicotine infused into the nucleus

accumbens increases synaptic dopamine as measured by in vivo microdialysis. Brain

Research, 478(2), 365–367.

Millar, N. S., & Gotti, C. (2009). Diversity of vertebrate nicotinic acetylcholine receptors.

Neuropharmacology, 56(1), 237–246.

Mucha, R. F., Geier, A., & Pauli, P. (1999). Modulation of craving by cues having differential

overlap with pharmacological effect: evidence for cue approach in smokers and social

drinkers. Psychopharmacology, 147(3), 306–313.

Muir, J. L., Everitt, B. J., & Robbins, T. W. (1994). AMPA-induced excitotoxic lesions of the

basal forebrain: a significant role for the cortical cholinergic system in attentional

function. The Journal of Neuroscience, 14(4), 2313–2326.

Muir, J. L., Everitt, B. J., & Robbins, T. W. (1996). The cerebral cortex of the rat and visual

attentional function: dissociable effects of mediofrontal, cingulate, anterior dorsolateral,

and parietal cortex lesions on a five-choice serial reaction time task. Cerebral Cortex,

6(3), 470–481.

Museo, E., & Wise, R. A. (1990a). Locomotion induced by ventral tegmental microinjections of

a nicotinic agonist. Pharmacology Biochemistry & Behavior, 35, 735–737.

Museo, E., & Wise, R. A. (1990b). Microinjections of a nicotinic agonist into dopamine

   

   

57

terminal fields: effects on locomotion. Pharmacology Biochemistry & Behavior, 37,

113–116.

Museo, E., & Wise, R. A. (1994). Place preference conditioning with ventral tegmental

injections of cytisine. Life Sciences, 55(15), 1179–1186.

National Institute on Drug Abuse. (2007). Drugs, Brains, and Behavior The Science of

Addiction. Retrieved from

https://d14rmgtrwzf5a.cloudfront.net/sites/default/files/soa_2014.pdf

Niaura, R. S., Rohsenow, D. J., Binkoff, J. A., Monti, P. M., Pedraza, M., & Abrams, D. B.

(1988). Relevance of cue reactivity to understanding alcohol and smoking relapse.

Journal of Abnormal Psychology, 97(2), 133–152.

Nisell, M., Marcus, M., Nomikos, G. G., & Svensson, T. H. (1997). Differential effects of acute

and chronic nicotine on dopamine output in the core and shell of the rat nucleus

accumbens. Journal of Neural Transmission, 104, 1–10.

Nisell, M., Nomikos, G. G., Hertel, P., Panagis, G., & Svensson, T. H. (1996). Condition-

independent sensitization of locomotor stimulation and mesocortical dopamine release

following chronic nicotine treatment in the rat. Synapse, 22(4), 369–381.

Nisell, M., Nomikos, G. G., & Svensson, T. H. (1994a). Infusion of nicotine in the ventral

tegmental area or the nucleus accumbens of the rat differentially

affects accumbal dopamine release. Pharmacology & Toxicology, 75, 348–352.

Nisell, M., Nomikos, G. G., & Svensson, T. H. (1994b). Systemic nicotine-induced dopamine

release in the rat nucleus accumbens is regulated by nicotinic receptors in the ventral

tegmental area. Synapse, 16, 36–44.

Olausson, P., Jentsch, J. D., & Taylor, J. R. (2003). Repeated nicotine exposure enhances

reward-related learning in the rat. Neuropsychopharmacology, 28, 1264–1271.

   

   

58

Olausson, P., Jentsch, J. D., & Taylor, J. R. (2004a). Nicotine enhances responding with

conditioned reinforcement. Psychopharmacology, 171(2), 173–178.

Olausson, P., Jentsch, J. D., & Taylor, J. R. (2004b). Repeated nicotine exposure enhances

responding with conditioned reinforcement. Psychopharmacology, 173, 98–104.

Palmatier, M. I., Liu, X., Matteson, G. L., Donny, E. C., Caggiula, A. R., & Sved, A. F. (2007).

Conditioned reinforcement in rats established with self-administered nicotine and

enhanced by noncontingent nicotine. Psychopharmacology, 195(2), 235–243.

Panlilio, L. V., & Goldberg, S. R. (2007). Self-administration of drugs in animals and humans as

a model and an investigative tool. Addiction, 102(12), 1863-1870.

Passetti, F., Chudasama, Y., & Robbins, T. W. (2002). The frontal cortex of the rat and visual

attentional performance: dissociable functions of distinct medial prefrontal subregions.

Cerebral Cortex, 12(12), 1254–1268.

Paxinos, G., & Watson, C. (6th Ed.) (2006). The rat brain in stereotaxic coordinates. San Diego:

Academic Press.

Payne, T. J., Schare, M. L., Levis, D. J., & Colletti, G. (1991). Exposure to smoking-relevant

cues: effects on desire to smoke and topographical components of smoking behavior.

Addictive Behaviors, 16(6), 467–479.

Picciotto, M. R. (2003). Nicotine as a modulator of behavior: beyond the inverted U. Trends in

Pharmacological Sciences, 24(9), 493–499.

Picciotto, M. R., Zoli, M., Rimondini, R., Léna, C., Marubio, L. M., Pich, E. M., … Changeux,

J. P. (1998). Acetylcholine receptors containing the β2 subunit are involved in the

reinforcing properties of nicotine. Nature, 391(6663), 173–177.

Pidoplichko, V. I., Noguchi, J., Areola, O. O., Liang, Y., Peterson, J., Zhang, T., & Dani, J. A.

   

   

59

(2004). Nicotinic cholinergic synaptic mechanisms in the ventral tegmental area

contribute to nicotine addiction. Learning & Memory, 11(1), 60–69.

Pierce, R. C., & Kumaresan, V. (2006). The mesolimbic dopamine system: The final common

pathway for the reinforcing effect of drugs of abuse? Neuroscience and Biobehavioral

Reviews, 30(2), 215–238.

Prus, A. J., James, J. R., & Rosecrans, J. A. (2009). Conditioned Place Preference. In J. J.

Buccafusco (Ed.), Methods of Behavior Analysis in Neuroscience (2nd ed.). Boca Raton,

FL: CRC Press.

Quick, S. L., Olausson, P., Addy, N. A., & Taylor, J. R. (2014). Repeated nicotine exposure

during adolescence alters reward-related learning in male and female rats. Behavioural

Brain Research, 261, 171–176.

Raftery, M. A., Hunkapiller, M. W., Strader, C. D., & Hood, L. E. (1980). Acetylcholine

receptor: complex of homologous subunits. Science, 208(4451), 1454–1456.

Ranaldi, R., & Beninger, R. J. (1993). Dopamine D1 and D2 antagonists attenuate

amphetamine-produced enhancement of responding for conditioned reward in rats.

Psychopharmacology, 113(1), 110–118.

Rapier, C., Lunt, G. G., & Wonnacott, S. (1990). Nicotinic modulation of [3H]dopamine release

from striatal synaptosomes: pharmacological characterisation. Journal of

Neurochemistry, 54(3), 937–945.

Reavill, C., & Stolerman, I. P. (1990). Locomotor activity in rats after administration of

nicotinic agonists intracerebrally. British Journal of Pharmacology, 99, 273-278.

Rezvani, A. H., & Levin, E. D. (2001). Cognitive effects of nicotine. Biological Psychiatry,

49(3), 258–267.

Risinger, F. O., & Oakes, R. A. (1995). Nicotine-induced conditioned place preference and

   

   

60

conditioned place aversion in mice. Pharmacology Biochemistry & Behavior, 51, 457–

461.

Robbins, T. W., Watson, B. A., Gaskin, M., & Ennis, C. (1983). Contrasting interactions of

pipradrol, d-amphetamine, cocaine, cocaine analogues, apomorphine and other drugs

with conditioned reinforcement. Psychopharmacology, 80(2), 113–119.

Rose, J. E., & Levin, E. D. (1991). Inter-relationships between conditioned and primary

reinforcement in the maintenance of cigarette smoking. British Journal of Addiction,

86(5), 605–609.

Rossi, M. A., Hayrapetyan, V. Y., Maimon, B., Mak, K., Je, H. S., & Yin, H. H. (2012).

Prefrontal cortical mechanisms underlying delayed alternation in mice. Journal of

Neurophysiology, 108(4), 1211–1222.

Segal, M., Dudai, Y., & Amsterdam, A. (1978). Distribution of an α-bungarotoxin-binding

cholinergic nicotinic receptor in rat brain. Brain Research, 148(1), 105–119.

Sellings, L. H. L., Baharnouri, G., McQuade, L. E., & Clarke, P. B. S. (2008). Rewarding and

aversive effects of nicotine are segregated within the nucleus accumbens. The European

Journal of Neuroscience, 28(2), 342–352.

Shaham, Y., Adamson, L. K., Grocki, S., & Corrigall, W. A. (1997). Reinstatement and

spontaneous recovery of nicotine seeking in rats. Psychopharmacology, 130(4), 396–

403.

Sharma, A., & Brody, A. L. (2009). In vivo brain imaging of human exposure to nicotine and

tobacco. Handbook of Experimental Pharmacology, (192), 145-171.

Shoaib, M., Stolerman, I. P., & Kumar, R. C. (1994). Nicotine-induced place preferences

following prior nicotine exposure in rats. Psychopharmacology, 113, 445–452.

Sine, S. M., & Engel, A. G. (2006). Recent advances in Cys-loop receptor structure and

   

   

61

function. Nature, 440(7083), 448–455.

Snell, L. D., & Johnson, K. M. (1989). Effects of nicotinic agonists and antagonists on N-

methyl-D-aspartate-induced 3H-norepinephrine release and 3H-(1-[1-(2-

thienyl)cyclohexyl]-piperidine) binding in rat hippocampus. Synapse, 3(2), 129–135.

Spina, L., Fenu, S., Longoni, R., Rivas, E., & Di Chiara, G. (2006). Nicotine-conditioned single-

trial place preference: selective role of nucleus accumbens shell dopamine D1 receptors

in acquisition. Psychopharmacology, 184, 447–455.

St. Peters, M., Demeter, E., Lustig, C., Bruno, J. P., & Sarter, M. (2011). Enhanced control of

attention by stimulating mesolimbic-corticopetal cholinergic circuitry. The Journal of

Neuroscience, 31(26), 9760–9771.

Stolerman, I. P., Chandler, C. J., Garcha, H. S., & Newton, J. M. (1997). Selective antagonism

of behavioural effects of nicotine by dihydro-β-erythroidine in rats.

Psychopharmacology, 129(4), 390–397.

Stolerman, I. P., Mirza, N. R., & Shoaib, M. (1995). Nicotine psychopharmacology: addiction,

cognition and neuroadaptation. Medicinal Research Reviews, 15(1), 47-72.

Tan, H., Bishop, S. F., Lauzon, N. M., Sun, N., & Laviolette, S. R. (2009). Chronic nicotine

exposure switches the functional role of mesolimbic dopamine transmission in the

processing of nicotine's rewarding and aversive effects. Neuropharmacology, 56(4),

741–751.

Taylor, J. R., & Robbins, T. W. (1984). Enhanced behavioural control by conditioned

reinforcers following microinjections of d-amphetamine into the nucleus accumbens.

Psychopharmacology, 84(3), 405–412.

Taylor, J. R., & Robbins, T. W. (1986). 6-Hydroxydopamine lesions of the nucleus accumbens,

   

   

62

but not of the caudate nucleus, attenuate enhanced responding with reward-related

stimuli produced by intra-accumbens d-amphetamine. Psychopharmacology, 90(3), 390–

397.

Tribollet, E., Bertrand, D., Marguerat, A., & Raggenbass, M. (2004). Comparative distribution

of nicotinic receptor subtypes during development, adulthood and aging: an

autoradiographic study in the rat brain. Neuroscience, 124(2), 405–420.

U.S. Department of Health and Human Services, Department of Health and Human Services,

Centers for Disease Control and Prevention, National Center for Chronic Disease

Prevention and Health Promotion, Office on Smoking and Health. (2004). The health

consequences of smoking: A report of the surgeon general. Retrieved from

http://www.cdc.gov/tobacco/data_statistics/sgr/2004/complete_report/index.htm

U.S. Department of Health and Human Services, Department of Health and Human Services,

Centers for Disease Control and Prevention, National Center for Chronic Disease

Prevention and Health Promotion, Office on Smoking and Health. (2010). How tobacco

smoke causes disease: The biology and behavioral basis for smoking-attributable

diasease: A report of the surgeon general. Retrieved from

http://www.ncbi.nlm.nih.gov/books/NBK53017/

Vetter, D. E., Katz, E., Maison, S. F., Taranda, J., Turcan, S., Ballestero, J., … Boulter, J.

(2007). The α10 nicotinic acetylcholine receptor subunit is required for normal synaptic

Sciences of the United States of America, 104(51), 20594–20599.

Walaas, I., & Fonnum, F. (1980). Biochemical evidence for γ-aminobutyrate containing fibres

from the nucleus accumbens to the substantia nigra and ventral tegmental area in the rat.

Neuroscience, 5(1), 63–72.

Wallace, T. L., & Bertrand, D. (2013). Importance of the nicotinic acetylcholine receptor system

   

   

63

in the prefrontal cortex. Biochemical Pharmacology, 85, 1713-1720.

Walters, C. L., Brown, S., Changeux, J. P., Martin, B., & Damaj, M. I. (2006). The β2 but not

α7 subunit of the nicotinic acetylcholine receptor is required for nicotine-conditioned

place preference in mice. Psychopharmacology, 184, 339–344.

Watkins, S. S., Epping-Jordan, M. P., Koob, G. F., & Markou, A. (1999). Blockade of nicotine

self-administration with nicotinic antagonists in rats. Pharmacology Biochemistry &

Behavior, 62(4), 743–751.

Welzl, H., Bättig, K., & Berz, S. (1990). Acute effects of nicotine injection into the nucleus

accumbens on locomotor activity in nicotine-naive and nicotine-tolerant rats.

Pharmacology Biochemistry & Behavior, 37, 743–746.

Whiteaker, P., Marks, M. J., Grady, S. R., Lu, Y., Picciotto, M. R., Changeux, J. P., & Collins,

A. C. (2000). Pharmacological and null mutation approaches reveal nicotinic receptor

diversity. European Journal of Pharmacology, 393, 123–135.

Wilkie, G. I., Hutson, P. H., Stephens, M. W., Whiting, P., & Wonnacott, S. (1993).

Hippocampal nicotinic autoreceptors modulate acetylcholine release. Biochemical

Society Transactions, 21(2), 429–431.

Williams, B. A. (1994). Conditioned reinforcement: experimental and theoretical issues. The

Behavior Analyst, 17(2), 261-285.

Wise, R. A. (2004). Dopamine, learning and motivation. Nature Reviews Neuroscience, 5(6),

483–494.

Wise, R. A., & Bozarth, M. A. (1987). A psychomotor stimulant theory of addiction.

Psychological Review, 94(4), 469–492.

Wonnacott, S. (2014). Nicotinic ACh Receptors. Tocris Bioscience Scientific Review Series, 1-

31.

   

   

64

Wonnacott, S., Kaiser, S., Mogg, A., Soliakov, L., & Jones, I. W. (2000). Presynaptic nicotinic

receptors modulating dopamine release in the rat striatum. European Journal of

Pharmacology, 393, 51–58.

Wonnacott, S., Sidhpura, N., & Balfour, D. J. K. (2005). Nicotine: from molecular mechanisms

to behaviour. Current Opinion in Pharmacology, 5(1), 53–59.

World Health Organization. (2006). Tobacco: Deadly in Any Form or Disguise. Retrieved from

http://www.who.int/tobacco/communications/events/wntd/2006/Report_v8_4May06.pdf

Zoli, M., Moretti, M., Zanardi, A., McIntosh, J. M., Clementi, F., & Gotti, C. (2002).

Identification of the nicotinic receptor subtypes expressed on dopaminergic terminals in

the rat striatum. The Journal of Neuroscience, 22(20), 8785–8789.

   

  65

6, Table captions

Table 1. Distribution of [3H]-nicotine and [125I]-α-Bgt binding sites in most regions of the rat

brain. Note. Bgt = Bungarotoxin.

   

  66

7. Tables

Table 1

Section [3H]-nicotine [125I]-α-Bgt

Amygdala Low or none (overall) High (overall)

Anterior cortical amygdaloid nucleus Low or none High

Posterior cortical amygdaloid nucleus Low or none High

Basolateral amygdaloid nucleus Low or none Moderate to high

Basomedial amygdaloid nucleus Low or none High

Posteromedial amygdaloid nucleus Low or none High

Brainstem Variable High to moderate (overall)

Interpeduncular nucleus High High (outer shell of nucleus)

Inferior colliculus Low or none High

Superior colliculus High High

Tegmentum High High to moderate

Raphe Moderate High

Nucleus tegmenti pontis - High

Nuclei of the lateral lemniscus - High

Dorsal cochlear nucleus Moderate to low High

Thalamus High overall Low or none (overall)

Lateral Habenula Low Moderate to low

Medial Habenula High Low or none

Anterior nuclear group High Low or none

Mediodorsal thalamic nucleus High Low or none

Lateral nuclear group High Low or none

Midline nuclear group High Low or none

Ventral group High Low or none

Posterior thalamic group Low or none Low or none

Geniculate body High High

   

   

67

Intralaminar nuclear group Low or none -

Reticular nucleus Low Low or none

Zona incerta High Moderate to high

Basal ganglia Variable Variable

Substantia nigra High Moderate to high

Ventral tegmental area High Low or none

Caudate putamen Moderate Low or none

Nucleus accumbens Moderate Low or none

Globus pallidus Moderate Low or none

Subthalamus (subthalamic nucleus) Low or none High

Ventral pallidum Low or none High

Hippocampus Low (overall) High (overall)

Presubiculum High High

Dentate gyrus High High

Field CA1 of the hippocampus Low Moderate to high

Field CA2 of the hippocampus Low Moderate to high

Field CA3 of the hippocampus Low Moderate to high

Cerebral cortex Moderate (overall) Moderate (overall)

Cerebellum Moderate (overall) Low or none (overall)

Hypothalamus Low or none (overall) High to moderate (overall)

Preoptic area Low or none Moderate to high

Bed nucleus of the stria terminalis Moderate High

Anterior nucleus Low or none High

Paraventricular nucleus Low or none Moderate to high

Dorsomedial hypothalamic nucleus Low or none Moderate to high

Ventromedial hypothalamic nucleus Low or none High

Arcuate hypothalamic nucleus Low or none High

Medial mammillary nucleus Low or none Moderate to high (lateral and ventral)

   

   

68

Lateral mammillary nucleus Low or none Moderate

Supraoptic nucleus Low or none High

Lateral hypothalamus Low or none High

Pons High (overall) High (overall)

Medulla Variable Moderate to high (overall)

Spinal trigeminal nucleus Moderate to low High

Vestibular nuclei Moderate to low High

Inferior olivary nucleus Low or none High

Nucleus ambiguus Low or none High

Area postrema High to moderate Low or none

Septum Moderate (overall) -

Medial septal nucleus Moderate to low Low

Lateral septal nucleus Moderate High

   

  69

8. Figure Captions

Figure 1. Latency to make a receptacle entry following CS presentation decreased across

training. Graph depicts mean (± SEM) latency in sec for a receptacle entry following CS

presentation across 12 sessions of Pavlovian conditioning.

Figure 2. Rats had higher levels of receptacle entries during the 5-sec CS presentation relative

to the 5-sec prior to CS presentation (preCS), suggesting that paired presentations of the CS and

water resulted in the formation of a predictive relation between these two events. Graph depicts

mean (± SEM) receptacle entries that occurred during CS presentation (filled symbols) and

mean (± SEM) receptacle entries that occurred during the preCS (open symbols) across 12

sessions of Pavlovian conditioning.

Figure 3. Nicotine enhanced responding for a CRf and DHβE infused into the VTA attenuated

this enhancement. Graph depicts mean (± SEM) CR and NCR lever presses during tests of

responding for conditioned reinforcement (n = 12). Note. SAL = saline; NIC = nicotine; DHβE

= Dihydro-βeta-Erythroidine. *p < .05 for comparisons between CR lever presses in the

SAL/NIC condition and SAL/SAL condition. ^p < .05 for comparisons between CR lever

presses in the DHBE/NIC condition and SAL/NIC condition.

Figure 4. Location of injector tips within the boundaries of the VTA for experiment 1.

Figure 5. Nicotine enhanced responding for a CRf and DHβE infused outside of the VTA failed

to attenuate this enhancement. Graph depicts mean (± SEM) CR and NCR lever presses during

tests of responding for conditioned reinforcement (n = 4). Note. SAL = saline; NIC = nicotine;

   

   

70

DHβE = Dihydro-βeta-Erythroidine. *p < .05 for comparisons between CR lever presses in the

SAL/NIC condition and SAL/SAL condition.

Figure 6. Location of injector tips outside the boundaries of the VTA for experiment 1.

Figure 7. Nicotine enhanced responding for a CRf and DHβE infused into an anterior site of the

NAcc attenuated this enhancement. Graph depicts mean (± SEM) CR and NCR lever presses

during tests of responding for conditioned reinforcement (n = 14). Note. SAL = saline; NIC =

nicotine; DHβE = Dihydro-βeta-Erythroidine. *p < .05 for comparisons between CR lever

presses in the SAL/NIC condition and SAL/SAL condition. ^p < .05 for comparisons between

CR lever presses in the DHBE/NIC condition and SAL/NIC condition.

Figure 8. Location of injector tips within an anterior site of the NAcc for experiment 2A.

Figure 9. Nicotine enhanced responding for a CRf and DHβE infused into a more posterior site

of the NAcc attenuated this enhancement. Graph depicts mean (± SEM) CR and NCR lever

presses during tests of responding for conditioned reinforcement (n = 13). Note. SAL = saline;

NIC = nicotine; DHβE = Dihydro-βeta-Erythroidine. *p < .05 for comparisons between CR

lever presses in the SAL/NIC condition and SAL/SAL condition. ^p < .05 for comparisons

between CR lever presses in the DHBE/NIC condition and SAL/NIC condition.

Figure 10. Location of injector tips within a posterior site of the NAcc for experiment 2B.

   

   

71

Figure 11. Nicotine infused into the VTA dose-dependently enhanced responding for a CRf.

Graph depicts mean (± SEM) CR and NCR lever presses during tests of responding for

conditioned reinforcement following saline or nicotine infusion (left panel; 8, 16, 32 nmol) or a

nicotine injection (right panel; 0.2 mg/kg) (n = 11). Note. SAL = saline; NIC = nicotine; s.c. =

subcutaneous. *p < .05 for comparisons between CR lever presses following nicotine infusion

(32 nmol) and saline infusion. ^p < .05 for comparisons between CR lever presses following

nicotine infusion (32 nmol) and nicotine injection (0.4 mg/kg).

Figure 12. Location of injector tips within the boundaries of the VTA for experiment 3.

Figure 13. Nicotine infused outside of the VTA failed to dose-dependently enhance responding

for a CRf. Graph depicts mean (± SEM) CR and NCR lever presses during tests of responding

for conditioned reinforcement following saline or nicotine infusion (8, 16, 32 nmol) (n = 3).

Note. SAL = saline; NIC = nicotine.

Figure 14. Location of injector tips outside the boundaries of the VTA for experiment 3.

Figure 15. Nicotine infused into a more posterior site of the NAcc failed to dose-dependently

enhance responding for a CRf; however, nicotine injected systemically enhanced responding for

a CRf. Graph depicts mean (± SEM) CR and NCR lever presses during tests of responding for

conditioned reinforcement following saline or nicotine infusion (left panel; 8, 16, 32 nmol) or a

nicotine injection (right panel; 0.2 mg/kg) (n = 15). Note. SAL = saline; NIC = nicotine; s.c. =

subcutaneous. ^p < .05 for comparisons between CR lever presses following nicotine injection

(0.2 mg/kg) and nicotine infusion (8, 16, 32 nmol).

   

   

72

Figure 16. Location of injector tips within a posterior site of the NAcc for experiment 4.

   

  73

9. Figures

Figure 1

1 2 3 4 5 6 7 8 9 10 11 120

2

4

6

8

10

Sessions

Late

ncy

(sec

)

   

   

74

Figure 2

1 2 3 4 5 6 7 8 9 10 11 120

20

40

60

80

100

120

Sessions

Rec

epta

cle

Ent

ries

CSPreCS

   

   

75

Figure 3

SALSAL

SALNIC

DHBESAL

DHBENIC

0

20

40

60

80

100

120

140Le

ver p

ress

es*

^

CRNCR

   

   

76

Figure 4

   

   

77

Figure 5

SALSAL

SALNIC

DHBESAL

DHBENIC

0

20

40

60

80

100Le

ver p

ress

es

*

CRNCR

   

   

78

Figure 6

   

   

79

Figure 7

SALSAL

SALNIC

DHBESAL

DHBENIC

020406080

100120140160

Leve

r pre

sses

*

^

CRNCR

   

   

80

Figure 8

   

   

81

Figure 9

SALSAL

SALNIC

DHBESAL

DHBENIC

020406080

100120140160

Leve

r pre

sses

*

^

CRNCR

   

   

82

Figure 10

   

   

83

Figure 11

SALNIC

8 NIC 16

NIC 32

NIC s.c.

020406080

100120140160180

Dose

Leve

r pr

esse

s

*

CRNCR

^

   

   

84

Figure 12

   

   

85

Figure 13

SALNIC

8 NIC 16

NIC 32

020406080

100120140160180

Dose

Leve

r pr

esse

sCRNCR

   

   

86

Figure 14

   

   

87

Figure 15

SALNIC

8 NIC 16

NIC 32

NIC s.c.

0

40

80

120

160

200

240

280

Dose

Leve

r pr

esse

sCRNCR

^

   

   

88

Figure 16