Division of Pharmacology and Toxicology

Faculty of Pharmacy

University of Helsinki

DIFFERENT RESPONSES OF THE NIGROSTRIATAL AND MESOLIMBIC

DOPAMINERGIC PATHWAYS TO NICOTINIC RECEPTOR AGONISTS

Sanna Janhunen

ACADEMIC DISSERTATION

To be presented, with the permission of the Faculty of Pharmacy of the University of

Helsinki, for public criticism in Auditorium 1041 of Viikki Biocentre, on June 17th,

2005, at 12 o’clock noon.

Helsinki 2005

Supervisors: Professor Liisa Ahtee, M.D. Division of Pharmacology and Toxicology Faculty of Pharmacy University of Helsinki Professor Raimo Tuominen, M.D. Division of Pharmacology and Toxicology Faculty of Pharmacy University of Helsinki Reviewers: Professor Agneta Nordberg, M.D., Ph.D. Division of Molecular Neuropharmacology

Department of Clinical Neuroscience Karolinska Institute, Stockholm

Sweden Docent Pekka Rauhala, M.D. Institute of Biomedicine Faculty of Medicine University of Helsinki Opponent: Professor Ben Westerink, Ph.D.

Department of Biomonitoring and Sensoring University Center for Pharmacy University of Groningen

The Netherlands © Sanna Janhunen 2005 ISBN 952-10-2475-5 (paperback) ISBN 952-10-2476-3 (PDF, http://ethesis.helsinki.fi/) ISSN 1795-7079 Yliopistopaino, University Press Helsinki, Finland 2005

To my parents, Sinikka and Tapio

4

CONTENTS

ABSTRACT ..................................................................................................................................... 6

LIST OF ABBREVIATIONS ........................................................................................................ 7

LIST OF ORIGINAL PUBLICATIONS ..................................................................................... 8

1. INTRODUCTION .................................................................................................................. 9

2. REVIEW OF THE LITERATURE ...................................................................................... 11

2.1. Ascending dopaminergic pathways ......................................................................... 11 2.1.1. Functional implications of nigrostriatal and mesolimbic pathways ............. 13

2.2. Synthesis and release of dopamine .......................................................................... 14

2.3. Metabolism of dopamine............................................................................................ 16

2.4. Dopamine and the neural circuits of basal ganglia ............................................... 17

2.5. Characteristics of Parkinson’s disease ..................................................................... 20

2.6. Neuronal nicotinic acetylcholine receptors (nAChRs) ......................................... 22 2.6.1. Structure and classification of nAChRs ............................................................. 23 2.6.2. Distribution of nAChRs in the brain .................................................................. 25 2.6.3. Activation and desensitization of nAChRs ....................................................... 27 2.6.4. Up-regulation of nAChRs .................................................................................... 28 2.6.5. Influence of subunits on functional properties of nAChRs ............................ 30

2.7. Nicotinic modulation of dopamine release............................................................. 32 2.7.1. Different effects of nicotine on dopamine in the CPu and NAc..................... 34 2.7.2. nAChR-mediated modulation of dopaminergic transmission ....................... 34 2.7.3. Subtypes of presynaptic nAChRs involved in dopamine release .................. 36 2.7.4. Modulation of nicotine-induced dopamine release by other neuro-transmitters, glutamate and GABA .................................................................................... 38

2.8. Nicotine – a substance of abuse................................................................................. 40 2.8.1. Rewarding and reinforcing effects of nicotine in animal models .................. 42 2.8.2. The effects of nicotine on locomotor activity in rats ........................................ 43

2.9. Epibatidine .................................................................................................................... 45

2.10. Nicotinic compounds in Parkinson’s disease ..................................................... 48

2.11. Nicotinic compounds in other CNS disorders.................................................... 51

3. AIMS OF THE STUDY........................................................................................................ 54

4. MATERIALS AND METHODS ........................................................................................ 55

4.1. Animals .......................................................................................................................... 55

4.2. Drugs............................................................................................................................... 55

4.3. Analysis of epibatidine stock solutions................................................................... 56

4.4. In vivo microdialysis (I, II, III) .................................................................................. 57

4.5. Rotational behaviour (I, IV) ....................................................................................... 58

5

4.6. Locomotor activity monitoring (V) ........................................................................... 60

4.7. Place conditioning (V) ................................................................................................. 60

4.8. Statistical analysis ........................................................................................................ 61

5. RESULTS ............................................................................................................................... 62

5.1. Microdialysis studies................................................................................................... 62 5.1.1. Effects of nicotine on the extracellular concentrations of dopamine, DOPAC, HVA and 5-HIAA in the CPu in combination with various dopaminergic drugs (I)......................................................................................................... 62 5.1.2. Effects of nicotine and epibatidine on the extracellular concentrations of dopamine, DOPAC, HVA and 5-HIAA in the CPu and NAc (II) .................................. 64 5.1.3. Effects of nicotine and epibatidine on the extracellular concentrations of dopamine, DOPAC, HVA and 5-HIAA in the CPu and NAc of nomifensine-pretreated rats (III, previously unpublished data) ........................................................... 67

5.2. Behavioural studies...................................................................................................... 69 5.2.1. Effects of acute nicotine on the rotational behaviour of unilaterally 6-OHDA lesioned rats (I, previously unpublished data).................................................... 69 5.2.2. Effects of repeated nicotine and epibatidine on the rotational behaviour of unilaterally 6-OHDA-lesioned rats (IV) ........................................................................ 71 5.2.3. Effects of nicotine and epibatidine on locomotor activity (V, previously unpublished data) ................................................................................................................. 72 5.2.4. Effects of nicotine and epibatidine on conditioned place preference (V) ..... 75

6. DISCUSSION ....................................................................................................................... 77

6.1. Microdialysis studies................................................................................................... 77 6.1.1. Effects of nicotine and dopaminergic drugs on nigrostriatal dopamine (I) .................................................................................................................................. 77 6.1.2. Effects of nicotine and epibatidine on dopaminergic transmission (II) ........ 79 6.1.3. Effects of nicotine and epibatidine on dopamine in combination with nomifensine (III) .................................................................................................................... 83

6.2. Behavioural studies...................................................................................................... 85 6.2.1. Effects of acute nicotine on rotational behaviour in combination with nomifensine or levodopa (I, unpublished results) ........................................................... 85 6.2.2. Effects of repeated nicotine and epibatidine on rotational behaviour (IV) .................................................................................................................................. 86 6.2.2. Effects of nicotine and epibatidine on locomotor activity (V, unpublished results) ............................................................................................................. 88 6.2.3. Effects of nicotine and epibatidine on conditioned place preference (V) ..... 91

7. SUMMARY AND CONCLUSIONS..................................................................................... 93

8. ACKNOWLEDGEMENTS ................................................................................................. 95

9. REFERENCES ....................................................................................................................... 97 APPENDIX: ORIGINAL PUBLICATIONS I-V

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ABSTRACT

Ascending cerebral dopaminergic pathways, the nigrostriatal and mesolimbic/mesocortical pathways, are involved in both motor control and the rewarding and reinforcing effects of drugs of abuse, such as nicotine. The loss of nigrostriatal dopaminergic neurons is characteristic of Parkinson’s disease. The mesolimbic/mesocortical dopaminergic pathway mediates effects on locomotor activity, but its role is better established in the mediation of reward and reinforcement. Nicotine, the main psychoactive ingredient in tobacco, acts at neuronal nicotinic acetylcholine receptors (nAChRs) to induce release of dopamine in the caudate-putamen and nucleus accumbens, the nerve terminal areas of the nigrostriatal and mesolimbic pathways. Epidemiological studies have shown a reduced incidence of Parkinson’s disease in smokers and even former smokers, compared to those who have never smoked. Once Parkinson’s disease has already developed, there is evidence to suggest that nicotine alleviates both motor and cognitive symptoms of patients. Indeed, nAChRs represent an important modulator of dopaminergic function, both under normal conditions and in pathological states. Discovery of their multiplicity has lead to the search for compounds that selectively bind different neuronal nAChR subtypes and that might be beneficial in the treatment of various diseases. However, the role of nAChRs in the mesolimbic pathway in dependence and reward complicates the development of therapeutic drugs that lack addictive properties. Therefore, this study investigates the differential regulation of nAChRs on the two major dopaminergic pathways, the nigrostriatal and mesolimbic pathways. This is achieved by comparison of two nAChR agonists, nicotine and epibatidine, which differ in their affinity for nAChR subtypes. Our present findings suggest that epibatidine, in contrast to nicotine, stimulates dopamine release preferentially in the nigrostriatal pathway, compared with the mesolimbic pathway. When given repeatedly, epibatidine similarly to nicotine stimulates the nigrostriatal dopamine system sufficiently to activate rotational behaviour. While inhibition of dopamine uptake by nomifensine enhances nicotine-induced dopamine release in both the caudate-putamen and nucleus accumbens, epibatidine-induced dopamine release is enhanced by nomifensine in the caudate-putamen, but inhibited in the nucleus accumbens. The modest effects of epibatidine on mesolimbic dopamine release and on place preference, suggest a reduced abuse potential of epibatidine compared with that of nicotine. Differences between epibatidine and nicotine are probably due to their differing affinities for the nAChR subtypes that mediate their effects. In contrast to dopamine release in the mesolimbic pathway, the dose-response curves of the both nAChR agonists studied on nigrostriatal dopamine release are bell-shaped, suggesting desensitization of the nAChR subtypes that mediate their effects on striatal dopamine. These differences may be due to the variation of nAChR subtypes, as well as distinct localization of subtypes between these two pathways. In conclusion, the present findings suggest that it is possible to develop compounds that selectively affect specific nAChR subtypes located on different dopaminergic pathways. Such compounds alone, or in combination with a dopamine uptake inhibitor, may be beneficial in the treatment of diseases, such as Parkinson’s disease, tobacco abuse or drug addiction.

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LIST OF ABBREVIATIONS

α-Bgt α-bungarotoxin α-Ctx α-conotoxin ACh acetylcholine ANOVA analysis of variance CNS central nervous system COMT catechol-O-methyl transferase CPu caudate-putamen DAT dopamine transporter DHβE dihydro-β-erythroidine DMPP 1,1-dimethyl-4-phenylpiperazinium DOPA 3,4-dihydroxyphenylalanine DOPAC 3,4-dihydroxyphenylacetic acid GABA γ-aminobutyric acid 5-HIAA 5-hydroxyindoleacetic acid 5-HT 5-hydroxytryptamine, serotonin HPLC high-performance liquid chromatography HVA homovanillic acid i.p. intraperitoneal, intraperitoneally IPN interpeduncular nucleus LDT laterodorsal tegmental nucleus MAO monoamine oxidase MFB medial forebrain bundle MLA methyllycaconitine MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mRNA messenger ribonucleic acid 3-MT 3-methoxytyramine NAc nucleus accumbens nAChR nicotinic acetylcholine receptor NMDA N-methyl-D-aspartate 6-OHDA 6-hydroxydopamine s.c. subcutaneous, subcutaneously S.E.M. standard error of the mean SN substantia nigra SNc substantia nigra pars compacta SNr substantia nigra pars reticulata TPP tegmental pedunculopontine nucleus TTX tetrodotoxin VTA ventral tegmental area

8

LIST OF ORIGINAL PUBLICATIONS

This dissertation is based on the following publications, herein referred by their Roman

numerals (I-V):

I. Janhunen S, Mielikäinen P, Paldánius P, Tuominen RK, Ahtee L, Kaakkola S

(2005) The effect of nicotine in combination with various dopaminergic drugs on

nigrostriatal dopamine in rats. Naunyn-Schmiedeberg’s Archives of

Pharmacology, in press

II. Janhunen S, Ahtee L (2004) Comparison of the effects of nicotine and epibatidine

on the striatal extracellular dopamine. European Journal of Pharmacology 494 (2-

3), 167-177.

III. Janhunen S, Tuominen RK, Piepponen TP, Ahtee L (2005) Nicotine and

epibatidine alter differently nomifensine-elevated dopamine output in the dorsal

and ventral striatum. European Journal of Pharmacology 511 (2-3), 143-150.

IV. Janhunen S, Tuominen RK, Ahtee L (2005) Comparison of the effects of nicotine

and epibatidine in combination with nomifensine on rotational behaviour.

Neuroscience Letters 381 (3), 314-319.

V. Janhunen S, Linnervuo A, Svensk M, Ahtee L (submitted) The effects of nicotine

and epibatidine on locomotor activity and conditioned place preference in rats.

Reprints were made with permission from the publishers.

9

1. INTRODUCTION

Tobacco smoking is the most prevalent preventable cause of death in developed countries

and is rapidly becoming a significant health problem in developing countries. It is predicted

that every year 3 million people worldwide die from smoking related diseases and that the

rapid increase in smoking in developing countries will increase this number to 10 million by

the year 2025 (Peto et al., 1996; WHO, 2003). Although most smokers are aware of the

harmful effects of smoking, such as increased risks of cardiovascular and pulmonary

diseases and various forms of cancer, many continue to smoke. It has been estimated that

70% of regular smokers want to quit and the majority of them have even tried, but most have

failed. Less than 10% of those who try remain abstinent from smoking after one year

(Cinciripini et al., 1997). As recently as the beginning of the 90’s it was debated whether

smoking should be regarded as a habit or as a true disease (Ochoa, 1994). There is now little

doubt that the majority of tobacco smokers do so in order to experience the

psychopharmacological properties of nicotine that are present in the smoke and to which a

significant proportion of habitual tobacco users become addicted (US Department of Health

and Human Sciences, 1988; Balfour, 1994). In fact, nicotine is now used as a therapeutic

agent in smoking cessation.

The neural mechanisms underlying nicotine dependence are complex and not fully

understood. Similar to other drugs of abuse, such as cocaine and amphetamine, nicotine

stimulates the mesolimbic dopaminergic pathway, which appears to play a critical role in

mediating the reinforcing and rewarding effects of tobacco smoke (Wise and Bozarth, 1987;

Balfour et al., 1998). Smoking (nicotine) improves cognitive function, including learning and

memory, elevates mood, produces a subjective state of well-being, alleviates the stress of

everyday life and prevents the feared withdrawal symptoms caused by the cessation of

smoking (Benowitz, 1996). In animal models of smoking, animals can be trained to self-

administer nicotine and the cessation of regular nicotine can induce physical withdrawal signs

and symptoms (Corrigall, 1999).

The effects of nicotine are mediated by nicotinic acetylcholine receptors (nAChRs), which are

ligand-gated ion channels composed of five subunits. Recent findings on the structural and

functional diversity of nAChR subtypes in the brain have provoked intense research into the

10

role of these receptors in several brain functions and behaviours. The discovery that nAChRs

modulate release of various neurotransmitters, including dopamine, noradrenaline, serotonin,

γ-aminobutyric acid (GABA) and glutamate, has lead to the idea that these receptors could be

therapeutic targets for diverse neurodegenerative and mood disorders, including Parkinson’s

and Alzheimer’s diseases, schizophrenia, depression, anxiety, Tourette’s syndrome and adult

attention deficit hyperactivity disorder (Holladay et al., 1997; Lloyd et al., 1998; Lloyd and

Williams, 2000; Hogg and Bertrand, 2004). In fact, many of these diseases are associated with

a reduction of nAChR number in the brain. Furthermore, discovery of epibatidine, a potent

nAChR agonist with effective analgesic properties (Badio and Daly, 1994; Bannon et al.,

1995), gave rise to the possibility that novel nAChR agonists could also be used to treat pain.

The purpose of present studies was to clarify differences in the nAChR-mediated regulation

of two major ascending dopaminergic pathways, the nigrostriatal and mesolimbic pathways.

The nigrostriatal dopaminergic pathway is involved in motor control and destruction of this

pathway is the main cause of Parkinson’s disease, while the mesolimbic dopaminergic

pathway plays a role in reinforcement and dependence. It is important to clarify the

differences in the nicotinic regulation of these pathways in order to develop novel nicotinic

compounds that elicit therapeutic effects on brain diseases, but lack addictive properties.

Thus, we carried out a series of in vivo brain microdialysis studies in freely moving rats and

studied the effects of two nAChR agonists, nicotine and epibatidine, on dopaminergic

transmission in the nerve terminal regions of the nigrostriatal and mesolimbic pathways. The

affinity of epibatidine at different nAChR subtypes, along with its behavioural properties,

differ from those of nicotine (Sullivan et al., 1994; Reuben et al., 2000). We also studied the

effects of various dopaminergic drugs, including inhibitors of either dopamine reuptake

(nomifensine), dopamine metabolism (tolcapone and selegiline) or presynaptic dopamine

receptors (haloperidol), on striatal dopamine in combination with nicotine. In light of these

results, further studies on the effects of nicotine and epibatidine on dopaminergic transmission

of the nigrostriatal and mesolimbic pathways were carried out in combination with

nomifensine. Furthermore, we studied the effects of nicotine and epibatidine on different

behaviours associated with the two dopaminergic pathways. Rotational behaviour of rats with

unilateral nigrostriatal lesions is thought to mainly reflect an activation of the nigrostriatal

pathway. In contrast, locomotor activity and conditioned place preference preferentially

involve a stimulation of the mesolimbic pathway.

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2. REVIEW OF THE LITERATURE

2.1. Ascending dopaminergic pathways

Dopamine was thought only to be a precursor for noradrenaline and adrenaline, but not an

independent transmitter in the brain until Carlsson et al. (1958, 1959) found large amounts of

dopamine in the basal ganglia and implicated it in the parkinsonian-like symptoms induced by

reserpine. Findings that dopamine is involved in motor control and that decreased striatal

dopamine is the cause of extrapyramidal symptoms in Parkinson’s disease, resulted in the

clinically most important step in the treatment of Parkinson’s disease so far, the use

dopamine’s prodrug, levodopa.

As shown in Fig. 2-1, dopaminergic neurons form three major ascending pathways, the

nigrostriatal, mesolimbic/mesocortical and tuberohypophyseal pathways (Rang et al., 1999).

Cell bodies of these pathways are located in the midbrain, mainly in the substantia nigra pars

compacta (SNc, A9) and the ventral tegmental area (VTA, A10) (Dahlström and Fuxe, 1964;

Ungerstedt, 1971c). In addition, there are some local dopaminergic interneurons in the

olfactory cortex, medulla and retina (Rang et al., 1999; Cooper et al., 2003).

The nigrostriatal dopaminergic pathway originates from the SNc and terminates in the

dorsal part of the striatum, the caudate-putamen (CPu), also known as the dorsal striatum

(Dahlström and Fuxe, 1964; Ungerstedt, 1971c). A minor part of the nigrostriatal pathway

consists of axons from cell bodies in the A8 area (dopaminergic neurons caudal to A9 and

dorsal to A10) projecting to ventral putamen (Ungerstedt, 1971c). Axons of the nigrostriatal

pathway run alongside fibres containing noradrenaline and 5-hydroxytryptamine (5-HT,

serotonin) through the medial forebrain bundle (MFB) and internal capsule to the striatum.

The nigrostriatal pathway contains about 75% of the dopamine in the brain and it is involved

in the control of posture and motor behaviour, as well as learning of motor programs and

habits.

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Fig. 2-1. Main dopaminergic pathways in the rat brain. Dopaminergic cell bodies are represented by triangles and axonal fibres by solid lines. (a) Nigrostriatal pathway. (b) Mesolimbic pathway. (c) Mesolimbic/mesocortical and mesothalamic pathways. (d) Tuberohypophyseal pathway and other short dopaminergic pathways. CPu = caudate-putamen, MFB = medial forebrain bundle, NAc = nucleus accumbens, SNc = substantia nigra pars compacta, SNr = substantia nigra pars reticulata. Modified from Feldman et al. (1997), with permission.

The mesolimbic/mesocortical dopaminergic pathway has cell bodies located in the VTA

and its axons ascend medially to the axons of the nigrostriatal pathway through the MFB to

limbic and cortical structures (Dahlström and Fuxe, 1964; Ungerstedt, 1971c). One branch of

these neurons, the mesolimbic pathway, innervates the nucleus accumbens (NAc), amygdala,

hippocampus, septum and olfactory tubercle, while the other, the mesocortical pathway,

innervates the limbic cortex, such as medial prefrontal, cingulate and entorhinal cortices

(Cooper et al., 2003). Some functional properties of the mesolimbic pathway, such as basal

rate of physiological activity and firing pattern differ from those of the mesocortical pathway.

The mesolimbic pathway is involved in the control of motor behaviour as well as motivation,

emotions and reward, while the mesocortical pathway is more involved in the higher

cognitive functions, learning and reward behaviour.

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The terminal field of the mesolimbic pathway, the NAc, is subdivided into two regions

according to its anatomical projections (Heimer et al., 1991). The NAc shell is part of the

extended amygdala and represents the ventromedial region, while the NAc core represents the

dorsolateral area. Evidence from pharmacological, anatomical and physiological studies

suggest that the shell and core regions are differentially involved in specific behaviours, such

as feeding behaviour and motor activity (Maldonado-Irizarry and Kelley, 1995; Baldo et al.,

2002; Mendoza et al., 2005). The NAc core is linked to the extrapyramidal motor system as it

projects to the dorsolateral ventral pallidum which, in turn, projects to the motor system

through the substantia nigra (SN) and subthalamic nucleus. The pattern of its efferent

connections closely resembles those of the CPu (Heimer et al., 1991) and it is involved in

somatomotor functions. In contrast, the NAc shell is suggested to be more limbic in nature,

because its efferent projections are closely connected to limbic output structures such as the

VTA, amygdala and lateral hypothalamus (Heimer et al., 1991). The shell is involved in

integration and expression of emotions and the projections between the shell and lateral

hypothalamus are relevant for the control of feeding behaviour (Stratford and Kelley, 1997,

1999). Both subdivisions also play a pivotal role in learnt behaviours and Pavlovian

conditioning. The shell is involved in the acquisition of responding for motivationally

significant stimuli, while the core is involved in responding that is elicited, or reinforced, by

conditioned stimuli (Di Chiara, 2002; Ito et al., 2004).

The tuberohypophyseal pathway is a group of short neurons that project from the arcuate

nucleus of the hypothalamus to the median eminence and pituitary gland. This pathway

regulates the function of the pituitary gland. Disruptions in the regulatory control of the

tuberohypophyseal pathway caused by drugs, such as haloperidol that block dopamine D2

receptors, may result in hyperprolactinemia and even lactation.

2.1.1. Functional implications of nigrostriatal and mesolimbic pathways

Both the nigrostriatal and mesolimbic dopaminergic pathways are involved in the control of

movement. Dopamine release in the CPu plays an integral role in the complex circuitry of the

basal ganglia and is crucial for voluntary movement. If this dopaminergic transmission is

disturbed, as occurs in Parkinson’s and Huntington’s diseases, movement generation is

14

damaged. The mesolimbic dopaminergic pathway is involved in locomotor activity, but the

role of the NAc is better established in the mediation of reward and reinforcement.

Recently, in addition to its general role in the initiation and patterning of a variety of

behaviours, the CPu appears to be a brain centre for habit formation as it stores information

about fixed action patterns (reviewed by Gerdeman et al., 2003). While synaptic plasticity in

the NAc appears to play a role in the early stages of drug abuse, the CPu seems to contribute

to the formation of persistent drug-related habits (Berke and Hyman, 2000; Koob and Le

Moal, 2001; Everitt and Wolf, 2002; Gerdeman et al., 2003). In this instance, casual drug-use

progresses towards compulsive, habitual drug-use that exhibits narrowly defined behavioural

repertoires directed mostly at drug-seeking. Recent findings support the idea that participation

of the CPu in the development of addiction is related to mechanisms of long-term synaptic

plasticity, which might occur in combination with similar processes in the NAc (Gerdeman et

al., 2003). Moreover, drug-induced alterations in function of the NAc could subsequently

facilitate synaptic plasticity in the CPu by influencing the flow of information through the

basal ganglia.

2.2. Synthesis and release of dopamine

Dopamine, like other catecholamines, is synthesized from an amino acid precursor, tyrosine

(Cooper et al., 2003). Dietary tyrosine is actively transported across the blood-brain barrier

into dopaminergic neurons and hydroxylated in the cell bodies and nerve terminals by

tyrosine hydroxylase to form 3,4-dihydroxyphenylalanine (DOPA). DOPA is rapidly

converted to dopamine by a cytoplasmic enzyme, aromatic L-amino acid decarboxylase and

newly synthesized dopamine is actively transported into storage vesicles. The conversion of

tyrosine to DOPA is the rate-limiting step of dopamine synthesis and it depends upon the

activity of tyrosine hydroxylase which, in turn, is regulated by several factors (Cooper et al.,

2003). For example, dopamine synthesis is inhibited by dopamine and other catecholamines

that function as inhibitory end-products by competing for a binding site on tyrosine

hydroxylase (Feldman et al., 1997). End-product inhibition is maximal, when neuronal

activity and transmitter release are low and catecholamine concentration in storage vesicles is

high. In addition, the availability of tyrosine may modestly modulate the activity of tyrosine

15

hydroxylase, particularly at high firing rates. Released dopamine activates presynaptic

dopamine receptors on nerve terminals, which may also result in feedback inhibition of

dopamine synthesis (Cooper et al., 2003). The synthesis of dopamine is also dependent on the

rate of impulse flow in dopaminergic neurons so that during increased impulse flow the rate

of tyrosine hydroxylation is increased and results in increased biosynthesis of catecholamines.

Long-term regulation of tyrosine hydroxylase occurs, for example, via increased gene

transcription and synthesis of enzyme molecules (Feldman et al., 1997). In regard to the

nigrostriatal and mesolimbic pathways, the synthesis of dopamine has been shown to be

substantially higher in the limbic areas, compared with the dorsal striatal areas (Andén et al.,

1983).

Dopamine is released in a calcium-dependent way from storage vesicles as an action potential

invades the nerve terminal (Fig. 2-2). The extent of dopamine released depends upon the rate

and pattern of firing, which, in turn, is regulated by autoreceptors located in the

somatodendritic region and the nerve terminals of dopaminergic neurons (Cooper et al.,

2003).

Fig. 2-2. Dopaminergic nerve terminal. Modified from Cooper et al. (2003), with permission.

16

Dopamine cells have been observed to fire with two main firing patterns, a slow single

spiking pattern and a burst firing (Grace and Bunney, 1984a, b). The single spike firing

pattern is characterized by trains of spikes that discharge at steady, but irregular, intervals, and

it is the typical firing pattern of the majority of dopamine cells encountered in untreated,

anaesthetised rats. The burst-firing mode consists of consecutive spikes in a burst, displaying

progressively decreasing amplitude and increasing duration. The bursting pattern leads to an

enhanced release of dopamine. Dopamine cells in the VTA have been shown to elicit a higher

level of burst-firing than those in the SN (Grenhoff et al., 1986). Consistently, the release of

dopamine has been found to be more rapid in the NAc than in the CPu (Garris and Wightman,

1994).

The functional activity of released dopamine is primarily terminated through reuptake by a

specific dopamine transporter (DAT) into the nerve terminal. DAT plays a critical role in

maintaining the homeostasis of the dopaminergic systems. For example, mice lacking DAT

protein exhibit hyperdopaminergic state, despite multiple compensatory adaptations

(Gainetdinov et al., 1999). Dopamine reuptake is particularly efficient in the CPu and indeed,

in the CPu there has been found two- to three-fold more DAT binding sites than in the NAc

(Marshall et al., 1990; Garris and Wightman, 1994; Hoffman and Gerhardt, 1998).

2.3. Metabolism of dopamine

As shown in Fig. 2-2, dopamine metabolism can take place in the synaptic cleft, in the

cytoplasm of the nerve terminal and inside glial cells. Dopamine is metabolized by

monoamine oxidase (MAO) and catechol-O-methyl transferase (COMT) to 3,4-

dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 3-methoxytyramine (3-

MT). In humans and primates, the major brain metabolite is HVA, while in rat brain it is

DOPAC (Cooper et al., 2003). In rat striatum, the major part of HVA is formed from DOPAC

and the minor part from 3-MT (Westerink and Spaan, 1982a). Accumulation of DOPAC,

HVA and 3-MT in the brain or the cerebrospinal fluid may be used as an index of the

functional activity of dopaminergic neurons (Cooper et al., 2003). Increased dopamine

turnover or electrical stimulation of dopaminergic neurons increases the amount of HVA and

17

3-MT in the brain. It has been suggested that 3-MT is a better indicator of decreased

dopamine release than HVA or DOPAC (Kehr, 1976; Westerink and Spaan, 1982b). There is

also an excellent correlation between changes in the impulse flow of dopaminergic neurons

and changes in the level of DOPAC. Furthermore, DOPAC is thought to reflect the

intraneuronal dopamine metabolism, rather than dopamine release (Roffler-Tarlov et al.,

1971).

COMT is found in soluble and membrane-bound forms in glial cells and postsynaptic neurons

(Rivett et al., 1983; Kaakkola et al., 1987; Männistö and Kaakkola, 1999). Inhibitors of

COMT increase the level of dopamine in the synaptic cleft and thus prolong dopamine

receptor activation. These inhibitors can be coadministered with levodopa to enhance

dopaminergic function in parkinsonian patients. MAO exists in intracellular and extracellular

compartments and inhibition of MAO thus increases presynaptic intracellular concentrations

of dopamine and prolongs the availability of released dopamine. MAO has two forms, MAO-

A and MAO-B, of which MAO-A displays strong affinities for noradrenaline, 5-HT and

dopamine, while MAO-B exhibits the highest affinities for β-phenylethylamine and

dopamine.

2.4. Dopamine and the neural circuits of basal ganglia

The basal ganglia are deep-lying structures of the cerebral hemisphere, including the striatum,

globus pallidus and amygdala, which act in conjunction with allied nuclei, such as the SNc,

substantia nigra pars reticulata (SNr), subthalamic nucleus and endopeduncular nucleus (Fig.

2-3a; Hauber, 1998). The basal ganglia collect inputs from the neocortex, integrate them with

each other and with inputs from the limbic areas and focus the integrated outputs to regions of

the frontal lobes and brainstem involved in motor planning and motor memory (Graybiel,

1990). In fact, abnormalities of the basal ganglia and their allied nuclei can result in disorders

of movement programming, certain forms of drug addiction and some mental disorders, such

as schizophrenia-like states and obsessive-compulsive disorder. The predominant

neurotransmitter in the basal ganglia is GABA and the primary site of functional interactions

in the basal ganglia is the striatum. The GABAergic medium spiny neurons of the striatum

receive dense glutamatergic inputs from the cortex and thalamus, dopaminergic inputs from

18

(a)

Glu

Glu

Glu

GABA

GABA

GABA

GABA

DA

Dorsal striatum

Cortex

Thalamus SNc/VTA

STN

GP

SNr GABA

GABA

ACh

Glu

Glu

ACh

GABA

GABA

TPP/LTD

Endopeduncular nucleus

ACh

(b)

Glu

Glu

Glu

GABA GABA

GABA

AGABA

GABA

GABA

ACh

ACh

DA

DA

NAc

PFC VTA TPP/LTD

Interneuron

Projection neuron

Non-α7 nAChR

α7 nAChR Fig. 2-3. Nigrostriatal (a) and mesolimbic/mesocortical dopaminergic pathways (b), associated neural circuitry and nicotinic acetylcholine receptor (nAChR) subtypes. A simplified scheme of afferent and efferent connections between the basal ganglia and allied structures is depicted (a). The major pathways using dopamine (DA), γ-aminobutyric acid (GABA), acetylcholine (ACh) and glutamate (Glu), including the locations of α7 and non-α7 (most likely β2-containing) nAChRs, are illustrated. GP = globus pallidus, LDT = laterodorsal tegmental nucleus, NAc = nucleus accumbens, PFC = prefrontal cortex, SNc = substantia nigra pars compacta, SNr = substantia nigra pars reticulata, STN = subthalamic nucleus, TPP = tegmental pedunculopontine nucleus, VTA = ventral tegmental area. Figure (b) modified from Wonnacott et al. (2005), with permission.

19

the midbrain (SNc and VTA) and cholinergic inputs from the tegmental pedunculopontine

nucleus (TPP)(Fig. 2-3a; for reviews e.g. Graybiel, 1990; Parent, 1990; Bolam et al., 2000;

Parent et al., 2000; Smith and Kieval, 2000). The major output from the striatum is provided

by two populations of GABAergic neurons. One projects to the globus pallidus and the other

to the SNr and entopeduncular nucleus and onwards to the thalamus and superior colliculus.

Dopaminergic neurons in the striatum and midbrain interact with neurotransmitter inputs

originating from several brain regions (Fig. 2-3), including glutamatergic projections from the

cortex and subthalamic nucleus, GABA- and peptide-containing inputs from the striatum, a

GABAergic input from the globus pallidus, a serotonergic input from the dorsal raphe

nucleus, a noradrenergic input from the locus coeruleus and a cholinergic input from the

brainstem (Usunoff et al., 1982; Nitsch et al., 1984; Kita and Kitai, 1987; Wirtshafter et al.,

1987; Gariano and Groves, 1988; Smith and Parent, 1988; Gould et al., 1989; Bolam and

Smith, 1990; Smith and Bolam, 1990). In addition, dopaminergic neurons interact with local

GABAergic and cholinergic interneurons (Woolf and Butcher, 1981; Nitsch and Riesenberg,

1988; Koós and Tepper, 1999). Activation of N-methyl-D-aspartate (NMDA)-type glutamate

receptors or excitatory glutamatergic afferents projecting from the frontal cortex, subthalamic

nucleus and TPP, increase the burst firing of dopaminergic neurons and secondarily, the

release of dopamine from nerve terminals (Grenhoff et al., 1988; Svensson and Tung, 1989;

Asencio et al., 1991, Gariano and Groves, 1988; Charléty et al., 1991; Suaud-Chagny et al.,

1992; Westerink et al., 1992b; Chergui et al., 1993; Karreman et al., 1996; Schilström et al.,

1998b). Mesolimbic dopaminergic neurons appear to be more sensitive than nigrostriatal

neurons to the stimulation mediated by NMDA glutamate receptors (Westerink et al., 1992b,

1996).

The TPP ascend excitatory cholinergic and glutamatergic projections to the midbrain (Fig. 2-

3). More specifically, neurons from the TPP project to dopaminergic neurons in the SNc and

run through the VTA. However, the activity of dopaminergic neurons of the VTA is

predominantly regulated by neurons from the laterodorsal tegmental nucleus (LDT)(Satoh and

Fibiger, 1986; Clarke et al., 1987; Bolam et al., 1991; Futami et al., 1995; Blaha et al., 1996).

The cholinergic interneurons in the striatum are tonically active and release acetylcholine

(ACh)(Aosaki et al., 1995; Bennett and Wilson, 1999), which provides an important control

of striatal dopamine release (Zhou et al., 2001). Inhibitory action of a nAChR antagonist

20

mecamylamine, on the activity of dopaminergic neurons suggests that the midbrain

dopaminergic neurons may be physiologically controlled by cholinergic nicotinic mechanisms

(Ahtee and Kaakkola, 1978; Clarke et al., 1985a; Grenhoff et al., 1986; Haikala and Ahtee,

1988). It is suggested that the mesolimbic dopaminergic cells are under a phasic, rather than a

tonic, cholinergic control (Grenhoff and Svensson, 1992).

The burst firing of dopaminergic neurons is tonically inhibited by GABAergic neurons from

the SNr (Fig. 2-3). Inhibition of these inputs by activation of the globus pallidus decreases the

tonic inhibition and facilitates the burst firing of dopaminergic neurons (Tepper et al., 1998).

Pharmacological blocking of GABA receptors in the VTA or SN increases spontaneous

release of dopamine in the nerve terminal areas (NAc or CPu), whereas activation of these

GABA receptors reduces the firing rate and burst firing of dopaminergic neurons (Westerink

et al., 1992a, 1996; Dewey et al., 1999; Cobb and Abercrombie, 2002; Erhardt et al., 2002).

Although the dorsal and ventral striata are very similar with respect to neuronal

cytoarchitecture and neurotransmitter content (i.e. most of the neurons are GABAergic), they

vary with regard to their afferent and efferent circuitry. Dopaminergic and GABAergic

neurons of the CPu are preferentially innervated by neurons from various associative and

sensorimotor cortical areas, while the NAc receives projections from the limbic structures,

prefrontal cortex, amygdala and hippocampus (Parent, 1990). Thus, there are differences in

the interactions of dopaminergic, GABAergic and cholinergic transmission between the

ventral and dorsal striata (Girault et al., 1986). It has been reported that the stimulation of

GABAA receptors in the region of dopaminergic cell bodies stimulates nigrostriatal neurons,

but inhibits mesolimbic neurons, while stimulation of GABAB receptors inhibits both

nigrostriatal and mesolimbic neurons (Santiago and Westerink, 1992; Westerink et al., 1996).

2.5. Characteristics of Parkinson’s disease

Parkinson’s disease was first described in the beginning of the 19th century by James

Parkinson, in whose honour the disease is named (Parkinson, 1817). The major symptoms of

Parkinson’s disease are tremor at rest, rigidity, bradykinesia and even akinesia (reviewed by

Obeso et al., 2000; Olanow, 2004). Postural instability and gait disturbances usually occur as

the disease progresses. The most debilitating of the late-onset symptoms are cognitive defects,

21

such as dementia, general confusion, disorientation and sleepiness. Depression can occur at

any stage of the disease. Pathologically, Parkinson’s disease is characterized by a progressive

preferential degeneration of melanized neurons of the SNc coupled with intracellular protein

aggregates known as Lewy bodies (Jellinger, 1999). Changes in the SNc are most pronounced

in the ventrolateral region. Dopamine deficiency produces dysfunction in the striatum which,

in turn, leads to decreased activity of the direct pathway from striatal GABAergic neurons to

the internal segment of the globus pallidus and the SNr and to increased activity of the

indirect pathway involving the external segment of the globus pallidus and the subthalamic

nucleus (Hamani and Lozano, 2003). As a consequence, the activities in the basal ganglia

output structures are disrupted, which, leads to disruption in the activity of brainstem motor

areas, such as the TPP and thalamocortical motor system. This leads to difficulties in the

initiation of movement and poverty of motion.

The loss of neurons in the SNc and other subcortical nuclei is proportional to striatal

dopamine deficiency and both the duration and clinical severity of the disease. Parkinsonian

motor symptoms appear when dopaminergic neuronal death exceeds a critical threshold: 70-

80% loss of striatal nerve terminals and 50-60% loss of SNc perikarya (Agid, 1991). Tyrosine

hydroxylase activity is decreased parallel to the loss of dopamine (Birkmeyer and Riederer,

1989). Decreased activity of tyrosine hydroxylase is also found in the limbic system and it

may result in psycho-pathological disturbances. In addition, there are deficits in several other

neurotransmitter systems, such as subcortical noradrenergic, serotonergic and cholinergic

ascending pathways (Table 2-1; Nordberg et al., 1985; Jellinger, 1997, 1999). For example,

the loss of two thirds of the cholinergic neurons in the basal ganglia contributes to dementia at

the late stage of Parkinson’s disease. The loss of cortical nAChRs positively correlates with

the degree of cognitive deficits (Paterson and Nordberg, 2000; Quik and Jeyarasasingam,

2000). Many patients have a deficiency of noradrenaline and a reduction of 40-50% of 5-HT

in several brain regions, but these reductions tend not worsen as the disease progresses.

GABA and glutamate decarboxylase in the SN are also found to be altered.

22

Table 2-1. Synopsis of the most important biochemical findings in Parkinson’s disease

Changes in Parkinson’s disease (advanced stage) compared with normal ageing Dopamine HVA Tyrosine hydroxylase Biopterin Aromatic amino acid decarboxylase MAO COMT cAMP-dependent protein kinase D1 dopamine receptors D2 dopamine receptors Noradrenaline

↓↓↓ ↓↓ ↓↓↓ ↓↓

↓(↓) =

= =

= ↑↓ = ↑↓ = ↓(↑)

Dopamine-β-hydroxylase Phenylethanolamine-N-methyltransferase 5-HT (serotonin) 5-HT decarboxylase 5-HIAA 5-HT receptors GABA Glutamate dehydrogenase GABA receptors Substance P Leu-enkephalin binding nAChRs

↓(↓) ↓ ↓(↓) ↓ = ↓(↓) ↓ = ↓(↓) ↓(↓) ↓(↓) ↓(↓) ↓ = ↑ ↓ = (↑)

↓↓↓ greatly diminished; ↓↓ moderately diminished; ↓ slightly diminished; = unchanged; ↑ increased. cAMP = cyclic adenosine monophosphate, COMT = catechol-O-methyl transferase, GABA = γ-aminobutyric acid, 5-HIAA = 5-hydroxyindoleacetic acid, 5-HT = 5-hydroxytryptamine, HVA = homovanillic acid, nAChR = nicotinic acetylcholine receptor. Modified from Birkmeyer and Riederer (1985, 1989), with permission.

The gradual appearance of clinical signs in Parkinson’s disease is due to compensatory

mechanisms that first mask existence of the disease and then delay onset and aggravation of

motor abnormalities (Zigmond et al., 1990). The compensatory processes include the intrinsic

properties of nigrostriatal dopaminergic neurons, an up-regulation of postsynaptic dopamine

receptors, neuronal plasticity, volume transmission of dopamine in the striatum and a different

input to the SNc (reviewed by Bezard and Gross, 1998; Moore, 2003). Also activation of

glutamatergic inputs from the subthalamic nucleus and globus pallidus to the SNc play a

crucial role in the compensation during the presymptomatic period (Bezard et al., 1997, 1998;

Bezard and Gross, 1998; Bezard et al., 1999).

2.6. Neuronal nicotinic acetylcholine receptors (nAChRs)

ACh is an important neurotransmitter in the brain that is involved in the excitation of central

nervous system (CNS). The effects of ACh are mediated via two main types of receptors,

muscarinic and nicotinic receptors (Rang et al., 1999). The G-protein coupled muscarinic

receptors can be divided into five subtypes of which the neural subtypes mediate slow

23

excitatory effects in the CNS that contribute, for instance, to memory. Nicotinic acetylcholine

receptors (nAChRs) are located at the neuromuscular junction, in autonomic ganglia and in

the CNS. Neuronal nAChRs subtypes are involved in fast excitatory transmission in the brain.

2.6.1. Structure and classification of nAChRs

All nAChRs are members of a supergene family of ligand-gated ion channels that also

includes GABAA, glycine and 5-HT3 receptors (reviewed by Karlin and Akabas, 1995;

Lindstrom et al., 1995; McGehee and Role, 1995). They have a pentameric cylindrical

structure, composed of five subunits organised around a central pore that is permeable to Na+,

K+ and Ca2+ ions (Fig. 2-4; Cooper et al., 1991). The muscle-type nAChRs in vertebrate

skeletal muscles and in electric organs of electric eels (Electrophorus electricus) and rays

(Torpedo californica or marmorata) have provided a model for understanding structural,

molecular and biophysical properties of neuronal nAChRs. Muscle nAChRs consist of two

subtypes, either fetal α1β1γδ or adult α1β1εδ subtypes (Karlin, 1993). The first neuronal

nAChR subunit cDNA, ultimately termed as α3, was cloned by Heinemann, Patrick and

colleagues (Boulter et al., 1986). Since this report, eleven homologous genes encoding

neuronal nAChR subunits have been cloned in mammals (α2-α7 and β2-β4 in the brain, α9

and α10 in mechanosensory hair cells)(Elgoyhen et al., 1994; Lukas et al., 1999; Elgoyhen et

al., 2001). In addition, α8 subunit has been identified in avian species (Gotti et al., 1997b).

Neuronal nAChRs are composed of α- and β-subunits (Fig. 2-3d). The α subunits contain two

adjacent cysteines at positions homologues to α1 192-193 in loop C, whereas the β (non-α)

subunits lack these cysteine residues (Le Novere and Changeux, 1995).

24

Fig 2-4. Structure of nAChRs. (a) Transmembrane topology of a single subunit. (b) Pentameric composition of subunits constituting a nAChR (in this case, α4β2 receptor) and ACh binding site at the extracellular interface between two adjacent subunits. (c) Allosteric transitions of nAChR: resting (B), active (A) and desensitized (I and D). Various ligands bind to different sites as indicated; CB = competitive blocker, NCB = non-competitive blocker. (d) Putative organization of three different types of neuronal nAChRs: α7, α4β2, and α4β2α5. Modified from Lena and Changeux (1997a) and Berkovic et al. (1999), with permission.

Neuronal nAChRs can be divided into subtypes according to their ability to bind the snake

venom α-bungarotoxin (α-Bgt)(Deneris et al., 1991; Le Novere and Changeux, 1995;

McGehee and Role, 1995; Colquhoun and Patrick, 1997). Neuronal nAChRs that do not bind

α-Bgt comprise of both α and β subunits and they can form several different nAChR subtypes

(Boulter et al., 1987; Sargent, 1993). The α2, α3 and α4 subunits can form functional nAChRs

in pairwise combinations with β2 or β4 subunits (Boulter et al., 1987; Luetje and Patrick,

1991; Elliott et al., 1996). While the α5 and β3 subunits do not participate in pairwise

combinations, they may form functional channels when they are coexpressed with other

functional αβ-subunit combinations, such as α4, β2, and β4 as well as α3 and α4 subunits

(Deneris et al., 1989; Ramirez-Latorre et al., 1996; Wang et al., 1996; Gerzanich et al., 1998;

25

Groot-Kormelink et al., 1998). The α6 subunit can form a functional nAChR in combination

with the β4 subunit (Gerzanich et al., 1997), but its expression is more efficient when

coexpressed with at least two other subunits, including β3 (Fucile et al., 1998; Kuryatov et al.,

2000).

Neuronal nAChRs that are inhibited by nanomolar concentrations of α-Bgt consist of α7, α8,

α9 and α10 subunits (Boulter et al., 1987; Sargent, 1993). Only the α7 subunit is widely

distributed in the mammalian CNS. The α7, α8 and α9 subunits are capable of forming robust

functional channels when expressed as homooligomers in Xenopus oocytes (Couturier et al.,

1990b; Elgoyhen et al., 1994; Gerzanich et al., 1994). Some evidence exists that α7 subunits

can also be expressed in heteromeric combinations with α5 and β2 subunits in heterologous

systems (Lukas, 1995; Alkondon et al., 1997; Guo et al., 1998; Yu and Role, 1998; reviewed

by Girod et al., 1999). α7α8 heteromeric nAChRs occur in avian species (Elgoyhen et al.,

1994; Gotti et al., 1994). The α10 subunit does not form functional homomeric channels, but

it can form heteromeric α9α10 nAChRs when coexpressed with α9 subunit (Elgoyhen et al.,

2001; Sgard et al., 2002). The α9 and α10 subunits are expressed in mammals in the cochlea,

pituitary gland, keratinocytes and lymphocytes (Sgard et al., 2002; Peng et al., 2004).

2.6.2. Distribution of nAChRs in the brain

A single neuron may express more than one nAChR subtype, with each subtype localised

separately and serving different functions. This makes predicting the distribution of nAChR

subtypes based purely on subunit gene expression a difficult task. In situ hybridisation and

immunohistochemical studies have shown that α4, β2 and α7 subunit mRNA are the most

abundantly expressed nAChR genes in the mammalian brain (Wada et al., 1989; Marks et al.,

1992; Hill et al., 1993; Seguela et al., 1993). α subunit genes as a group are expressed in most

regions of the brain, but each individual α subunit mRNA is expressed in a unique, although

partly overlapping, set of neuronal structures (Wada et al., 1989). The most predominant α

subunit, α4, is strongly expressed in a large number of brain areas, including the thalamus,

habenula, cerebral cortex and dopaminergic nuclei. The α4 subunit, together with the β2

subunit, accounts for more than 90% of high-affinity nicotine binding sites in the rat brain

(Whiting and Lindstrom, 1986; Wada et al., 1989; Flores et al., 1992). The α7 subunit mRNA

26

accounts for approximately 90% of [125I]α-Bgt binding sites in the brain (Clarke et al., 1985b;

Couturier et al., 1990a; Schoepfer et al., 1990; Seguela et al., 1993). α7 subunits are

distributed throughout the brain with dense localisations in cortical and limbic areas (Orr-

Urtreger et al., 1997). In contrast to α7, the α2, α3 and α5 subunit mRNA are found in limited

areas of the mammalian brain (Wada et al., 1990; Lena and Changeux, 1997a). α2 mRNA is

expressed in the interpeduncular nucleus (IPN), hippocampal formations and olfactory bulb.

α3 and α5 mRNA are expressed in dopaminergic nuclei and cortical, hippocampal and

thalamic areas. Lastly, α6 mRNA, which is often shown to colocalise with β3 mRNA, has

been found in limited regions of the rat brain, including the SN, VTA and locus coeruleus (Le

Novere et al., 1996; Lena and Changeux, 1997a).

The β2 subunit has been shown by both mRNA and protein level, to be widely expressed

throughout the brain, largely, but not exclusively in combination with the α4 subunit (Hill et

al., 1993; Lena and Changeux, 1997a). Their distribution parallels high-affinity nicotine

binding sites in the brain, being strong in the thalamus, intermediate in the striatum and

cortex, and weak in the hypothalamus (Clarke et al., 1985b). β3 and β4 subunit mRNA are

expressed in a limited number of brain areas, such as the habenula, IPN and locus coeruleus

(Lena and Changeux, 1997a). β3 subunits are also expressed in dopaminergic nuclei.

Several radiolabelled nAChR ligands, including agonists such as ACh, nicotine, and more

recently epibatidine and cytisine, as well as antagonists such as α-Bgt, are used to study the

distribution of nAChRs (Clarke et al., 1985b; Marks et al., 1986, 1998). The distribution of

high-affinity binding sites, measured by nAChR agonist binding, differ from that of low-

affinity ([125I]α-Bgt) binding sites, because these ligands apparently bind to different nAChR

subunits (α4β2 vs. α7)(Clarke et al., 1985b; Schoepfer et al., 1990; Flores et al., 1992;

Seguela et al., 1993). Distribution of [3H]nicotine binding is generally consistent with that of

[3H]ACh, [3H]epibatidine or [3H]cytisine, but [3H]epibatidine can also detect nAChRs that

have lower a affinity for nicotine and are not labelled by [3H]nicotine or [3H]ACh (Perry and

Perry, 1995; Marks et al., 1998; Zoli et al., 1998). [3H]nicotine binding sites are distributed

throughout the mammalian brain, with highest levels in the thalamus and nucleus basalis of

Meynert, and lower levels in the IPN, hippocampus, CPu, cortex and superior colliculus

(Clarke et al., 1985b; Cimino et al., 1992; Gotti et al., 1997a; Paterson and Nordberg, 2000;

Han et al., 2003). In comparison with [3H]nicotine binding, [125I]α-Bgt binding is lower in

27

thalamic areas and higher in the hippocampus (Clarke et al., 1985b; Cimino et al., 1992; Han

et al., 2003). [125I]α-Bgt binding is dense in sympathetic ganglia, hypothalamus, cerebellum,

cortex and superior colliculus.

In conclusion, nAChRs seem to be located in brain areas that receive cholinergic innervation,

as measured by immunohistochemistry for ACh-synthesizing enzyme, choline

acetyltransferase (Woolf, 1991). The striatal complex, including the CPu and NAc contains

cholinergic interneurons. Cholinergic neurons of the basal forebrain project to the

hippocampus, limbic cortex, amygdala, olfactory bulbs and neocortex, while those of the

hypothalamic areas and medial habenula project to the IPN. In addition, the thalamus, globus

pallidus, SN, locus coeruleus and raphe nuclei receive cholinergic innervation. The

cholinergic transmission mediated by nAChRs is best characterised in the brain in

dopaminergic nuclei, cerebral cortex, medial habenula and IPN (Clarke, 1993b).

2.6.3. Activation and desensitization of nAChRs

Nicotine and other nAChR agonists can activate and desensitize nAChRs. When a nAChR is

activated upon agonist binding, it rapidly undergoes an allosteric transition from a closed,

resting conformation to an open channel that allows a flow of ions through it and may lead to

depolarisation of the neuron (Fig. 2-3c). Instead of activation, nAChRs can also become

desensitized in response to prolonged exposure to agonists, which has been shown in vitro

(Ogden and Colquhoun, 1985; Bertrand et al., 1990) and in vivo (James et al., 1994). The

desensitization of nAChRs was first described following ACh application in the

neuromuscular junction of the frog by Katz and Thesleff (1957). They introduced a cyclic

model postulating interconvertible high- and low-affinity agonist binding states to explain

desensitization following exposure to non-stimulating and stimulating concentrations of

agonists. Their model consists of two paths, each leading to the nAChR in the desensitized

state. The first path shows receptor activation, but prolonged presence of an agonist induces

ion channel closure and the transition of the nAChR to its desensitized confirmation, which is

refractory to activation, but displays higher affinity for agonists. The second path to

desensitization occurs without significant nAChR activation at low agonist concentrations and

when an agonist binds to an unbound, desensitized nAChR. This is possible because the

28

desensitized form of the nAChR displays higher affinity for agonists than the ground state

form of the receptor and because the unbound ground state and desensitized state are

interconvertible. Therefore, exposure to non-stimulating or stimulating concentrations of

agonists may result in bound desensitized receptors that are refractory to activation.

The cyclic model proposed by Katz and Thesleff was later modified into a more complex

model that includes two ligand bound states (Dilger and Liu, 1992), an inactivated state in

addition to resting, activated and desensitized states (Changeux et al., 1990; Lena and

Changeux, 1993) and multiple desensitized states of nAChRs (Changeux and Edelstein,

1998). Desensitization is characterised by a rapid onset and a quick and fully reversible loss

of nAChR function. As the exposure to an agonist ends, the agonist dissociates from a

receptor and the desensitized form returns to a form that is can be activated (Lena and

Changeux, 1998). The longer-lasting loss of nAChR function (Fig. 2-3c; inactive state) has

been called “specific chronic desensitization” (Ochoa et al., 1990; Collins et al., 1994),

“functional down-regulation” (Marks et al., 1993), “long-lasting inactivation” (Kawai and

Berg, 2001) or “persistent inactivation” (Ke et al., 1998). The desensitized states of nAChRs

are thought to underlie some of the long-term addictive properties of nicotine (Marks et al.,

1983; Schwartz and Kellar, 1985; Kawai and Berg, 2001; Buisson and Bertrand, 2002).

2.6.4. Up-regulation of nAChRs

Generally, chronic agonist administration elicits a decrease in the number of receptors, i.e.

downregulation, accompanied by tolerance to the effects of agonist. On the other hand,

chronic administration of antagonist produces an up-regulation of receptors and enhances

sensitivity to agonistic effects. However, chronic nicotine administration, such as smoking has

been found to increase the number of nAChRs in the human brain post mortem (Benwell et

al., 1988; Breese et al., 1997; Teaktong et al., 2004). Smoking during pregnancy alters the

expression of α4 and α7 subunits in the human fetal brain (Falk et al., 2005). Up-regulation of

nAChRs depends on the number of cigarettes smoked per day (Breese et al., 1997) and a two-

month abstinence from tobacco decreases a number of nAChRs to the levels found in non-

smoking subjects (Breese et al., 1997). Thus, up-regulation of nAChRs seems to be a

consequence of smoking rather than smoking being a consequence of higher number of

29

nAChRs. Smoking up-regulates nAChRs at diverse densities in different brain regions and

up-regulation is highest in the hippocampus, cortical areas and cerebellum, and lowest in the

striatum (Court et al., 1998; Perry et al., 1999). Acutely, tobacco smoke up-regulates α4*

nAChRs (asterisk represents unidentified subunits; Lukas et al., 1999) in axon terminals and

dendrites, as well as α7* nAChRs in perikarya, while chronically it down-regulates α7

expression on astrocytes (Teaktong et al., 2004).

Chronic nicotine administration, either by experimenter or by an animal itself, has been found

to dose-dependently increase the number of high-affinity binding sites (mainly α4β2 nAChRs)

in rodent brain (Marks et al., 1983; Schwartz and Kellar, 1983; Ksir et al., 1985; Schwartz

and Kellar, 1985; Flores et al., 1992, 1997; Pietilä et al., 1998; Nuutinen et al., 2005) and in

transfected cells (Peng et al., 1994a; Bencherif et al., 1995; Zhang et al., 1995). Up-regulation

of nAChRs is consistent with the localization of nAChR subtypes in the brain so that up-

regulation of high-affinity nicotine binding sites is highest in the thalamus, while low-affinity

nicotine binding sites up-regulate mainly in the hippocampus (Schwartz and Kellar, 1985; el-

Bizri and Clarke, 1994b). The functional consequences of nAChR up-regulation are not fully

resolved. In animals, up-regulation is accompanied by tolerance to some of nicotine’s effects,

for instance, on rotarod performance, heart rate and body temperature (Marks et al., 1983).

However, up-regulation is also associated with enhancement of some of the behavioural

effects of nicotine, for example, stimulation of locomotor activity (Ksir et al., 1985) and

responses of frontal cortical neurons to nicotine (Abdulla et al., 1995). Up-regulation is

thought to have implications for nicotine withdrawal, rather than the development of nicotine

addiction (Wonnacott et al., 2005).

Up-regulation has been suggested to result from the conversion of some of the low-affinity

nAChRs into high-affinity nAChRs (Romanelli et al., 1988), but also [125I]α-Bgt binding sites

(mainly α7 nAChRs) are up-regulated, although at higher nicotine concentrations and to a

lesser extent than the high-affinity nicotine binding sites (Marks et al., 1983; Pauly et al.,

1991; el-Bizri and Clarke, 1994b; Nuutinen et al., 2005). Studies in transfected cell lines have

shown that higher nicotine concentrations are needed to up-regulate α3* and α7* than α4β2

nAChRs (Peng et al., 1997; Fenster et al., 1999b). Differences in the regulation of nAChR

subtypes might be due to differences in subtype-specific sensitivities of active and

desensitized receptor states (Fenster et al., 1997; Ke et al., 1998; Wang et al., 1998). Up-

30

regulation of nAChRs following chronic nicotine treatment has been proposed to result from

receptor desensitization, resulting in nicotine as a functional antagonist (Wonnacott, 1990).

However, several studies imply that up-regulation requires neither receptor activation,

desensitization nor an ion flow through the ion channel. Up-regulation requires higher agonist

concentrations than those activating nAChRs and the same or even higher concentrations than

those that desensitize nAChRs (Rowell and Li, 1997; Whiteaker et al., 1998; Fenster et al.,

1999a). After prolonged exposure to nicotine most receptors are permanently unable to open

their ion channels in response to nicotine binding. Although several in vitro studies have

shown that up-regulation is related to loss of nAChR function, there is also evidence that up-

regulated nAChRs, such as α4β2*, α3β2*, and even more α7*, remain functional (Wang et

al., 1998; Buisson and Bertrand, 2001, 2002). Furthermore, an ion channel blocking

antagonist mecamylamine fails to inhibit the nicotine-elicited up-regulation of α3*, α7* and

α4β2* nAChRs in isolated cell lines (Peng et al., 1997) and can even up-regulate [3H]nicotine

binding sites itself in vitro (Peng et al., 1994a) and in vivo (Collins et al., 1994; Abdulla et al.,

1996; Pauly et al., 1996). In addition, other nAChR antagonists, such as the α4β2-selective

dihydro-β-erytroidine (DHβE) and the α7-selective methyllycaconitine (MLA), have been

found to up-regulate α4β2 nAChRs and MLA also up-regulates [125I]α-Bgt binding sites in

transfected cell lines (Molinari et al., 1998; Buisson and Bertrand, 2001). Mechanisms of

nAChR up-regulation do not appear to be related to increased transcription of nAChRs

(Marks et al., 1992; Ke et al., 1998), but rather to a decrease of the turnover of receptor

breakdown (Peng et al., 1994a; Zhang et al., 1995), an increased affinity of the receptors as a

result from their conformational change (Sallette et al., 2004) or an incorporation of

intracellular nAChRs into the cell membrane (Bencherif et al., 1995; Whiteaker et al., 1998).

2.6.5. Influence of subunits on functional properties of nAChRs

Functional properties of nAChRs, primarily permeability for cations and desensitization

properties, vary with nAChR subunit composition. Homomeric nAChRs that consist of either

α7, α8 or α9 subunits exhibit high Ca2+ permeability and they desensitize very rapidly in

response to high concentrations of nAChR agonists (Couturier et al., 1990b; Revah et al.,

1991; Seguela et al., 1993; Elgoyhen et al., 1994; Gerzanich et al., 1994). For example, the

relative permeability value of α7* nAChRs for Ca2+ and Na+ ions is 15 while that of subtypes

31

including α2 to α6 and β2 to β4 subunits ranges between 0.5 and 2.5 (Role, 1992; Role and

Berg, 1996). In heteromeric nAChRs, the α subunit is thought to regulate ligand binding and

determine the apparent agonist affinity of active and desensitized states of a nAChR (Cachelin

and Jaggi, 1991; Fenster et al., 1997). Also the β subunit may modulate the apparent affinity

for some nAChR subtypes, but it is mainly thought to modulate the overall time course of

desensitization of a nAChR and the rate at which agonists and antagonists dissociate from a

receptor, or ion channel opening (Cachelin and Jaggi, 1991; Luetje and Patrick, 1991; Fenster

et al., 1997). The α4* subtypes are more sensitive to desensitizing levels of nicotine and

recover more slowly from desensitization than the α3* subtypes (Fenster et al., 1997). The

β2* subtypes desensitize rapidly, but they also seem to recover faster than β4* subtypes

(Cachelin and Jaggi, 1991; Fenster et al., 1997). Presence of an α5 subunit in α3β2 or α3β4

nAChR complexes alters apparent sensitivity to nAChR agonists and increases Ca2+

permeability as well as the rate and magnitude of desensitization (Wang et al., 1996;

Gerzanich et al., 1998). The α6 subunit alters the properties of α3β2 nAChRs more than those

of α4β2 (Kuryatov et al., 2000).

nAChRs with different subunit compositions show significant pharmacological diversity. This

diversity includes subtype-specific sensitivity to agonists, but also the action elicited by a

ligand may vary, ranging from a full agonist to a partial agonist or even to an antagonist. For

example, nicotine’s affinity to nAChR subtypes varies. Nicotine shows highest affinity for

α4*, moderate for α3* and lowest for α7* subtypes (Table 2-2 on page 46; Fenster et al.,

1997). α7* subtypes are the only nAChRs activated by choline (Alkondon et al., 1997). The

β2 subunit exhibits higher potency for ACh and nicotine than β4 subunits (Gerzanich et al.,

1998) and nicotine is a partial agonist on α3β2 nAChRs, but a full agonist on α3β4 nAChRs

(Wang et al., 1996; Gerzanich et al., 1998). Cytisine is a full agonist on β4* subtypes, but a

potent inhibitor of ACh-induced currents in β2* subtypes (Papke and Heinemann, 1994). The

nAChR antagonist, α-conotoxin-MII (α-CtxMII) competitively inhibits α3β2 nAChRs, but not

α3β4 nAChRs (Cartier et al., 1996). An individual nAChR subtype may also possess different

properties in different brain areas (Wooltorton et al., 2003), which further complicates the

pharmacological characterization of these receptors. Moreover, the pharmacology of nAChRs

also varies between different species. For example, rat α3β4 is more sensitive to ACh than

α3β2 subtype, but the opposite situation exists in corresponding chick nAChR subtypes. 1,1-

dimethyl-4-phenylpiperazinium (DMPP) is a full agonist in rat and human α7 subtypes, but

32

only a partial agonist for chick α7 nAChR (Bertrand et al., 1992; Seguela et al., 1993; Peng et

al., 1994b).

2.7. Nicotinic modulation of dopamine release

Nicotine increases dopamine release in the terminal fields of nigrostriatal, mesolimbic and

mesocortical dopaminergic pathways, to a greater extent in the CPu and NAc and to a lesser

extent in the cerebral cortex and amygdala (Summers and Giacobini, 1995; Marshall et al.,

1997; Rowell, 2002). Nicotine has been reported to stimulate dopamine release in vitro in

rodent striatal slices (Westfall, 1974; Arqueros et al., 1978; Giorguieff-Chesselet et al., 1979;

Sacaan et al., 1995; Teng et al., 1997), accumbal slices (Rowell et al., 1987; Fung, 1989),

hypothalamic slices (Goodman, 1974) and in striatal and frontal cortical synaptosomes

(Sakurai et al., 1982; Rapier et al., 1988; Grady et al., 1992; el-Bizri and Clarke, 1994a;

Rowell, 1995; Whiteaker et al., 1995). In vivo, nicotine-induced dopamine release has been

indirectly shown by increased HVA and DOPAC levels in the dorsal striatum (Nose and

Takemoto, 1974; Lichtensteiger et al., 1976, 1982; Roth et al., 1982; Haikala et al., 1986) and

by increased DOPAC contents in limbic, primarily accumbal, regions (Grenhoff and

Svensson, 1988). In vivo microdialysis studies have shown that acute nicotine increases

extracellular levels of dopamine and its metabolites in the CPu and NAc (Imperato et al.,

1986; Damsma et al., 1988, 1989; Toth et al., 1992; Benwell and Balfour, 1997; Marshall et

al., 1997; Seppä and Ahtee, 2000). Similar to other drugs of abuse, nicotine acutely increases

dopamine overflow in the shell, but not in the core of the NAc (Pontieri et al., 1996; Nisell et

al., 1997; Di Chiara, 2000; Ferrari et al., 2002). Nicotine-induced elevation of dopamine and

its metabolites is inhibited both in vitro and in vivo by a brain penetrating nAChR antagonist

mecamylamine, but not by a peripheral nAChR antagonist hexamethonium (Roth et al., 1982;

Imperato et al., 1986). Nicotine-induced dopamine release is accompanied by stimulation of

dopaminergic cell firing. In electrophysiological studies, nicotine in vivo accelerates the firing

frequency and increases the amount of burst firing in dopaminergic cell bodies of the SN and

VTA (Lichtensteiger et al., 1976, 1982; Clarke et al., 1985a; Grenhoff et al., 1986; Calabresi

et al., 1989; Nisell et al., 1996).

33

Dopamine release induced by chronic nicotine administration is dependent upon the dosing

regimen, brain area studied, species and even conditioning during nicotine administration.

Prolonged exposure to nicotine at low non-activating or higher activating doses attenuates the

nicotine-induced release of dopamine from rat and mouse synaptosomes (Grady et al., 1994;

Rowell and Hillebrand, 1994). Continuous infusion of nicotine at larger doses, that maintain

plasma nicotine concentrations similar to those commonly found in smokers, or repeated

intrastriatal administration of nicotine, have been found to attenuate the acute elevating effect

of nicotine on extracellular levels of dopamine and its metabolites both in the CPu and NAc

(Benwell et al., 1994, 1995; Benwell and Balfour, 1997; Lecca et al., 2000). This tolerance to

nicotine’s acute effects is thought to be due to desensitization of nAChRs mediating effects on

dopamine release. However, repeated nicotine injections to rats have also failed to produce

tolerance to the nicotine-induced increase in the accumbal dopaminergic transmission

(Damsma et al., 1989; Nisell et al., 1996). Indeed, daily nicotine pretreatment has been also

found to enhance nicotine-elevated endogenous dopamine content, dopamine release and

dopamine formation from tyrosine in accumbal slices (Fung, 1989). Repeated, intermittent

administration of nicotine sensitizes the acute elevating effect of nicotine on dopamine release

in vitro in rat striatal slices (Yu and Wecker, 1994) and in vivo in the CPu and to a greater

extent in the NAc in rats (Benwell and Balfour, 1992; Reid et al., 1996; Marshall et al., 1997;

Shim et al., 2001). The subdivisions of the NAc, the shell and core, respond differently to

repeated, discontinuous nicotine exposure. In the core, nicotine-induced increase of dopamine

overflow is thought to become sensitized (Cadoni and Di Chiara, 2000), although another

study failed to show such sensitization (Nisell et al., 1997). In the shell, the dopamine

overflow that nicotine acutely increases is reduced after repeated nicotine administration

(Cadoni and Di Chiara, 2000; Di Chiara, 2000).

The sensitization of dopamine release evoked by daily nicotine injections has been suggested

to relate to a regionally selective downregulation of the control of mesoaccumbens

dopaminergic neurons by inhibitory autoreceptors and to depend upon co-stimulation of

NMDA-type glutamate receptors (Shoaib et al., 1994; Balfour et al., 1998; Ferrari et al.,

2002). Also up-regulation of nAChRs requires stimulation of NMDA receptors, suggesting

that it may relate to enhanced responsiveness to repeated nicotine (Shoaib et al., 1997). The

sensitized responses of the mesolimbic dopaminergic pathway elicited by nicotine as well as

34

other drugs of abuse are suggested to be critical for the mechanisms underlying development

of addiction to these drugs (Robinson and Berridge, 1993).

2.7.1. Different effects of nicotine on dopamine in the CPu and NAc

Nicotine stimulates the firing rate of dopaminergic neurons more effectively in the VTA than

in the SNc in locally anaesthesized rats (Mereu et al., 1987; Westfall et al., 1989), whereas in

generally anaesthesized rats the response to nicotine does not differ between these two brain

areas (Grenhoff et al., 1986; Mereu et al., 1987; Westfall et al., 1989). Nicotine-evoked

currents in dopaminergic neurons have been found to be of a larger amplitude in the VTA

than in the SN (Klink et al., 2001). Nicotine preferentially increases dopamine metabolism

and synthesis in limbic areas, compared with striatal areas (Clarke et al., 1988; Grenhoff and

Svensson, 1988). The nicotine-induced increase of dopamine overflow is comparatively

greater and occurs at lower doses in the NAc than in the CPu (Imperato et al., 1986; Benwell

and Balfour, 1997). Furthermore, the dose-response curves of dopamine overflow in response

to nicotine differ between the CPu and NAc. In the CPu, the dose-response curve for nicotine

is bell-shaped so that maximal elevation of dopamine overflow occurs at the 0.4 mg/kg dose

and increase in the dose attenuates the elevation of dopamine. In the NAc however, the

dopamine overflow is nearly maximally elevated already at the 0.1 mg/kg dose and increase

in the nicotine dose prolongs the elevation of dopamine (Benwell and Balfour, 1997). Also,

when nicotine is given repeatedly, desensitization of dopamine overflow in the CPu requires

higher doses of nicotine than is needed in the NAc (Benwell and Balfour, 1997). Thus, there

appears to be physiological and functional differences between the nigrostriatal and

mesolimbic dopaminergic pathways and the mechanisms mediating nicotine’s effects on the

nigrostriatal pathway differ from those of the mesolimbic pathway.

2.7.2. nAChR-mediated modulation of dopaminergic transmission

Despite the wide distribution of nAChRs, these receptors do not play a major role in direct

mediation of central synaptic transmission, but rather modulate release of several

neurotransmitters, including dopamine, GABA, glutamate, noradrenaline and ACh (Role and

35

Berg, 1996). nAChRs can be divided into somatodendritic, preterminal and presynaptic

receptors on the basis of their location in the neuron (Wonnacott, 1997). Stimulation of

somatodendritic or preterminal nAChRs results in Na+ influx and local depolarisation of a

neuron, sufficient to activate voltage-sensitive Ca2+ channels that, in turn, provide calcium for

exocytosis of transmitter, such as dopamine, into the synaptic cleft (Harsing et al., 1992;

Prince et al., 1996; Soliakov and Wonnacott, 1996). Somatodendritic nAChRs have little

influence in the modulation of transmitter release once it has been initiated, while presynaptic

nAChRs appear to function as synaptic or non-synaptic receptors and effectively modulate

chemical transmission at the release site (Vizi and Lendvai, 1999). Stimulation of presynaptic

nAChRs can either modulate transmitter release initiated by axonal firing or itself induce a

sufficient Na+ and Ca2+ influx to depolarise the neuron and release transmitter. Transmitter

release mediated by either somatodendritic or presynaptic nAChRs is Ca2+-dependent, but

while the modulation by somatodendritic nAChRs is sensitive to tetrodotoxin (TTX), the

modulation by presynaptic nAChRs is mostly insensitive to TTX.

On the nigrostriatal and mesolimbic dopaminergic neurons, nAChRs are located at cell bodies

in the SN and VTA, as well as on nerve terminals in the CPu and NAc (Clarke and Pert, 1985;

Clarke et al., 1985b). Local administration of nicotine into the SN increases the firing of

nigrostriatal dopaminergic neurons and concentrations of dopamine metabolites in the dorsal

striatum (Lichtensteiger et al., 1976, 1982). Nicotine administered into the VTA increases

extracellular level of dopamine in the NAc (Yoshida et al., 1993), and this increase in the

accumbal dopamine overflow is significantly greater when nicotine is given into the VTA

rather than into the NAc (Nisell et al., 1994a; Ferrari et al., 2002). Indeed, nicotine-evoked

accumbal dopamine release is thought to be predominantly mediated by somatodendritic

nAChRs, because when the nAChR antagonist mecamylamine, is infused into the VTA,

dopamine release is abolished, but this does not occur when mecamylamine is infused into the

NAc (Nisell et al., 1994b). In addition, dopamine release is inhibited by TTX in the VTA

(Benwell et al., 1993; Marshall et al., 1996). However, nicotine infused directly into the

terminal field (CPu or NAc) does elevate dopamine release from the nerve terminals

(Marshall et al., 1997) and this effect is blocked by prior administration of the α7-selective

antagonist, MLA (Alkondon et al., 1992; Lecca et al., 2000). Thus, different types of nAChRs

located in the SN/VTA and CPu/NAc appear to be required for the full range of nicotine’s

effects on the dopaminergic pathways.

36

2.7.3. Subtypes of presynaptic nAChRs involved in dopamine release

Although there is strong evidence for the nAChR-mediated modulation of transmitter release,

the lack of knowledge about subunit compositions of native nAChRs and the limited number

of subtype-selective tools has restricted our understanding of nAChR subtypes involved in

this modulation. Moreover, precise localization of nAChRs in relation to synapses remains to

be elucidated. The array of subunit mRNA expressed by neurons in the SN and VTA has been

shown to be α3, α4, α5, α6, α7, β2, β3 and β4 (Wada et al., 1990; Göldner et al., 1997; Sgard

et al., 1999). In detail, 80-100% of midbrain dopaminergic neurons appear to express α4, α5,

α6, β2, and β3 subunits, 40-60% express α3 and α7 subunits, and a few neurons express β4

subunits (Le Novere et al., 1996; Klink et al., 2001; Azam et al., 2002). Characterization of

presynaptic nAChRs on striatal dopaminergic terminals has been significantly aided by the

discovery of the α-conotoxins, particularly α-CtxMII, the selective α3β2* and α6* receptor

antagonist (Cartier et al., 1996; Mogg et al., 2002). Presynaptic nAChRs of the striatum can

be divided into α-CtxMII-sensitive and α-CtxMII-resistant nAChRs (Kulak et al., 1997;

Kaiser et al., 1998). Immunoprecipitation and 6-OHDA lesioning studies identified the α-

CtxMII-sensitive nAChRs as α6β2(β3) and α6α4β2(β3) subtypes and the α-CtxMII-

insensitive nAChRs as α4β2 and α4α5β2 subtypes (Fig. 2-5; Zoli et al., 2002).

Fig 2-5. The subunit composition of striatal presynaptic nAChRs that mediate dopamine release. The α-CtxMII-sensitive receptors have one (α4α6β2β3) or two (α6β2β3) α-CtxMII-sensitive ACh-binding sites. The α-CtxMII-resistant receptors (α4β2, α4α5β2) each have two α-CtxMII-resistant ACh-binding sites. Modified from Luetje (2004), with permission.

37

In regard to the function of presynaptic nAChRs, it is suggested that striatal dopamine release

is mediated to a great extent by α4* nAChRs and to a lesser extent by non-α4* nAChRs

(Grady et al., 1992; Kulak et al., 1997; Kaiser et al., 1998; Sharples et al., 2000). Agonist-

stimulated dopamine release is almost completely removed both in vivo and in vitro in knock-

out mice lacking either α4 or α4α6 subunits (Champtiaux et al., 2003; Marubio et al., 2003).

Furthermore, nAChRs mediating striatal dopamine release seem to contain β2 subunit,

because nicotine-stimulated dopamine release is suppressed in mice lacking this subunit in

vivo (Picciotto et al., 1998) and in vitro (Grady et al., 2001; Zhou et al., 2001; Champtiaux et

al., 2003). The non-α4 subtype was first identified as the α3β2 subtype when it was observed

that the α3β2 antagonists, α-CtxMII (Cartier et al., 1996) and neuronal bungarotoxin (Luetje

et al., 1990), substantially inhibited dopamine release from striatal synaptosomes and slices

(Grady et al., 1992; Kulak et al., 1997; Kaiser et al., 1998). Shortly after this discovery α-

CtxMII, neuronal bungarotoxin and α-CtxPnIa, the toxins previously characterized as α3β2-

specific antagonists, were found to also block α6* nAChRs expressed in oocytes (Kuryatov et

al., 2000; Dowell et al., 2003; Everhart et al., 2003). Furthermore, both α-CtxMII binding in

dopaminergic nuclei and α-CtxMII-sensitive nicotine-stimulated [3H]dopamine release, were

abolished by deletion of the α6 (Whiteaker et al., 2002) or β3 subunit (Cui et al., 2003), but

not by deletion of the α3 subunit in knock-out mice (Champtiaux et al., 2002). The α6 and β3

subunits are often coexpressed with β2 in the SN and VTA (Le Novere et al., 1996), which

further supports the idea that the α-CtxMII-sensitive component of nicotine-induced

dopamine release is controlled by these subunits. Consistent with the localization of nAChR

subtypes in striatal dopaminergic terminals (Fig. 2-5; Zoli et al., 2002), the α-CtxMII-

sensitive dopamine release was recently suggested to be mediated by α6β2β3 and α4α6β2β3

subtypes and the α-CtxMII-resistant dopamine release by α4β2 and α4α5β2 subtypes

(Salminen et al., 2004). Thus, β2 is present in all these receptors, α4 in all resistant and some

sensitive receptors, and β3 in most or all sensitive, but no resistant receptors.

The α7 and β4 subunits are not significantly involved in the function of nAChRs on the

presynaptic terminals of dopaminergic projections to the striatum. Neither deletion of the β4

subunit in knock-out mice (Salminen et al., 2004) nor use of the α3β4 selective blocker, α-

CtxAuIB (Luo et al., 1998), altered agonist-stimulated dopamine release from synaptosomes

(Grady et al., 2001). Furthermore, β4 subunits are only expressed at low levels in the SN

(Klink et al., 2001). Both deletion of the α7 subunit (Salminen et al., 2004) and use of the α7

selective blockers, α-Bgt and α-CtxImI (Johnson et al., 1995; Pereira et al., 1996), failed to

38

suppress agonist-stimulated dopamine release in synaptosomal preparations (Rapier et al.,

1990; Kulak et al., 1997). However, synaptosomes represent isolated nerve terminals and

modulation of transmitter release reflects only direct effects via presynaptic receptors. On the

other hand, slice preparations allow the cross talk between nerve terminals that is present in

vivo to be observed. In striatal slices an additional component of dopamine release has been

observed. Dopamine release in striatal slices can be blocked by the α7-selective antagonists,

α-Bgt, α-CtxImI and MLA and also by glutamate receptor antagonists (Kaiser and Wonnacott,

2000; Wonnacott et al., 2000). Furthermore, in vivo infusion of MLA or NMDA-type

glutamate receptor antagonists into the VTA abolishes nicotine-induced accumbal dopamine

overflow, implying that this dopamine release is also indirectly controlled by presynaptic α7*

nAChRs, as well as by NMDA receptors located in dopaminergic nuclei (Schilström et al.,

1998a, b). While activation of nAChRs located in the VTA is required for the accumbal

dopamine release induced by systemic nicotine, nAChRs located in the NAc modulate this

response (Fu et al., 2000a). This fine tuning of the mesolimbic dopaminergic pathway through

α7* nAChRs in the NAc may amplify the release of dopamine, allowing a subthreshold brain

concentration of nicotine to become an effective stimulus for dopamine release. Furthermore,

α7 nAChRs are preferentially expressed in the VTA, compared with the SN (Wooltorton et

al., 2003), suggesting that these nAChRs might preferentially modulate dopaminergic

transmission in the mesolimbic pathway.

2.7.4. Modulation of nicotine-induced dopamine release by other neuro-transmitters, glutamate and GABA

The effects of nicotine on dopaminergic transmission are modulated by several other

neurotransmitters, including glutamate and GABA. Intra-striatally administered nicotine

elevates extracellular levels of both dopamine and glutamate and blocking glutamate receptors

diminishes nicotine-induced dopamine overflow in the striatum (Toth et al., 1992, 1993).

Both glutamate and α7* nAChRs seem to be involved in nicotine-induced dopamine release

in the NAc, because the release of dopamine is attenuated by intrategmental administration of

NMDA or α7* nAChR antagonists (Schilström et al., 1998a, b; Svensson et al., 1998; Fu et

al., 2000a, b; Grillner and Svensson, 2000). Indeed, glutamatergic neurotransmission and α7*

nAChRs are thought to amplify nicotine’s effects on dopamine secretion particularly in the

reward-mediating mesolimbic pathway. The α7* receptors can be localised to glutamatergic

39

nerve terminals (Fig. 2-3b on page 18; Marchi et al., 2002) and are thought to evoke

glutamate release, which, in turn, acts on glutamate receptors located on dopaminergic

terminals to stimulate dopamine release (Kaiser and Wonnacott, 2000; Nomikos et al., 2000).

However, in vivo glutamate release may be dependent on the nicotine dose. At low nicotine

doses, tonic activation of NMDA receptors in the VTA may be necessary for dopamine

release, while at higher doses the phasic release of glutamate in the VTA is involved in the

accumbal dopamine response (Fu et al., 2000b). There may also be somatodendritic or

postsynaptic α7* nAChRs that modulate glutamatergic transmission (Alkondon et al., 1996b;

Fisher and Dani, 2000).

Pharmacological stimulation of GABA receptors inhibits nicotine-induced dopamine release

in the NAc (Dewey et al., 1999; Schiffer et al., 2000), more precisely, in the NAc shell (Fadda

et al., 2003). In vitro, nicotine concentration-dependently stimulates GABA release in

synaptosomes, particularly at concentrations also found in smokers (Wonnacott et al., 1989;

Lu et al., 1998) and in slices prepared from rabbit caudate nucleus (Limberger et al., 1986),

rat IPN (Lena et al., 1993), rat hippocampus (Alkondon et al., 1997; Chiodini et al., 1999), rat

SN and globus pallidus (Kayadjanian et al., 1994) and mouse thalamus (Lena and Changeux,

1997b). It is thought that higher concentrations of nicotine fail to release GABA due to

desensitization of nAChRs (Chiodini et al., 1999). Nicotine also promotes the release of

GABA in vivo (Yang et al., 1996; Bertolino et al., 1997). It is proposed that nicotine directly

stimulates release of GABA via activation of nAChRs located on GABAergic neurons (Fig.

2-3 on page 18; Wonnacott et al., 1989; Lena et al., 1993; Alkondon et al., 1996a; Lena and

Changeux, 1997b). However, nicotine may also release other neurotransmitters, such as

dopamine and 5-HT, which indirectly modulate nicotine-stimulated GABA release in vivo

(Kayadjanian et al., 1994; Bianchi et al., 1995; Neal et al., 2001). The nAChRs that mediate

nicotine’s effects on GABA release are thought to be located at a preterminal site on

GABAergic axons (Lena et al., 1993). Expression of α2, α4, β2 or β3 subunits are not affected

by 6-OHDA lesioning of dopaminergic neurons, which suggests that these subunits may

preferentially be located on non-dopaminergic neurons including GABAergic neurons or

interneurons in the SN and VTA (Fig. 2-3; Göldner et al., 1997; Charpantier et al., 1998;

Kaiser et al., 1998; Sgard et al., 1999; Zoli et al., 2002). Recently, α7 and β2 nAChR mRNAs

have been found in the majority of GABAergic neurons (Fig. 2-3; Azam et al., 2003). Indeed,

the effects of nicotine on GABA release appear to be mediated via both α7* and α4β2*

40

receptors (Alkondon et al., 1997; Lena and Changeux, 1997b; Whiteaker et al., 2000;

Alkondon and Albuquerque, 2001). Consistent with this, deletion of the β2 subunit in knock-

out mice eliminates nicotine-induced GABA release (Lu et al., 1998).

Glutamate and GABA might offer an explanation for the controversial finding that a single

nicotine exposure is sufficient to increase dopamine level in the mesolimbic reward system

for hours (Nisell et al., 1994a; Ferrari et al., 2002), although nicotine concentrations

experienced by smokers desensitize nAChRs on dopaminergic neurons in seconds, or perhaps

minutes (Wonnacott et al., 1990; Alkondon et al., 2000; Mansvelder et al., 2002). Nicotine at

low concentrations increases the firing rate of dopaminergic neurons and transiently enhances

GABAergic transmission (Fisher et al., 1998) before these inhibitory GABAergic inputs are

persistently inhibited by nAChR desensitization (Mansvelder et al., 2002). The non-

dopaminergic, probably GABAergic neurons, in the VTA have been found to respond

robustly to nicotine at non-activating concentrations, but to desensitize (Lu et al., 1999; Yin

and French, 2000; Pidoplichko et al., 2004). Simultaneously, nicotine enhances glutamatergic

transmission via nAChRs that have a slower desensitization rate than than those mediating

GABA release. The released glutamate activates NMDA receptors and induces long-term

potentiation of excitatory inputs in the reward-related mesolimbic pathway (Mansvelder and

McGehee, 2000). Hence, spatial and temporal differences in nAChR activity on both

excitatory and inhibitory neurons in reward areas might coordinate to reinforce nicotine

reward and self-administration (Mansvelder et al., 2003).

2.8. Nicotine – a substance of abuse

The terms drug addiction or drug dependence are usually described as a compulsion to take a

drug with a loss of control over apparently voluntary acts of drug seeking and drug taking

(Wise and Bozarth, 1987). Addiction is a chronic disorder since even after treatment and

extended periods of drug abstinence, the risk of relapse to active drug use remains high.

Craving, intensely wanting drugs, is fundamental to addiction (Robinson and Berridge, 1993).

A drug that is able to support drug-seeking behaviour is a reinforcer (Stolerman and Shoaib,

1991). Phenomena that are pivotal to dependence-producing drugs are tolerance and

sensitization. Tolerance (attenuation of acute effects, such as aversive effects of a drug) and

41

sensitization (progressive increase in an acute effect with repeated treatment) may be

important for the dependence-producing properties. Withdrawal signs and symptoms result

from physiological adaptations induced by chronic drug exposure and are relieved when the

drug is used again.

Nicotine is known to play an essential role in maintaining a tobacco smoking habit and many

habitual smokers become dependent upon the drug (US Department of Health and Human

Sciences, 1988; Stolerman and Shoaib, 1991; Benowitz, 1996). Addiction to nicotine depends

on the rate and route of nicotine intake. Inhalation of nicotine via cigarette smoke appears to

be the most addictive method of intake (Benowitz, 1996). After inhalation, nicotine passes

rapidly to the arterial bloodstream and into the brain within 10-19 s (Russell et al., 1978). This

exposure results in relatively intense CNS effects that occur in close temporal proximity to

smoking, an ideal situation for behavioural reinforcement (Benowitz, 1996). Smoking is

characterized by intermittent peak concentrations of nicotine with each cigarette smoked,

followed by a low, steady-state baseline level until the next cigarette is smoked (Benowitz et

al., 1990; Clarke, 1993a). The level of nicotine in venous blood after smoking a single

cigarette ranges from 5 to 30 ng/ml depending upon how the cigarette is smoked (Herning et

al., 1983). During the day, the baseline levels of nicotine rise so that the influence of peak

levels becomes less important (Benowitz et al., 1990). In the afternoon, blood or plasma

concentrations of smokers generally range from 10 to 50 ng/ml and if a smoker smokes until

bedtime, measurable nicotine levels persist all night. The prolonged exposure to nicotine leads

to desensitization of nAChRs, which is though to explain tachyphylaxis (short-term tolerance

of rapid onset) and perhaps tolerance to nicotine associated with up-regulation of nAChRs in

the brain of a smoker (Benwell et al., 1988). Decline in the brain nicotine level between

cigarettes and especially overnight, provide an opportunity for nAChRs to return from a

desensitized state to a resting state, so positive reinforcement can, to some extent, occur with

successive cigarettes, despite the development of tolerance (Clarke, 1993a; Benowitz, 1996).

In smokers, nicotine self-administration appears to be motivated by both positive and negative

reinforcement (Benowitz, 1996; Koob, 1996). Positive reinforcing effects include relaxation,

reduced stress, enhanced vigilance, improved cognitive function, mood modulation and lower

body weight. Negative reinforcement refers to the relief of nicotine withdrawal symptoms,

such as nervousness, restlessness, irritability, anxiety, impaired concentration and cognitive

42

function. In addition to its reinforcing properties, nicotine is particularly effective in

establishing or magnifying the incentive properties and reinforcing the effects of associated,

non-pharmacological stimuli, such as the predictive visual and auditory cues typically found

in animal self-administration paradigms, or the taste and smell of tobacco smoke for smokers

(Rose and Corrigall, 1997; Balfour et al., 2000; Perkins, 2002).

2.8.1. Rewarding and reinforcing effects of nicotine in animal models

In habitual smokers, nicotine acts as a reinforcer and it is self-administered by volunteers

deprived of cigarettes (Henningfield and Goldberg, 1983). In rats, nicotine can act as a

discriminative stimulus (cue) so that rats can differentiate the effects of nicotine from those of

saline in order to obtain a food reinforcement (Rosecrans and Meltzer, 1981). Nicotine has

been found to effectively reinforce operant responding at doses comparable to those taken in

by human smokers in a self-administration model in several species, such as squirrel monkeys

(Goldberg et al., 1981), mice (Picciotto et al., 1998; Epping-Jordan et al., 1999) and rats

(Corrigall and Coen, 1989; Corrigall et al., 1992; Donny et al., 1995, 1998; Corrigall, 1999;

Donny et al., 1999). In self-administration studies in rats, the dose-response curve for nicotine

is bell-shaped, with the maximal response occurring at the dose range 30-60 µg/injection

(Corrigall and Coen, 1989). The lack of self-administration at higher nicotine doses may be

due to either aversive effects produced by these doses or desensitization of nAChRs

mediating the rewarding effects of nicotine (Benwell et al., 1995).

In the place conditioning paradigm, the primary motivational properties of a drug or non-drug

treatment serve as an unconditioned stimulus that is repeatedly paired with neutral

environmental stimuli (Tzschentke, 1998). During the conditioning the neutral stimuli

acquires motivational properties and may thus act as conditioned stimuli and elicit approach

(or withdrawal, if the primary motivational properties of the treatment were aversive) when

the animal is subsequently exposed to these stimuli. Nicotine has also been reported to

produce conditioned place preference in rats (Fudala et al., 1985; Fudala and Iwamoto, 1986;

Acquas et al., 1989; Carboni et al., 1989a; Calcagnetti and Schechter, 1994; Vastola et al.,

2002; Le Foll and Goldberg, 2005). However, the opposite results have also been obtained,

suggesting that nicotine-induced conditioned place preference is effected by the experimental

43

paradigm used (Clarke and Fibiger, 1987; Fudala and Iwamoto, 1987; Jorenby et al., 1990;

Calcagnetti and Schechter, 1994; Le Foll and Goldberg, 2005).

Similar to other drugs of abuse, the reinforcing and rewarding effects of nicotine appear to

depend upon direct and/or indirect stimulation of the mesolimbic dopaminergic pathway,

because lesions or pharmacological blockade of these neurons attenuate self-administration

(Singer et al., 1982; Corrigall and Coen, 1991; Corrigall et al., 1992). The pattern of increased

immediate-early gene expression exhibited in the terminal fields of the mesolimbic

dopaminergic neurons is similar for cocaine and nicotine self-administration in rats (Pagliusi

et al., 1996; Pich et al., 1997). Nicotine’s reinforcing effects seem to be mediated by nAChRs

located on dopaminergic neurons in the VTA, because they are diminished by intra-

ventricular administration of a nAChR antagonist chlorisondamine (Corrigall et al., 1992) or

by intrategmental administration of the nAChR antagonist DHβE (Corrigall et al., 1994).

These nAChR subtypes are likely to contain β2 subunit, because mice lacking this subunit

will not lever-press for nicotine, but they do lever-press for cocaine and food (Picciotto et al.,

1998; Epping-Jordan et al., 1999).

Additional neuronal connections to the VTA are thought to be important in drug reward.

Nicotine injected into the TPP supports place preference conditioning (Iwamoto, 1990) and

cholinergic lesions of the TPP or DHβE infused into this nucleus attenuate nicotine self-

administration (Lança et al., 2000b). Also GABA receptor agonists administered into the

VTA or TPP attenuate nicotine self-administration in rats (Corrigall et al., 2000, 2001; Fattore

et al., 2002; Markou et al., 2004; reviewed by Cousins et al., 2002). Furthermore, systemic

nicotine has been found to increase expression of immediate-early genes in the TPP and LDT

and this effect is possibly mediated via nAChRs located in non-cholinergic, presumably

GABAergic neurons that modulate the activity of cholinergic cells in the TPP, which, in turn,

regulates dopamine release in the mesolimbic system (Lança et al., 2000a).

2.8.2. The effects of nicotine on locomotor activity in rats

In man, nicotine is considered to have both depressant and stimulant actions (Gilbert, 1979).

In rats also, acute nicotine can induce either depressant or stimulant effects on locomotor

44

activity. The depressant effect occurs particularly in experimentally naïve rats at high nicotine

doses (Stolerman et al., 1973; Clarke and Kumar, 1983b). Depression of activity is readily

followed by stimulation as rats habituate to the test environment (Morrison and Stephenson,

1972; Clarke and Kumar, 1983a, b) and furthermore, if the rats have been previously

habituated to test apparatus, nicotine preferentially stimulates activity (O'Neill et al., 1991;

Benwell and Balfour, 1992). Repeated, intermittent administration of nicotine rapidly

produces tolerance to depressant effects and dose-dependently enhances stimulant effects, i.e.

produces behavioural sensitization (Morrison and Stephenson, 1972; Stolerman et al., 1973;

Bättig et al., 1976; Clarke and Kumar, 1983b; Ksir et al., 1985; Benwell and Balfour, 1992;

Whiteaker et al., 1995; Nisell et al., 1996). The sensitized locomotor response has been found

to be accompanied by the up-regulation of brain nAChRs (Ksir et al., 1985; Marks et al.,

1985). However, when nicotine is infused continuously, the stimulant effect is attenuated

(Benwell et al., 1994, 1995), which is suggested to be a consequence of down-regulation of

nAChR function (Marks et al., 1993).

The mesolimbic dopaminergic pathway is thought to play a pivotal role in mediating the

stimulant effects of nicotine, because lesions or pharmacological blockade of these neurons

attenuates the stimulation of locomotor activity (Clarke et al., 1988; Corrigall and Coen,

1991; Louis and Clarke, 1998). Stimulation of GABA receptors in the NAc reduces activity,

possibly by inhibiting accumbal dopamine release (Wachtel and Andén, 1978), and inhibits

hyperactivity induced by intra-accumbal injection of dopamine or amphetamine (Pycock and

Horton, 1979). Systemic nicotine elevates accumbal dopamine release at doses that stimulate

locomotion (Imperato et al., 1986; Clarke et al., 1988; Di Chiara and Imperato, 1988).

Repeated, intermittent administration of nicotine that results in the sensitized locomotor

stimulant response, also causes sensitization of its effects on dopamine overflow in the NAc

(Benwell and Balfour, 1992; Reid et al., 1996) and the prefrontal cortex (Nisell et al., 1996)

and possibly also in the CPu (Shim et al., 2001). Behavioural sensitization to nicotine is

associated with a reduction in dopamine overflow in the NAc shell and with an increase in

dopamine overflow in the core (Cadoni and Di Chiara, 2000).

Like nicotine’s actions on accumbal dopamine release (Nisell et al., 1994a; Ferrari et al.,

2002), the locomotor stimulant effect of nicotine appears to be primarily mediated by its

binding to nAChRs in the somatodendritic region (VTA), rather than in the terminal area

45

(NAc)(Museo and Wise, 1990; Reavill and Stolerman, 1990; Leikola-Pelho and Jackson,

1992; Panagis et al., 1996; Ferrari et al., 2002). However, nicotine’s actions on the

mesolimbic dopaminergic terminal fields in the NAc probably modulate its effects on

locomotor activity (Welzl et al., 1990). The α6* nAChR appears to mediate nicotine’s

stimulant effects on locomotor activity, because an infusion of α6 antisense oligonucleotides

reduces the effect (Le Novere et al., 1999). DHβE inhibits the locomotor stimulant effect of

nicotine at doses that do not antagonize its locomotor depressant effects, which suggests that

these two effects are mediated via different mechanisms (Stolerman et al., 1997).

Furthermore, deletion of the β2 subunit reduces the locomotor activity of habituated mice, but

not the exploratory activity of naïve mice (Picciotto et al., 1998).

2.9. Epibatidine

Epibatidine is an alkaloid originally purified from the skin of the Ecuadorian, poisonous frog

Epipedobates tricolor (Spande et al., 1992). It is a potent nAChR agonist that has proved to

be 80 to 2000 times more potent in preventing nociception than morphine (Badio and Daly,

1994; Sullivan et al., 1994). Epibatidine-induced analgesia is not blocked by the opioid

receptor antagonist naloxone, but it is abolished by mecamylamine and slightly reduced by

DHβE, which suggests that the effect is mediated by nAChRs (Badio and Daly, 1994; Damaj

et al., 1994; Bannon et al., 1995), particularly α4β2 nAChRs (Marubio et al., 1999). Indeed,

epibatidine displaces [3H]cytisine and [3H]nicotine binding in rat brain preparations, but fails

to displace ligands for several other CNS receptors, including opioid, muscarinic, adrenergic,

dopamine, 5-HT and GABA receptors (Qian et al., 1993; Badio and Daly, 1994). It displays

higher affinity at heteromeric nAChRs than at homomeric nAChRs (Table 2-2; Alkondon and

Albuquerque, 1995; Gerzanich et al., 1995; Bertrand et al., 1999). In contrast to nicotine,

epibatidine desensitizes α4β2 nAChRs at concentrations that do not activate the receptor and

up-regulates α4β2 nAChRs to a lesser extent than nicotine (Whiteaker et al., 1998; Buisson et

al., 2000). It also attenuates nicotine-induced up-regulation of α4β2 nAChRs, suggesting that

it might function as a partial up-regulatory agonist (Whiteaker et al., 1998).

46

Table 2-2. Binding affinities and functional potencies (measured by ion efflux) for acetylcholine (ACh), nicotine (NIC) and epibatidine (EPI) at native or recombinant mammalian nAChR subunits, and potency (EC50) for nicotine and epibatidine to release dopamine from rat striatal slices

Binding affinity Ki (nM)

Functional potency EC50 (µM) References

nAChR subtype ACh NIC EPI ACh NIC EPI

α4β2 8.6-34 1.7-8.1 0.01-0.09 3-270 0.59-14

0.014-0.043

a,b,c,e,f,g,k,l,n,p, q,u,v,x,y,z,,ac

α7 4000-19.000

400-14.000

20-233 79-322 40-83 0.17-1.3 d,h,l,m,o,p, q,s,t,w,y,z

α3β2 29 16 0.014-

0.035 26-126 6.6-7.7 0.1-0.19 g,h,l,r,t, u,v,x,aa

α6β2 8.0 2.5-3.8 0.034 n.d. n.d. n.d. f,ac

α3β4 560-881 183-475 0.15-0.59 160-217 7.5-110

0.07-0.128

a,l,p,t,u,v, x,aa,ab,ac

α4β4 72 26 kd 0.085 19 0.63-0.74 0.017 a,k,u,x

α2β2 1.8 0.81 kd 0.010 87-500 n.d. 0.29 a,t,u

α2β4 110 70 kd 0.087 82.6-44 1.95 n.d. u,x

α6β4 n.d. n.d. n.d. 18 7.1 0.0097 r

α6β2, α4α6β2 16.6 2.8 kd 0.046 n.d. n.d. n.d. f

Release of (-)-Nicotine (±)-Epibatidine

dopamine 60 nM 0.4 nM z

n.d. = not detected, a(Avalos et al., 2002), b(Bencherif et al., 1996), c(Bencherif et al., 1998), d(Briggs et al., 1995), e(Buisson et al., 1996), f(Champtiaux et al., 2003), g(Chavez-Noriega et al., 2000), h(Davies et al., 1999), i(de Fiebre et al., 1995), j(Free et al., 2002), k(Gentry et al., 2003), l(Gerzanich et al., 1995), m(Gopalakrishnan et al., 1995), n(Gopalakrishnan et al., 1996), o(Grottick et al., 2000), p(Hahn et al., 2003), q(Kem et al., 1997), r(Kuryatov et al., 2000), s(Meyer et al., 1998), t(Papke et al., 1997), u(Parker et al., 1998), v(Perry et al., 2002), w(Quik et al., 1997), x(Rao et al., 2003), y(Sharples et al., 2000), z(Sullivan et al., 1994), aa(Wang et al., 1996), ab(Xiao et al., 1998), ac(Zoli et al., 2002)

Epibatidine enhances 86Rb+ efflux in clonal cell lines and elicits calcium- and concentration-

dependent dopamine release from rat striatal and cortical slices, as well as noradrenaline

release from hippocampal and thalamic slices (Sullivan et al., 1994; Sacaan et al., 1996;

47

Jacobs et al., 2002). Epibatidine-evoked currents are characterized by late onset and offset,

compared with those evoked by ACh (Gerzanich et al., 1995; Bertrand et al., 1999; Buisson et

al., 2000). Epibatidine is also more efficacious in enhancing 86Rb+ efflux and dopamine

release from striatal synaptosomes than nicotine (Sullivan et al., 1994; Reuben et al., 2000),

but appears to be less efficacious in enhancing dopamine release from dendrosomal

preparations of the SN/VTA (Reuben et al., 2000). In slice preparations, it has been found to

evoke striatal dopamine release 150-240-fold more potently and cortical dopamine release

400-fold more potently than nicotine (Table 2-2; Sullivan et al., 1994; Puttfarcken et al.,

2000). It is also 180-fold more potent and more efficacious in releasing GABA from mouse

synaptosomes (Lu et al., 1998). Unlike nicotine, in vivo epibatidine preferentially increases

the extracellular dopamine level in the nigrostriatal pathway, compared with the mesolimbic

pathway (Seppä and Ahtee, 2000). In fact, it may even decrease dopamine overflow in the

NAc and frontal cortex (Bednar et al., 2004). However, epibatidine increases dopamine

metabolism in these brain areas (Seppä and Ahtee, 2000; Bednar et al., 2004). Prior treatment

with repeated nicotine attenuates the epibatidine-induced decrease in dopamine overflow, but

not the increase in dopamine metabolism in the NAc (Bednar et al., 2004).

Epibatidine provokes nicotine-like responding in the discrimination test in rats with 100-fold

greater potency than nicotine (Damaj et al., 1994). In a similar way to nicotine, it improves

attentional performance, and response rate and speed in rats (Hahn et al., 2003). In contrast to

nicotine, epibatidine lacks reinforcing effects in a self-administration test in mice (Rasmussen

and Swedberg, 1998) and anxiolytic activity on a plusmaze test (Sullivan et al., 1994). It

decreases locomotor activity and body temperature in mice, and prevents nociception 300 to

1000 times more potently and somewhat more efficaciously than nicotine (Damaj et al., 1994;

Sullivan et al., 1994; Bannon et al., 1995; Bonhaus et al., 1995). Like nicotine, acute

epibatidine decreases locomotor activity in naïve, unhabituated rats, but increases the activity

after a delay in habituated rats (Sacaan et al., 1996; Menzaghi et al., 1997b; Reuben et al.,

2000). Epibatidine increases locomotor activity to a lesser extent than nicotine and the

maximal increase by it occurs at the 3.0 µg/kg dose and that by nicotine at the 0.4 mg/kg dose

(Clarke and Kumar, 1983a; Menzaghi et al., 1997b; Reuben et al., 2000). In addition, prior

repeated treatment with nicotine enhances the acute locomotor stimulant effect of nicotine,

but attenuates that of epibatidine (Reuben et al., 2000).

48

Apart from ACh and nicotine, no other agent has had such an impact on nAChR research as

has epibatidine had (Dukat and Glennon, 2003). Its picomolar affinity for α4β2 nAChR

subtype and potent antinociceptive properties have stimulated interest in developing novel

nicotinic agents and nonopioid analgesics. Nowadays, several nicotinic compounds inspired

by epibatidine and nicotine are under evaluation in preclinical and clinical trials as possible

new treatments for neurodegenerative and mood disorders (Lloyd and Williams, 2000; Dukat

and Glennon, 2003; Hogg and Bertrand, 2004).

2.10. Nicotinic compounds in Parkinson’s disease

Although it is well-documented that nicotine (the main component in tobacco) has many

harmful effects, it has also been shown to have a number of positive effects in relation to

Parkinson’s disease. Several epidemiological studies have shown that people who smoke or

who have smoked during their lifetime, are 50% less likely to develop Parkinson’s disease

than those who have never smoked (Baron, 1986; Morens et al., 1995; Gorell et al., 1999;

Fratiglioni and Wang, 2000; Allam et al., 2004; Scott et al., 2005; Wirdefeldt et al., 2005).

This negative association between smoking and Parkinson’s disease is reproducible, dose-

related and not a result of selective mortality. However, the risk of Parkinson’s disease among

smokers is increased by other factors, such as old age and family history of Parkinson’s

disease (Elbaz et al., 2000). The mechanisms responsible for this decline in the incidence of

Parkinson’s disease are not known and several compounds in tobacco are able to alter

biological processes. One explanation for this may be the alteration of metabolic enzyme

activity. For example, activities of MAO-A and B are decreased in the brains of smokers,

which may attenuate the enzymatic oxidation of dopamine and thereby reduce oxidative stress

in dopaminergic neurons, or block MAO-mediated toxic xenobiotics (Yong and Perry, 1986;

Fowler et al., 1996, 2003). Another explanation may be nicotine’s interaction with

dopaminergic systems. Indeed, dopaminergic activity in the caudate and putamen is elevated

in smokers, compared with non-smokers (Salokangas et al., 2000).

Nicotine has been shown to have neuroprotective effects in several in vitro primary culture

models, for example, in striatal, cortical, cerebellar and mesencephalic neurons against several

toxic insults, including NMDA (Akaike et al., 1994; Marin et al., 1994; Sullivan et al., 1997;

49

Meyer et al., 1998; Minana et al., 1998) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

(MPTP), a toxin that selectively destroys dopaminergic neurons (Quik and Jeyarasasingam,

2000; Jeyarasasingam et al., 2002). In vivo, chronic nicotine administration to rats protects

dopaminergic cell bodies in the SN and nerve terminals in the striatum from mechanical

lesioning (Janson et al., 1988; Fuxe et al., 1990; Janson et al., 1991; Janson and Moller,

1993), restores glucose utilization in lesioned areas (Owman et al., 1989), counteracts lesion-

induced dopamine receptor up-regulation (Janson et al., 1994) and increases the rate of

dopamine turnover (Fuxe et al., 1990). In addition, smoking and acute and chronic nicotine

treatment have been shown to protect the nigrostriatal dopaminergic neurons from MPTP or

6-hydroxydopamine (6-OHDA)(Carr and Rowell, 1990; Janson et al., 1992; Maggio et al.,

1998; Costa et al., 2001; Ryan et al., 2001; Parain et al., 2003). However, at high

desensitizing doses nicotine fails to protect neurons and may even increase MPTP-mediated

toxicity (Behmand and Harik, 1992; Janson et al., 1992; Hadjiconstantinou et al., 1994).

Studies into the mechanisms of the neuroprotective actions of nicotine (reviewed by Picciotto

and Zoli, 2002) suggest that it may exert its effects by inducing expression of neurotrophic

factors that are known to promote survival of dopaminergic neurons (Maggio et al., 1998;

Roceri et al., 2001). Epibatidine is also shown to up-regulate the expression of neurotrophic

factors (Belluardo et al., 2000). The α7* nAChR appears to be involved in cell survival and

apoptosis (Shimohama et al., 1996; Kaneko et al., 1997; Li et al., 1999). Consistent with this,

α7-selective nAChR agonists (Briggs et al., 1997) have been found to elicit neuroprotective

effects in cultured cells (Buccafusco et al., 2004; Marrero et al., 2004).

Nicotine may also play a role in alleviating the symptoms of Parkinson’s disease once it has

developed. Smoking/nicotine has been found to ameliorate parkinsonian motor symptoms,

including tremor, bradykinesia, rigidity and lack of energy (Moll, 1926; Marshall and

Schnieden, 1966; Ishikawa and Miyatake, 1993; Fagerström et al., 1994; Kelton et al., 2000).

Motor symptoms are reduced shortly after smoking a cigarette for 10-30 min and this

reduction is more prominent after smoking, rather than chewing nicotine gum (Ishikawa and

Miyatake, 1993; Fagerström et al., 1994). One of the least understood areas of Parkinson’s

disease are the cognitive deficits, such as problems of attention, planning and cognitive

flexibility, and these symptoms also respond least to current pharmacological treatments.

Levodopa has been shown to alleviate (Pillon et al., 1989), fail to effect (Schneider et al.,

1999) or even exacerbate some cognitive deficits (Gotham et al., 1988; Lange et al., 1995).

50

Furthermore, levodopa has been reported to reduce several nAChR subtypes in monkey

striatum at doses ameliorating parkinsonian symptoms in both humans and monkeys (Quik et

al., 2003). Nicotine is known to improve attention and cognitive and memory performances

both in healthy subjects (Warburton et al., 1992; Levin et al., 1998; Levin and Rezvani, 2000,

2002) and in young and old animals (Brioni and Arneric, 1993; Levin and Torry, 1996;

Zarrindast et al., 1996; Grilly et al., 2000; reviewed by Decker et al., 1995; Levin and Simon,

1998). Furthermore, nicotine improves cognitive performance in parkinsonian patients

(Fagerström et al., 1994; Kelton et al., 2000), and it is the hippocampal α4β2 nAChRs that

have been proposed to mediate nicotine’s effects on memory functioning (Bancroft and

Levin, 2000). Consistent with this, nicotine and several novel β2-selective nAChR agonists,

such as altinicline and its racemates (Cosford et al., 2000), have been found to improve

MPTP-induced cognitive deficits (Schneider et al., 1999) and reverse cognitive and motor

deficits in combination with the parkinsonian drugs, levodopa and benserazide, in reserpine-

treated rats (Menzaghi et al., 1997a), as well as in MPTP-treated monkeys (Schneider et al.,

1998a, b; Domino et al., 1999). These nAChR agonists also stimulate locomotor activity and

dopamine turnover in rats (Menzaghi et al., 1997b; Sacaan et al., 1997). Potentiation of

levodopa’s effects by nAChR agonists may allow patients to reduce their levodopa dose and

eliminate complications of long-term levodopa treatment (Schneider et al., 1998b).

Another important aspect of Parkinson’s disease with respect to nAChRs, is the loss of high-

affinity nicotine binding sites in the caudate, putamen and SN, as well as in the cortex and

hippocampus (Aubert et al., 1992; Lange et al., 1993; Perry et al., 1995; Court et al., 2000b;

Paterson and Nordberg, 2000; Guan et al., 2002). The loss of nAChRs in the parkinsonian

brain is accompanied by a loss of choline acetyltransferase, indicating reduced cholinergic

transmission preferentially in cortical areas as compared with striatal areas (Nordberg et al.,

1985; Rinne et al., 1991; Aubert et al., 1992; Lange et al., 1993; Perry et al., 1993).

Furthermore, the loss of nAChRs parallels a decline in dopaminergic neurons (Piggott et al.,

1999; Quik et al., 2004) and is not associated with an overall loss of synaptic density, as

measured with synaptophysin immunoreactivity (Martin-Ruiz et al., 2000), but a specific loss

of nigrostriatal dopaminergic neurons (Court et al., 2000b). To date, the exact nAChR

subtypes lost in the human brain in Parkinson’s disease patients are not fully resolved. Low-

and high-affinity α-CtxMII binding sites are known to be present in the striatum and they are

both decreased in Parkinson’s disease (Quik et al., 2004). Furthermore, expression of α4, α7

51

and β2 subunits in putamen appear unaltered, both in parkinsonian subjects (Martin-Ruiz et

al., 2000) and in monkeys with dopaminergic lesions (Quik et al., 2000, 2005), suggesting

that these subunits are preferentially located on non-dopaminergic neurons. However,

expression of α3* subtypes has been found to be reduced in the caudate and temporal cortex

in parkinsonian patients (Guan et al., 2002). There appear also to be reduced expression of

β2* subtypes in the hippocampus and cortex, but an increased expression of α7 subtypes in

the cortex (Guan et al., 2002).

In rodent and monkey brain, destruction of the SN neurons by 6-OHDA or MPTP leads to

reduction in number of high-affinity nicotine binding sites in the SN and striatum (Schwartz

et al., 1984; Clarke and Pert, 1985), particularly those including α3, α5, α6, β3 and β4

subunits (Charpantier et al., 1998; Elliott et al., 1998; Quik et al., 2001, 2002). Consistent

with this, these nAChR subunits are reported to be located on dopaminergic neurons in

dopaminergic nuclei (Le Novere et al., 1996; Klink et al., 2001; Azam et al., 2002; Zoli et al.,

2002). The α6 subunit also appears to play a key role in the mediation of locomotor activity

(Le Novere et al., 1999), which further supports the importance of this nAChR subunit in

Parkinson’s disease. The α7 subunit may also be important in Parkinson’s disease (Quik and

Kulak, 2002), because it is implicated in neuromodulation, synaptic plasticity and

development (Role and Berg, 1996; Wonnacott, 1997; Dani, 2001), as well as in

neuroprotection (Shimohama et al., 1996; Kaneko et al., 1997; Li et al., 1999). The α7 subunit

has also been suggested to be involved in the cognition enhancing activity of nicotine via

modulation of glutamate release in the cerebral cortex (Levin and Simon, 1998).

2.11. Nicotinic compounds in other CNS disorders

nAChRs in the brain are suggested to play a role in attention, memory, sensation and

perception. If not causal, the involvement of nAChR function is likely to be important in

several neurodegenerative and mood disorders. Different nAChR subtypes seem to be

selectively implicated in different brain diseases in which nAChRs are shown to be modified.

For example, autosomal nocturnal frontal lobe epilepsy is related to a mutation in the α4

subunit, which reduces α4β2 subtype function and may cause nAChR-mediated seizures via

GABAergic systems (Steinlein et al., 1995; Weiland et al., 1996; Kuryatov et al., 1997;

52

Bertrand et al., 1998; Dobelis et al., 2003; Fonck et al., 2003). Abnormalities in the size and

number of nAChRs have been found in the cortex and cerebellum of autistic patients (Perry et

al., 2001; Lee et al., 2002; Martin-Ruiz et al., 2004) and in the hippocampus of older

individuals with Down’s syndrome (Court et al., 2000a). The status of nAChRs in Tourette’s

syndrome is not clear, but nicotine delivered by gum or transdermal patch can provide short-

term relief and may prevent symptoms of this syndrome from being exacerbated. However,

the use of nicotine is limited by frequent toxicity, primarily nausea (Dursun and Kutcher,

1999). Nicotine also improves attention in attention deficit and hyperactivity disorder in

adults (Levin et al., 2001).

Even though schizophrenic subjects are consistently found to smoke tobacco, which is likely

to up-regulate nAChRs, they were actually found to have reduced levels of both high- (α4β2)

and low-affinity (α7) nAChRs in several brain regions, including the striatum, hippocampus

and frontal cortex (Freedman et al., 1995; Guan et al., 1999; Breese et al., 2000; Durany et al.,

2000). These decreases are unlikely to result from neuroleptic medication, but they seem to be

associated with a high incidence of smoking among schizophrenics (Glassman, 1993; Dalack

et al., 1998). In fact, smoking is suggested to be a form of self-medication and nicotine

appears to normalize the sensory gating deficit associated with schizophrenia (Adler et al.,

1998). This deficit in schizophrenia is linked to a mutation in the α7 subunit (Stevens et al.,

1996; Leonard et al., 2002) and thus α7 nAChR agonists may be reasonable candidates for the

treatment of cognitive and perceptual disturbances in schizophrenia (Martin et al., 2004).

Indeed, in a similar way to nicotine, a novel partial α7-selective nAChR agonist has been

found to normalize an auditory evoked-potential deficit in mice expressing decreased levels of

α7 subunits (Stevens et al., 1998). In addition to schizophrenia, the incidence of smoking is

higher in people with bipolar disorder (manic depression) or with depression, and smoking

cessation therapies are less successful among these patients (Glassman, 1993; McEvoy and

Allen, 2002). Smoking may be a form of self-medication in subgroups of depressed

individuals that show a genetic predisposition (Lerman et al., 1998) and nicotine is thought to

modulate stress-induced responses that occur when a subject is not smoking (Balfour and

Ridley, 2000).

The loss of cholinergic neurons appears to result in learning and memory deficits associated,

for example, with Alzheimer’s disease. Drugs that enhance cholinergic neurotransmission,

53

such as cholinesterase inhibitors are used to compensate these deficits. The number of both

low- and high-affinity nAChRs decline with age (Nordberg, 1994; Court et al., 1997; Marutle

et al., 1998), but in Alzheimer’s disease and in dementia with Lewy bodies, there are

selective, region specific decreases in high-affinity nicotine binding sites post mortem

(Nordberg et al., 1992; Warpman and Nordberg, 1995; Nordberg et al., 1997; Court et al.,

2000b; Rusted et al., 2000). For example, in Alzheimer’s disease expression of α4 and

possibly α3 subunits is reduced in the neocortex (Hellström-Lindahl et al., 1999; Martin-Ruiz

et al., 1999; Guan et al., 2000), while expression of α7 subunits may increase (Hellström-

Lindahl et al., 1999) or decrease in the hippocampus (Guan et al., 2000), but appear to remain

unaltered in the cerebral cortex (Sugaya et al., 1990). Changes in expression of nAChRs are

shown to differ at various stages of Alzheimer’s disease and this may lead to inconsistencies

between reports (reviewed by Court et al., 2000a).

In addition to a loss of cortical innervation and basal forebrain neurons (Cummings and

Benson, 1987), the pathology of Alzheimer's disease is characterised by β-amyloid plaques

and neurofibrillary tangles, and inhibition of β-amyloid accumulation has been proposed to be

essential for effective therapy of this disease (Picciotto and Zoli, 2002). Nicotine has been

found to reduce β-amyloid aggregation, possibly via α7 nAChRs in the mouse brain

(Nordberg et al., 2002; Hellström-Lindahl et al., 2004b) and to antagonize β-amyloid-induced

memory deficits in mice (Maurice et al., 1996). Recently, smoking was reported to decrease

the level of β-amyloid in patients with Alzheimer’s disease (Hellström-Lindahl et al., 2004a).

However, the results of epidemiological studies into the protective effects of smoking in

Alzheimer’s disease are less consistent than those in Parkinson’s disease (Fratiglioni and

Wang, 2000; Sabbagh et al., 2002). Nicotine, as well as nAChR agonists showing selectivity

for nAChRs in the CNS, more precisely those consisting of α4β2 or α7 subunits, have been

found to improve overall cognitive performance, including memory in rats (Lippiello et al.,

1996; Meyer et al., 1997; Bencherif et al., 2000; Hahn et al., 2003), in monkeys (Bontempi et

al., 2003; Schneider et al., 2003) and in humans (Jones et al., 1992; Levin and Simon, 1998;

Gatto et al., 2004; Newhouse et al., 2004). The novel nAChR agonists appear to have less

harmful effects than nicotine which indicates a potential use for these compounds as treatment

for Alzheimer's disease (Lloyd et al., 1998; Meyer et al., 1998; Vernier et al., 1998; Cosford

et al., 2000; Gatto et al., 2004).

54

3. AIMS OF THE STUDY

The discovery of the multiplicity of nAChRs has stimulated interest in developing compounds

that selectively affect different nAChR subtypes in the brain and that might be therapeutically

beneficial in diseases, such as Parkinson’s and Alzheimer’s diseases, Tourette’s syndrome,

depression, schizophrenia, anxiety and pain. However, nAChRs also mediate effects on the

dopaminergic pathways involved in dependence and reward, which complicates the

development of compounds that affect nAChRs, but lack addictive properties. Thus, we

clarified differences in the nicotinic regulation of the nigrostriatal and mesolimbic

dopaminergic pathways by comparing the effects of two different nAChR agonists, nicotine

and epibatidine, on dopaminergic transmission in the CPu and NAc, and on dopamine-

mediated behaviours.

The detailed aims of the study were:

To clarify the mode of action of nicotine on dopaminergic transmission. Nicotine’s effects on

extracellular levels of dopamine and its metabolites in the CPu were studied in combination

with several drugs affecting dopaminergic transmission. These drugs affected endogenous

synaptic dopamine via inhibition of either dopamine reuptake (nomifensine), dopamine

metabolism (tolcapone, selegiline) or presynaptic dopamine receptors (haloperidol). (I)

To compare the effects of epibatidine and nicotine on dopamine overflow in the CPu and

NAc. The two nAChR agonists were given to rats acutely either alone or after pretreatment

with saline (II) or the dopamine uptake inhibitor nomifensine (III).

To explore if changes in striatal dopaminergic transmission result in altered behaviour of rats.

The effects of nicotine and epibatidine were studied in the well-known parkinsonian animal

model, rotational behaviour of unilaterally 6-OHDA-lesioned rats. Nicotine and epibatidine

were given acutely and repeatedly either alone or after pretreatment with saline or

nomifensine. (I, IV)

To explore if the effects of nicotine and epibatidine on dopaminergic transmission lead to

changes in locomotor activity and conditioned place preference. (V) Additionally, nicotine’s

effects on locomotor activity were studied at an elevated ambient temperature (+30-33ºC).

55

4. MATERIALS AND METHODS

4.1. Animals

Naïve male Wistar rats were used in all experiments. The rats were bred at the Laboratory

Animal Center, University of Helsinki (I-IV) or purchased from the Harlan Netherlands BV

(II, V). The rats used in the microdialysis and rotational behaviour studies weighed 250-380 g

and those used in the locomotion and place preference studies weighed 230-300 g. The animal

room was kept at an ambient temperature of 20-23°C and a 12-h light-dark cycle, with lights

on from 6:00 to 18:00. Standard rat chow and tap water were available ad libitum. The rats

were housed in groups of four, except after the stereotaxic surgeries. In the microdialysis

studies, the rats were housed one rat per cage during the recovery and experiment. In the

rotational behaviour studies, the rats lesioned with 6-OHDA were allowed to recover for 1-2

days in single cages and thereafter housed in the same group of four rats as before the surgery.

The experimental designs were approved by the Committee for Animal Experiments of the

University of Helsinki (I-V) and the chief veterinarian of the county administrative board (I-

IV). All experiments were conducted according to the European Convention for the

Protection of Vertebrate Animals used for Experimental and other Scientific Purposes.

4.2. Drugs

nAChR agonists, (-)-nicotine base (Fluka Chemie, Buchs, Switzerland) and (±)-epibatidine

hydrochloride (Sigma, MO, U.S.A.) were diluted with saline (0.9% NaCl solution). The pH of

the nicotine solution was adjusted to 7.0-7.4 with 0.05M HCl. nAChR antagonists,

hexamethonium chloride (Sigma, MO, U.S.A.) and mecamylamine hydrochloride (Merck, NJ,

U.S.A.) were dissolved in saline. Haloperidol (Orion-Pharma, Finland), selegiline (a gift from

Orion-Pharma, Finland) and tramadol hydrochloride (Orion-Pharma, Finland) were dissolved

in saline. The pH of the haloperidol solution was adjusted to 7.2 with 1M NaOH.

Nomifensine maleate (RBI, MA, U.S.A. or Sigma, MO, U.S.A.) and desmethylimipramine

hydrochloride (Sigma, MO, U.S.A.) were dissolved in sterile water. Tolcapone (a gift from

Orion-Pharma, Finland) was suspended in phosphate buffer (pH 7.4) containing a drop of

56

Tween 80. Carbidopa (a gift from Orion-Pharma, Finland) was suspended in 5% gum arabic.

Levodopa methyl ester hydrochloride (Sigma, MO, U.S.A.) was dissolved in distilled water.

6-OHDA hydrochloride (Sigma, MO, U.S.A.) was dissolved in saline containing 0.02%

ascorbic acid. Nicotine, epibatidine, haloperidol, selegiline and tramadol were administered

subcutaneously (s.c.) and the other drugs were given intraperitoneally (i.p.). The drugs were

mainly administered in a volume of 1 ml/kg, but nomifensine was given in a volume of 3

ml/kg and levodopa, carbidopa, selegiline and tolcapone in a volume of 2 ml/kg. All drug

doses refer to free base.

4.3. Analysis of epibatidine stock solutions

All together, three epibatidine batches and stock solutions with different concentrations (12,

30 and 50 µg/ml) were used in these experiments. Stock solutions were stored in small

aliquots at –80°C and on each experimental day an aliquot was defrosted and diluted with

saline to the required concentration (0.1-3.0 µg/ml). To ensure that the stability of epibatidine

was not affected by freezing, defrosting or dilution procedures, we analysed two stock

solutions using ultraviolet-spectrometry and thin-layer chromatography (TLC Silica gel 60

F254, 0.2 mm, Merck). One solution analysed was stored at –80°C for eight months and the

other solution was newly made. As shown in Fig. 4-1, in ultraviolet-spectrometry the maximal

absorbance occurred at the wavelength 270 nm and the absorbances of the stock solutions

were comparable (0.59 and 0.52, respectively). Thin-layer chromatography was also used to

detect any degradation of epibatidine solutions, but none was detected.

Fig. 4-1. The absorbance curves of two epibatidine stock solutions. The upper curve is from the solution stored at –80°C for eight months and the lower curve is from the newly made solution.

57

4.4. In vivo microdialysis (I, II, III)

The rats were under general halothane (3.5% induction, 2% maintenance) and local lidocaine

anaesthesia as microdialysis guides were implanted using stereotaxic guidance. In the CPu,

we used microdialysis probes with 2.00 mm (II) or 4.00 mm (I-III) membrane (BAS MD-

2200, 0.32 O.D., Bioanalytical Systems, IN, U.S.A.) and therefore, coordinates relative to

bregma and dura in the insertion of the BAS MD-2250 guide cannula (Bioanalytical Systems,

IN, U.S.A.) were A/P +1.0, L/M ±2.7, and D/V -2.0 or -4.0 (Paxinos and Watson, 1986).

Thus, final location of the tip of the microdialysis probe was D/V -6.0 for the CPu in all

experiments. In the NAc, coordinates of the guides were A/P +1.7, L/M ±1.4 and D/V -6.3

and final location of the tip of the probe was D/V -8.3. After the surgery, the rats were given

tramadol 1.0 mg/kg for postoperative pain, after which each rat was placed in its own test

cage and allowed to recover for five to eight days before conducting experiments.

The microdialysis experiments were performed in freely moving animals. In experiments

described in (I), we used a dummy probe to shorten the equilibration period on the

experimental day. The dummy probe was inserted through the guide cannula one day prior to

the dialysis experiment and it was replaced by an identical experimental probe the next

morning. In experiments (II, III), no dummy probe was used, but the experimental probe was

inserted between 7:00 and 8:00 on the experimental day. Input of the probe was connected to

a microperfusion pump and artificial cerebrospinal fluid (Ringer solution, pH 7.4 containing

147 mM NaCl, 1.2 mM CaCl2, 2.7 mM KCl, 1.0 mM MgCl2 and 0.04 mM ascorbic acid) was

infused through the probe at a flow rate of 2 µl/min. Ringer solution was infused for 2 h in

experiment (I) and for 3-3.5 hours in experiments (II, III) before collecting baseline samples.

When a stable outflow was shown by four consecutive samples of dopamine, the rats were

given drugs and microdialysate samples (30 µl) were collected in 15 min intervals for 2.5-4 h

after the last drug administration. When the nAChR agonists were studied in combination

with other drugs, the drugs were administered in 30 min intervals in all microdialysis

experiments, except for those studying selegiline. Microdialysate levels of dopamine,

DOPAC, HVA and 5-hydroxyindoleacetic acid (5-HIAA) were analysed immediately with

high-performance liquid chromatography (HPLC) with electrochemical detection.

58

The HPLC system consisted of a Coulochem II detector (ESA Inc., MA, U.S.A.) equipped

with a model 5014B microdialysis cell, a model 2248 HPLC pump (Pharmacia LKB,

Sweden) and a model LP-21 pulse damper (Scientific Systems Inc., PA, U.S.A.). The column

(Spherisorb ODS2, 3 µm, 4.6x100 mm, Waters, U.S.A.) was kept at 40°C with a CROCO-

CIL column heater (Cluzeau Info-Labo, France). The mobile phase was 0.1 M NaH2PO4

buffer, pH 4.0 (adjusted with 1.0 mM citric acid), 0.6-0.95 mM octane sulphonic acid, 10-

15% (v/v) methanol and 1.2 mM EDTA. A flow rate of the mobile phase was 1.0 ml/min. A

CMA/200 autoinjector (CMA/Microdialysis, Stockholm, Sweden) was utilized for injecting

20 µl of the microdialysate sample into the HPLC system. Dopamine was reduced with an

amperometric detector (potential –100 mV) after having been oxidized with a coulometric

detector (+300 mV). Chromatograms were processed with a C-R5A chromatointegrator

(Shimadzu Co., Kyoto, Japan). Values were not corrected for in vitro probe recovery

efficiency, which was approximately 25% for dopamine and 9-12% for DOPAC, HVA and 5-

HIAA. The average concentration of the first four stable dialysis samples (< 20% variation)

was determined as baseline (= 100%) and data are expressed as percentage change of the

baseline values.

At the end of the experiments, the rats were anaesthetised with CO2, decapitated and brains

were rapidly removed and frozen on dry ice (-80ºC). Positions of the probes in the CPu and

NAc were examined visually or microscopically from 100-µm coronal sections stained with

thionine.

4.5. Rotational behaviour (I, IV)

The rats were under general halothane (3.5% induction, 2% maintenance) and local lidocaine

anaesthesia as 6-OHDA was injected using stereotaxic guidance. The rats were pretreated

with a noradrenaline uptake inhibitor, desmethylimipramine (15 mg/kg i.p.) 30 min before

injecting 6-OHDA in order to protect noradrenergic nerves from 6-OHDA. The 6-OHDA

solution (2 µg/µl) was prepared just before the surgery. It was kept on ice throughout the

surgery and if the solution developed a reddish colour, a sign of oxidation of 6-OHDA, a new

solution was prepared. 6-OHDA solution was injected with a self-made 30 gauge stainless

steel cannula attached by polyethylene tubing to a 10 µl Hamilton syringe driven by a syringe

59

pump. The injection cannula filled with 6-OHDA solution was carefully inserted through a 1

mm hole in the skull into the MFB using coordinates relative to bregma and dura (A/P –4.2,

L/M ±1.4, D/V –8.2) according to the rat brain atlas (Paxinos and Watson, 1986). A total of 8

µg of 6-OHDA base was infused slowly at a flow rate of 1 µl/min during 4 min and the

injection cannula was kept in place for an additional minute to prevent the backflow of

solution. After the surgery, the rats were allowed to recover in individual cages for 1-2 days

and thereafter in groups of four up to a total of 14 days before conducting experiments.

The rotational experiments were conducted for each group of rats every day at the same time

(±1 h). Rotational behaviour of the rat was measured in a circular metal bowl (35 cm

diameter, 15 cm high) surrounded by a transparent 40 cm high cylinder. The rat was attached

to a rotation sensor with a spring steel tether connected to a plastic collar around the neck of

the rat. The rotation sensor detected full (360°) clockwise (in this case, ipsilateral) and counter

clockwise (contralateral) turns, and the rotations were counted in 15 min intervals for 3-4 h.

When nAChR agonists were studied in combination with other drugs, the drugs were

administered in 15 min intervals in all rotational experiments. In experiments (IV) the drugs

were administered repeatedly on five consecutive days and once more on the day 8. The

rotations were measured on test days 1, 3 and 5 and challenge day 8.

In order to verify depletion of dopamine in the CPu, the rats were decapitated two days after

the rotational experiments. The brains were removed from the skull and placed into the rat

brain mould. A 3x3 mm sized sample was punched out of the dorsal striatum and the

dissected samples were immediately frozen on dry ice and stored at –80 °C until the assay

was carried out. The samples were homogenized and purified as described earlier (Haikala,

1987) and thereafter assayed for dopamine concentration with the HPLC system using a C-18

reverse-phase column (Spherisorb ODS2, 3 µm, 4.6x250 mm, Waters, U.S.A.) and

electrochemical detection (+780 mV, Waters Model 464 detector, Millipore, MA, U.S.A.).

Only rats with dopamine depletion of more than 95% in the lesioned right dorsal striatum,

compared with the intact left dorsal striatum, were included in the final data analysis. With

this criterion, about 90% of the rats were qualified and the mean dopamine depletion of these

rats was 100%. Except for the experiments with levodopa, all the qualified rats rotated

predominently in the ipsilateral direction and thus the few contralateral rotations seen were

excluded from the data.

60

4.6. Locomotor activity monitoring (V)

Drugs were administrated and the locomotor activity of each group of rats was monitored

every day at the same time (±1 h) between 8:00-13:00. The rats were individually placed in

43x43x30 cm3 Plexiglas boxes (MED Associates ENV-515, Vermont, GA, U.S.A.). The

computer registered and analysed infrared photo beam interruptions. The rats were first

habituated to the activity monitoring boxes on two consecutive days for 2 h each. On the first

habituation day, the rats were placed in the boxes and their activity was measured, while on

the second day the rats were given s.c. saline before placing them in the boxes and measuring

the activity. On the following day, test day 1, the rats were given drugs and the locomotor

activity was measured in 5 min intervals for 2 h. The drugs were given repeatedly on five

consecutive days, and the locomotor activity was measured on test days 1, 3 and 5. On days 2

and 4 the drugs were given in the home cages. The rats repeatedly given epibatidine (or saline

as control) were given an additional challenge dose of epibatidine (or saline) on test day 7 and

the locomotor activity was monitored similarly to previous test days. The locomotion

experiments were normally carried out at an ambient temperature of 20-23°C, but some

experiments were conducted at the higher ambient temperature of 30-33°C and for those

experiments, the rats were habituated to 30-33°C for two days before conducting the

experiments.

4.7. Place conditioning (V)

The place conditioning apparatus consisted of identical Plexiglas chambers divided into two

equally sized compartments (41x41x28 cm3) by a separating black wall with guillotine door

(MED Associates, Vermont, GA, U.S.A.). In one compartment there was a grid rod floor and

the walls were covered outside with a white layer (“white” compartment), whereas in the

other compartment there was a wire mesh floor and the walls were covered with a black layer

(“black” compartment). The computer registered interruptions of infrared photo beams, which

were used both to measure the locomotor activity and to determine the rat’s position in the

compartments. The rats were accustomed to experimenter by handling and weighing them for

5 min on 2-3 days before experiments. On test days, the experiments were conducted each

day at the same time (±1 h) during 8:00-14:00 and the rats were habituated to the experiment

61

room for 30 min before the experiments. White noise was used to cover background noise.

Each experiment was performed over six consecutive days and consisted of three phases:

habituation (days 1 and 2, one session per day), conditioning (days 3, 4 and 5, two sessions

per day), and testing (day 6, one session).

Habituation. The rats were in the test chambers with free access to both compartments (door

open). We used a biased conditioning design, in which rats initially prefer one compartment,

and thus, only the rats that initially spent more time in the black compartment with a wire

mesh floor were included in the data. The time that the rats spent in the non-preferred (white)

compartment during the first 15 min on the second habituation day was used as an initial

preference level for each rat (preconditioning time).

Conditioning. In the morning, the rats were given saline and immediately exposed to the

preferred black compartment (door closed). Three to four hours later, the rats were given

drugs or saline (controls) and immediately exposed to the non-preferred white (drug-paired)

compartment (door closed). The conditioning time was either 20 min (nicotine) or 60 min

(epibatidine).

Testing. On day 6, the preference for compartments was tested without any drug

administration similarly to the testing on the second habituation day. The rats had free access

to both compartments (door open) and the time in the drug-paired (white) compartment

(postconditioning time) was measured. The differences in the postconditioning and

preconditioning times spent in the drug-paired compartment were calculated. When the

change was positive, a drug was considered to induce place preference.

4.8. Statistical analysis

All data are expressed as means ± standard errors of the mean (S.E.M.). The microdialysis,

rotational behaviour and locomotor activity data (I-V) were analysed with one-, two- or three-

way analysis of variance (ANOVA) for repeated measures (Statview 5.0, SAS Institute Inc.,

U.S.A. or GraphPad Prism 3.0 for Windows, GraphPad Software, San Diego, CA, U.S.A.).

When appropriate (P < 0.05), multiple comparisons were conducted by using Student

Newman Keuls (I, IV, V) post hoc tests or contrast analysis with Bonferroni levels (II, III).

The basal concentrations in the microdialysis samples and part of the rotational data were

analysed with Student’s t-test (I-IV).

62

5. RESULTS

5.1. Microdialysis studies

5.1.1. Effects of nicotine on the extracellular concentrations of dopamine,

DOPAC, HVA and 5-HIAA in the CPu in combination with various dopaminergic drugs (I)

Nicotine (0.5 mg/kg s.c.) was given in combination with either the DAT inhibitor nomifensine

(3 mg/kg i.p.), the COMT inhibitor tolcapone (10 mg/kg i.p.), the MAO-B inhibitor selegiline

(5 x 1 mg/kg s.c.) or the dopamine receptor blocker haloperidol (0.1 mg/kg s.c.). The effects

of nicotine, nomifensine, tolcapone and haloperidol on the extracellular concentrations of

dopamine and its metabolites DOPAC and HVA in the CPu are summarised in Table 5-1.

Table 5-1. The maximal peak changes in the extracellular concentrations of dopamine, DOPAC and HVA in the caudate-putamen of rats given acute saline or nicotine (0.5 mg/kg) either alone or in combination with various dopaminergic drugs

Treatment Dopamine% DOPAC% HVA%

Saline controls 88 ± 12 105 ± 5 105 ± 5

Nicotine (0.5 mg/kg) 140 ± 16 ↑ 125 ± 4 ↑ 137 ± 6 ↑↑

Nomifensine (3 mg/kg) + saline

201 ± 28 ↑

89 ± 4

125 ± 8

Nomifensine (3 mg/kg) + nicotine (0.5 mg/kg)

370 ± 55 ↑↑↑ * #

90 ± 5 *

124 ± 11

Tolcapone (10 mg/kg) + saline

88 ± 10

142 ± 13 ↑↑

13 ± 4 ↓↓↓

Tolcapone (10 mg/kg) + nicotine (0.5 mg/kg)

134 ± 8 ↑ #

177 ± 11 ↑↑↑ ** #

21 ± 5 ↓↓↓ ***

Saline + haloperidol (0.1 mg/kg)

182 ± 15

206 ± 8 ↑↑↑

272 ± 15 ↑↑↑

Nicotine (0.5 mg/kg) + haloperidol (0.1 mg/kg)

185 ± 17 ↑

228 ± 11 ↑↑↑ ***

292 ± 19 ↑↑↑ ***

Data on repeated dialysate samples were analysed with 2-way ANOVA followed by Student Newman Keuls test: ↑ P < 0.05, ↑↑ P < 0.01, ↑↑↑ P < 0.001, ↓↓↓ P < 0.001 vs. saline controls, * P < 0.05, ** P < 0.01, *** P < 0.001 vs. nicotine alone, # P < 0.05 vs. corresponding dopaminergic drug + saline. Data are presented as percentage change values (means ± S.E.M., n = 6-8 in all groups) of the baseline values (= 100%).

63

Nicotine (0.5 mg/kg) significantly elevated the extracellular levels of dopamine, DOPAC and

HVA in the CPu (P < 0.05). The elevations were inhibited by the brain penetrating nAChR

antagonist mecamylamine (5 mg/kg i.p., P < 0.05), but not by the peripheral nAChR

antagonist hexamethonium (5 mg/kg i.p.). Nomifensine (3 mg/kg) significantly increased the

extracellular level of dopamine (P < 0.05), but did not alter dopamine metabolites in the CPu.

When nicotine was given after nomifensine, the dopamine level was further elevated,

compared with the elevation induced by either nomifensine or nicotine alone (P < 0.05).

Tolcapone (10 mg/kg) did not alter the dopamine level, but increased the level of DOPAC (P

< 0.01) and decreased that of HVA (P < 0.001). When nicotine was given after tolcapone, the

dopamine level was elevated to the same degree as when nicotine was given alone. However,

the DOPAC level was further elevated, compared with the elevation induced by either

tolcapone or nicotine alone (P < 0.05). The level of HVA was decreased to the same extent as

after tolcapone alone. Although haloperidol (0.1 mg/kg) did not significantly elevate the

dopamine level, it significantly elevated the levels of DOPAC and HVA (P < 0.001). When

nicotine was given before haloperidol, the elevation in the levels of dopamine, DOPAC and

HVA was the same as after haloperidol alone. None of the drugs studied altered the

extracellular 5-HIAA concentration in the CPu (data not shown). Furthermore, when the

nAChR antagonist mecamylamine (5 mg/kg i.p.) was given in combination with the

dopaminergic drugs, it did not alter the effects of nomifensine, tolcapone or haloperidol on the

levels of dopamine, DOPAC, HVA or 5-HIAA (unpublished data; data not shown).

Repeated daily pretreatment with selegiline (5 x 1 mg/kg s.c.) elevated the basal extracellular

dopamine concentration and decreased the basal level of dopamine metabolites, particularly

that of DOPAC, in the CPu (Table 5-2; P < 0.05). Selegiline did not alter the basal

extracellular 5-HIAA concentration in the CPu. Nicotine did not significantly alter the

extracellular levels of dopamine, DOPAC, HVA or 5-HIAA when given acutely 3 h after the

fifth selegiline administration (Table 5-2).

64

Table 5-2. The basal extracellular concentrations of dopamine, DOPAC, HVA and 5-HIAA in the baseline samples of the caudate-putamen after daily repeated saline or selegiline (5 x 1 mg/kg) pretreatment and the maximal peak changes from these baseline levels (= 100%) after the selegiline-pretreated rats were given acute saline or nicotine (0.5 mg/kg)

Baseline levels Treatment

Dopamine (fmol/20 µl)

DOPAC (pmol/20 µl)

HVA (pmol/20 µl)

5-HIAA (pmol/20 µl)

Saline (5 x 1 ml/kg) 72 ± 23 21.2 ± 3.8 14.1 ± 3.2 3.5 ± 0.6

Selegiline (5 x 1 mg/kg) 394 ± 98 ↑ 7.9 ± 0.9 ↓ 8.2 ± 0.9 3.6 ± 0.3

Peak changes Treatment Dopamine% DOPAC% HVA% 5-HIAA%

Selegiline (5 x 1 mg/kg) + saline

50 ± 15

63 ± 12

42 ± 7

60 ± 11

Selegiline (5 x 1 mg/kg) + nicotine (0.5 mg/kg)

67 ± 8

88 ± 9

64 ± 12

73 ± 11

Selegiline data on the baseline samples were analysed with Student’s t-test: ↑ P < 0.05, ↓ P < 0.05 vs. saline pretreatment (mean ± S.E.M., n = 6-10). The baseline dialysate samples were collected 2 h after the fifth selegiline or saline administration. Data on peak changes are presented as percentage change values (means ± S.E.M., n = 4-6) of the baseline values (= 100%). This data were analysed with 2-way ANOVA (P > 0.10).

5.1.2. Effects of nicotine and epibatidine on the extracellular concentrations of dopamine, DOPAC, HVA and 5-HIAA in the CPu and NAc (II)

The effects of nicotine (0.5 mg/kg s.c.) and epibatidine (0.1, 0.3, 0.6, 1.0, 2.0 and 3.0 µg/kg

s.c.) on the extracellular dopamine concentration in the CPu and NAc are shown in Fig. 5-1.

Nicotine (0.5 mg/kg) significantly elevated the dopamine level in the CPu and even more so

in the NAc (P < 0.0001). Epibatidine significantly reduced the dopamine level in the CPu at

0.3 µg/kg and increased it at 0.6 and 1.0 µg/kg (P < 0.0001). The larger doses of epibatidine

(2.0 and 3.0 µg/kg) failed to alter the dopamine level in the CPu. However, the accumbal

dopamine level was significantly elevated at the 3.0 µg/kg dose of epibatidine (P < 0.0001),

but not at the lower epibatidine doses studied (0.1-2.0 µg/kg).

65

-60 0 60 120 180 240

NIC ↑↑↑

SAL

EPI 0.6 ↑↑↑

75

100

125

150

175D

A%

of t

he b

asel

ine

-60 0 60 120 180 240

SALNIC ↑↑↑

EPI 0.6

75

100

125

150

175

-60 0 60 120 180 240

SAL

EPI 3.0

EPI 2.0

EPI 1.0 ↑↑↑

75

100

125

150

175

DA

% o

f the

bas

elin

e

-60 0 60 120 180 240

SAL

EPI 3.0 ↑↑↑

EPI 2.0

EPI 1.0

75

100

125

150

175

-30 0 30 60 90 120 150

EPI 0.1

75

100

125

150

175

EPI 0.3 ↓↓↓

SAL

Time (min)

DA

% o

f the

bas

elin

e

-30 0 30 60 90 120 150

EPI 0.1

75

100

125

150

175

EPI 0.3

SAL

Time (min)

Caudate-putamen Nucleus accumbens

Fig. 5-1. The extracellular dopamine (DA) concentrations in the caudate-putamen and nucleus accumbens of the rats given acute nicotine (NIC, 0.5 mg/kg s.c.), epibatidine (EPI, 0.1, 0.3, 0.6, 1.0, 2.0 or 3.0 µg/kg s.c.) or saline (SAL, 1 ml/kg s.c.). The arrows indicate the time points of drug injections: some rats were first pretreated i.p. with saline (first arrow of the two arrows) and then given s.c. the nAChR agonists (second arrow), while some rats were not pretreated, but given s.c. epibatidine (0.1 or 0.3 µg/kg) or saline (single arrows in the figures below). Data on repeated dialysate samples were analysed with 2-way ANOVA followed by contrast analysis: ↑↑↑ P < 0.0001, ↓↓↓ P < 0.0001 vs. saline-treated controls. Data are presented as mean percentage change values (n = 6-8 in all groups) of the baseline values (= 100%).

66

The effects of nicotine and epibatidine on the extracellular concentrations of DOPAC, HVA

and 5-HIAA are summarised in Table 5-3. Neither nicotine nor epibatidine significantly

altered the DOPAC or HVA level in the CPu. However, the 3.0 µg/kg dose of epibatidine

significantly elevated the 5-HIAA level in the CPu (P < 0.0001). In the NAc, nicotine

elevated the levels of DOPAC and HVA (P < 0.0001), but not that of 5-HIAA. Epibatidine

dose-dependently elevated the accumbal DOPAC and HVA levels (P < 0.0001). It also

elevated the accumbal 5-HIAA level at all doses except 1.0 µg/kg (P < 0.0001).

Table 5-3. The maximal peak increases in the extracellular concentrations of DOPAC, HVA and 5-HIAA in the caudate-putamen and nucleus accumbens of rats given acute saline, nicotine (0.5 mg/kg) or epibatidine (0.1, 0.3, 0.6, 1.0, 2.0 or 3.0 µg/kg)

Treatment DOPAC% HVA% 5-HIAA%

Caudate-putamen

Saline 100 ± 7 105 ± 10 103 ± 6 Nicotine 0.5 mg/kg 109 ± 10 119 ± 18 108 ± 11 Epibatidine 0.1 µg/kg 103 ± 3 98 ± 14 99 ± 7 0.3 µg/kg 103 ± 5 98 ± 7 99 ± 5 0.6 µg/kg 112 ± 7 115 ± 9 113 ± 14 1.0 µg/kg 115 ± 3 119 ± 5 108 ± 3 2.0 µg/kg 116 ± 5 127 ± 3 114 ± 8 3.0 µg/kg 122 ± 11 141 ± 14 145 ± 13 ↑↑↑

Nucleus accumbens

Saline 95 ± 5 87 ± 10 88 ± 6 Nicotine 0.5 mg/kg 159 ± 10 ↑↑↑ 179 ± 25 ↑↑↑ 104 ± 11 Epibatidine 0.1 µg/kg 123 ± 6 ↑↑↑ 120 ± 6 ↑↑↑ 128 ± 7 ↑↑↑ 0.3 µg/kg 124 ± 7 ↑↑↑ 126 ± 6 ↑↑↑ 117 ± 5 ↑↑↑ 0.6 µg/kg 129 ± 7 ↑↑↑ 136 ± 5 ↑↑↑ 125 ± 8 ↑↑↑ 1.0 µg/kg 126 ± 17 ↑↑↑ 124 ± 14 ↑↑↑ 102 ± 8 2.0 µg/kg 160 ± 11 ↑↑↑ 168 ± 21 ↑↑↑ 113 ± 14 ↑↑↑ 3.0 µg/kg 177 ± 14 ↑↑↑ 188 ± 23 ↑↑↑ 150 ± 13 ↑↑↑

Data on repeated dialysate samples were analysed with 2-way ANOVA followed by contrast analysis: ↑↑↑ P < 0.0001 vs. saline controls. Data are presented as percentage change values (means ± S.E.M., n = 6-8 in all groups) of the baseline values (= 100%).

67

5.1.3. Effects of nicotine and epibatidine on the extracellular concentrations of dopamine, DOPAC, HVA and 5-HIAA in the CPu and NAc of nomifensine-pretreated rats (III, previously unpublished data)

The effects of nicotine (0.5 mg/kg s.c.) and epibatidine (0.6, 2.0 and 3.0 µg/kg s.c.) on the

extracellular dopamine concentrations in the CPu and NAc of nomifensine-pretreated rats are

shown in Fig. 5-2. Nomifensine (3 mg/kg) significantly elevated the extracellular dopamine

level in the CPu and even more so in the NAc as compared with the rats correspondingly

pretreated with saline (P < 0.0001). In the CPu, nicotine (0.5 mg/kg) and epibatidine at 0.6

µg/kg further increased the nomifensine-elevated dopamine level (P < 0.0001). In the NAc,

however, the nomifensine-elevated dopamine level was further elevated by nicotine (P <

0.01), but decreased by epibatidine at the 0.6 µg/kg dose (P < 0.01). Neither of the larger

epibatidine doses studied (2.0 or 3.0 µg/kg) altered the nomifensine-elevated dopamine level

in the CPu or NAc (data on the 2.0 µg/kg dose previously unpublished).

-60 0 60 120 180 240

NOM + SAL •••

NOM + NIC ↑↑↑

NOM + EPI 0.6 ↑↑↑

100

200

300

400

500

600

NOM + EPI 3.0NOM + EPI 2.0

SAL + SAL

Time (min)

DA

% o

f the

bas

elin

e

-60 0 60 120 180 240

NOM + NIC ↑↑

NOM + SAL •••

NOM + EPI 0.6 ↓↓

100

200

300

400

500

600

NOM + EPI 3.0NOM + EPI 2.0

SAL+ SAL

Time (min)

Caudate-putamen Nucleus accumbens

Fig. 5-2. The extracellular dopamine (DA) concentrations in the caudate-putamen and nucleus accumbens of the rats given acute nomifensine (NOM, 3 mg/kg i.p) and nicotine (NIC, 0.5 mg/kg s.c.), epibatidine (EPI, 0.6, 2.0 or 3.0 µg/kg s.c.) or saline (SAL, 1 ml/kg s.c.). The arrows indicate the time points of drug injections: nomifensine (first arrow) and the nAChR agonists or saline (second arrow). Data on the 2.0 µg/kg dose of epibatidine were previously unpublished. Data on repeated dialysate samples were analysed with 2-way ANOVA followed by contrast analysis: ••• P < 0.0001 vs. saline pretreatment (Fig. 5-1), ↑↑ P < 0.01, ↓↓ P < 0.01, ↑↑↑ P < 0.0001, vs. NOM + SAL. Data are presented as mean percentage change values (n = 6-8 in all groups) of the baseline values (= 100%).

68

The effects of nicotine and epibatidine on the extracellular concentrations of DOPAC, HVA

and 5-HIAA in the CPu and NAc of nomifensine-pretreated rats are summarized in Table 5-4.

Nomifensine (3 mg/kg) significantly decreased the extracellular DOPAC level in the CPu (P

< 0.01) and to an even greater extent in the NAc (P < 0.0001). Nicotine or epibatidine did not

alter the nomifensine-decreased DOPAC level in the CPu, but significantly elevated that in

the NAc (P < 0.0001). Nomifensine alone or in combination with the nAChR agonists did not

alter the HVA levels in either brain area studied. Furthermore, nomifensine significantly

increased the 5-HIAA levels in both brain areas and two-way ANOVA for repeated measures

revealed a significant pretreatment-effect for nomifensine [previously unpublished data; F(1,21)

= 11.8, P = 0.0025].

Table 5-4. The maximal peak changes in the extracellular concentrations of DOPAC, HVA and 5-HIAA in the caudate-putamen and nucleus accumbens of the rats given acute saline, nicotine (0.5 mg/kg) or epibatidine (0.6, 2.0 or 3.0 µg/kg) after nomifensine (3 mg/kg)

Treatment DOPAC% HVA% 5-HIAA%

Caudate-putamen

Saline + saline controls 100 ± 7 105 ± 10 103 ± 6 Nomifensine + saline 81 ± 4 •• 104 ± 7 129 ± 12 ↑↑↑ Nomifensine + nicotine 0.5 mg/kg 102 ± 5 117 ± 6 129 ± 5 ↑↑↑ Nomifensine + epibatidine 0.6 µg/kg 96 ± 6 109 ± 9 117 ± 11

2.0 µg/kg 97 ± 4 112 ± 5 136 ± 7 ↑↑↑ 3.0 µg/kg 104 ± 7 121 ± 8 143 ± 8 ↑↑↑

Nucleus accumbens

Saline + saline controls 95 ± 5 87 ± 10 88 ± 6 Nomifensine + saline 69 ± 3 ••• 92 ± 7 142 ± 12 ↑↑↑ Nomifensine + nicotine 0.5 mg/kg 118 ± 8 ↑↑↑ 114 ± 12 118 ± 15 ↑↑↑ Nomifensine + epibatidine 0.6 µg/kg 99 ± 10 ↑↑↑ 109 ± 10 150 ± 16 ↑↑↑

2.0 µg/kg 110 ± 8 ↑↑↑ 114 ± 13 121 ± 21 ↑↑↑ 3.0 µg/kg 117 ± 10 ↑↑↑ 120 ± 2 126 ± 8 ↑↑↑

Data on repeated dialysate samples were analysed with 2-way ANOVA followed by contrast analysis: •• P < 0.01, ••• P < 0.0001 vs. saline + saline controls, ↑↑↑ P < 0.0001 vs. nomifensine + saline. Data on the 2.0 µg/kg dose of epibatidine and the 5-HIAA levels were not previously unpublished. Data are presented as percentage change values (means ± S.E.M., n = 6-8 in all groups) of the baseline values (= 100%).

69

Furthermore, nomifensine was observed to induce stereotypical behavior, such as sniffing,

gnawing and repetitive head and limb movements, and clear hyperactivity for 10-60 min after

its administration. Nicotine and epibatidine depressed this hyperactivity for 10 min, but after

that the hyperactivity and stereotypical behaviour were again observed and even enhanced by

nicotine and epibatidine 3.0 µg/kg. Interestingly, although the rats were calm and habituated

to handling, during the depression phase many of the rats given nomifensine + epibatidine 3.0

µg/kg were seemingly scared of handling and vocalized even in the presence of an observer

beside the test cage.

5.2. Behavioural studies

5.2.1. Effects of acute nicotine on the rotational behaviour of unilaterally 6-

OHDA lesioned rats (I, previously unpublished data)

The effects of acute nicotine (0.5 mg/kg s.c.) and nomifensine (3 mg/kg i.p.) on the rotational

behaviour of rats with unilateral nigrostriatal 6-OHDA lesions are shown in Fig. 5-3.

-30 0 30 60 90 120 150 180

0

10

20

30

40

50

SAL + NIC

NOM + SAL

NOM + NIC * #

Time (min)

Ipsi

late

ral r

otat

ions

/ 15

min

-30 0 30 60 90 120 150 180

0

10

20

30

40

50

MEC 2 + SAL + SAL

MEC 2 + NOM + NIC

MEC 5 + SAL + SAL

MEC 5 + NOM + NIC ↓

SAL + NOM + NIC

Time (min)

A B

Fig. 5-3. The ipsilateral rotational behaviour of 6-OHDA-lesioned rats given acute nomifensine (NOM, 3 mg/kg i.p.) or saline (SAL, 3 ml/kg i.p.) and nicotine (NIC, 0.5 mg/kg s.c.) or saline (SAL, 1 ml/kg s.c.) (A). Figure B presents data on the rats given mecamylamine (MEC, 2 or 5 mg/kg i.p.) or saline before nomifensine (or saline) and nicotine (or saline). The arrows indicate the time points of drug injections in 15 min intervals. Data on the 2 mg/kg dose of mecamylamine were previously unpublished. Data on repeated dialysate samples were analysed with 2-way ANOVA followed by Student Newman Keuls test: * P < 0.05 vs. SAL + NIC, # P < 0.05 vs. NOM + SAL, ↓ P < 0.05 vs. SAL + NOM + NIC. Data are presented as means ± S.E.M. (n = 6-8 for A, n = 3-14 for B).

70

The effect of nomifensine or nicotine alone on ipsilateral rotations was modest, whereas the

combination of these two drugs produced a strong ipsilateral rotational behaviour. Indeed, the

rats given both nomifensine and nicotine rotated ipsilaterally significantly more than those

given either nomifensine or nicotine alone (P < 0.05). Some of the rats were pretreated with

the nAChR antagonist mecamylamine (2 or 5 mg/kg i.p; data on the 2 mg/kg dose previously

unpublished). Mecamylamine did not induce rotational behaviour. However, mecamylamine

dose-dependently inhibited the rotational behaviour induced by the combination of

nomifensine and nicotine and this effect reached significance at the 5 mg/kg dose (P < 0.05).

As shown in Table 5-5, the effect of nicotine (0.5 mg/kg s.c.) on the rotational behaviour of 6-

OHDA-lesioned rats was also studied in combination with parkinsonian drugs (levodopa, 10

mg/kg i.p., the DOPA decarboxylase inhibitor carbidopa, 20 mg/kg i.p., and the COMT-

inhibitor tolcapone, 10 mg/kg i.p.; previously unpublished data). The rats given levodopa in

combination with carbidopa and tolcapone rotated mainly contralaterally. Nicotine (0.5

mg/kg) tended to enhance the rotational behaviour as compared with the rats correspondingly

given saline (Student’s t-test, P = 0.097).

Table 5-5. The cumulative contralateral and ipsilateral rotational behaviour of 6-OHDA-lesioned rats given saline or nicotine (0.5 mg/kg) in combination with levodopa treatment

Rotational behaviour/ 4 h Treatment

Contralateral Ipsilateral

Levodopa (10 mg/kg) + carbidopa (20 mg/kg) + tolcapone (10 mg/kg) +

Saline 203 ± 98 14 ± 6 Nicotine 0.5 mg/kg 710 ± 264 29 ± 13

The following drugs were given: carbidopa (10 mg/kg i.p.), followed 15 min later by tolcapone (10 mg/kg i.p.) and nicotine or saline, and another 15 min later by carbidopa (10 mg/kg i.p.) and levodopa (10 mg/kg i.p.), after which the rotations were counted in 15 min intervals up to 4 h. Data are presented as means ± S.E.M., n = 11-12.

71

5.2.2. Effects of repeated nicotine and epibatidine on the rotational behaviour of unilaterally 6-OHDA-lesioned rats (IV)

Fig. 5-4 shows the effects of repeated nicotine (0.5 mg/kg s.c.) and epibatidine (0.6 and 3.0

µg/kg s.c.), following saline or nomifensine (3 mg/kg i.p.) pretreatment, on the rotational

behaviour of the rats with unilateral, nigrostriatal 6-OHDA-lesions.

SAL + SAL SAL + NIC SAL + EPI 0.6 SAL + EPI 3.00

20

40

60

80

100

120

Test day 1 Test day 5 Test day 3 Challenge day

***

*°

**

*

°°#

#

Ipsi

late

ral r

otat

ions

/ 3

h

NOM + SAL NOM + NIC NOM + EPI 0.6 NOM + EPI 3.00

200

400

600

800

1000

1200

°°°#

###

Ipsi

late

ral r

otat

ions

/ 3

h

°

°°

° °°

°°

A

B

Fig. 5-4. The 3 h ipsilateral rotational behaviour of the 6-OHDA-lesioned rats given repeatedly either saline (A; SAL, 3 ml/kg i.p.) or nomifensine (B; NOM, 3 mg/kg i.p.) pretreatment and 15 min later saline (SAL, 1 ml/kg s.c.), nicotine (NIC, 0.5 mg/kg s.c.) or epibatidine (EPI, 0.6 or 3.0 µg/kg s.c.). The drug combinations were administered repeatedly on five consecutive days and once more on challenge day 8 and rotations were measured on test days 1, 3 and 5 and on the challenge day. Data were analysed with Student’s t-test: * P < 0.05, ** P < 0.01 vs. SAL, O P < 0.05, OO P < 0.01, OOO P < 0.001 vs. test day 1, # P < 0.05, ### P < 0.001 vs. test day 3. Data are presented as means ± S.E.M. (n = 3-6 for A, n = 8-11 for B).

72

In the saline-pretreated rats, acute nicotine and epibatidine produced only few ipsilateral

rotations. The effect of nicotine on rotational behaviour was gradually enhanced by repeated

administration and the rats rotated significantly more on the challenge day than on test day 1

(P < 0.01). The rotational effect of epibatidine at 0.6 µg/kg, but not at 3.0 µg/kg, was also

enhanced by repeated administration, although to a lesser extent than that of nicotine.

Nomifensine’s effect on the rotational behaviour was significantly enhanced by repeated

administration even by test day 3, compared with its acute effect on test day 1 (P < 0.05).

Nicotine and epibatidine at 0.6 µg/kg tended to enhance the effect of repeated nomifensine on

the rotational behaviour, but the higher dose of epibatidine (3.0 µg/kg) tended to prevent the

nomifensine-induced sensitization of rotational behaviour.

5.2.3. Effects of nicotine and epibatidine on locomotor activity (V, previously

unpublished data)

As shown in Fig. 5-5, the effects of repeated nicotine (0.5 and 0.8 mg/kg s.c.) on locomotor

activity were studied at a normal ambient temperature of 20-23°C, as well as at an elevated

ambient temperature of 30-33°C (data on the effects at 30-33°C previously unpublished).

Analysis of basal locomotor activity on the second habituation day revealed that the rats were

significantly more active at the ambient temperature of 30-33°C than at 20-23°C (Fig. 5-5

Insert; Student’s t-test, P = 0.0082). When the activity on test day 1 was analysed, two-way

ANOVA revealed that during 0-60 min there was a significant temperature(20-23 vs. 30-

33°C)-effect [F(1,90) = 5.6, P = 0.0198] and during 0-120 min a significant treatment-effect

was seen [0-60 min: F(2,90) = 6.6, P = 0.0021; 61-120 min: F(2,90) = 8.4, P = 0.0004], but there

were no significant treatment × temperature-interactions (P > 0.08). Thus, acute nicotine

increased the activity at 0.5 mg/kg during both test hours and at 0.8 mg/kg during the second

test hour at both ambient temperatures (Student Newman Keuls P < 0.05: 0-60 min: NIC 0.5

vs. SAL/NIC 0.8, 61-120 min: NIC 0.5/0.8 vs. SAL).

73

0-60 61-120 0-60 61-120 0-60 61-1200

1000

2000

3000

4000

20-23°C30-33°C

Test day 1

• * ****

*#Lo

com

otor

act

ivity

coun

ts20-23°C 30-33°C

0

1000

2000••

Cou

nts/

2 h

0-60 61-120 0-60 61-120 0-60 61-1200

1000

2000

3000

4000 Test day 3

***°°°

••

***°°°°***

**

** *****

°°°

#

Loco

mot

or a

ctiv

ityco

unts

0-60 61-120 0-60 61-120 0-60 61-1200

1000

2000

3000

4000 Test day 5

******°°°°°°

•

**°°°

* *

°°°

°

%

Loco

mot

or a

ctiv

ityco

unts

Saline NIC 0.5 mg/kg NIC 0.8 mg/kg

Saline NIC 0.5 mg/kg NIC 0.8 mg/kg

Saline NIC 0.5 mg/kg NIC 0.8 mg/kg

Fig. 5-5. The locomotor activities of the rats given repeated saline or nicotine (NIC, 0.5 or 0.8 mg/kg s.c.) at ambient temperatures of 20-23°C and 30-33°C. The insert shows the comparison of the 2 h locomotor activities of all rats (n = 48) at the two ambient temperatures after saline injection on the second habituation day. The drugs were administered repeatedly on five consecutive days and the figure shows the activities during time periods 0-60 min and 61-120 min on test days 1, 3 and 5. The rats kept at 30-33°C were habituated to the elevated ambient temperature two days before the experiments, after which they were kept at 30-33°C throughout the experiments. The data on 30-33°C were previously unpublished. Data were analysed with ANOVA followed by Student Newman Keuls test: * P < 0.05, ** P < 0.01, *** P < 0.001 vs. saline controls, # P < 0.05 vs. NIC 0.8 mg/kg, O P < 0.05, OOO P < 0.001 vs. the test day 1, % P < 0.05 vs. test day 3. Comparisons between different ambient temperatures were carried out using Student’s t-test: ●P < 0.05, ●●P < 0.01 vs. activity at 20-23°C. Data are presented as means ± S.E.M. (n = 15-18, except on test day 5 saline: n = 6).

74

When locomotor activities at the elevated ambient temperature of 30-33°C on test days 1, 3

and 5 were analysed, the two-way ANOVA for repeated measures revealed that during 0-120

min there were significant treatment-effects [Fig. 5-5; 0-60 min: F(2,66) = 21.4, P < 0.0001; 61-

120 min: F(2,66) = 6.8, P = 0.0021] and during 0-60 min a significant day(1, 3 and 5)-effect

[F(2,132) = 51.5, P < 0.0001] and treatment × day-interaction [F(4,132) = 8.1, P < 0.0001]. Thus,

when given repeatedly, nicotine increased the activity throughout the 2 h test at 0.5 and 0.8

mg/kg (Student Newman Keuls P < 0.05: NIC 0.5/0.8 vs. SAL) and the stimulant effects of

nicotine were significantly enhanced by repeated treatment as compared with the saline

controls. However, the elevated ambient temperature of 30-33°C did not significantly alter the

acute or sensitized effects of nicotine as compared with the effects at 20-23°C.

The rats given either saline or nicotine 0.5 mg/kg tended to be more active at 30-33°C than at

20-23°C, while the rats given nicotine 0.8 mg/kg tended to be less active at 30-33°C than at

20-23°C. Compared with the corresponding group at 20-23°C, the rats given nicotine 0.8

mg/kg at 30-33°C were less active during the second test hour on test day 1, but when

nicotine was given repeatedly the rats were less active also during the first test hour on test

days 3 and 5. In fact, on test day 5 the rats given 0.8 mg/kg at 30-33°C were significantly less

active during 0-60 min than the corresponding rats at 20-23°C (Student’s t-test, P = 0.0337).

The effects of repeated epibatidine (0.6 and 3.0 µg/kg s.c.) on locomotor activity are

presented in Fig. 5-6. Both acute doses of epibatidine significantly increased locomotor

activity during the period of 31-60 min on test day 1 (P < 0.05). The locomotor stimulant

effects of epibatidine were enhanced after the repeated administration, at the 3.0 µg/kg dose

more markedly than at the 0.6 µg/kg dose. The rats given epibatidine 0.6 µg/kg were more

active on test day 5 than on test days 1 and 3 (P < 0.05) and the rats given epibatidine 3.0

µg/kg were more active on test days 3 and 5 than on test day 1 (P < 0.05). On the challenge

day, the activity of the rats repeatedly treated with epibatidine 3.0 µg/kg was significantly

enhanced from that on the test day 1, compared with the corresponding saline controls.

75

Saline day Test day 1 Test day 3 Test day 5 Challenge 0

500

1000

1500

2000

2500

3000

3500

EPI 3.0

EPI 0.6

SAL

* *

*

Loco

mot

or a

ctiv

ity c

ount

s/ 6

0 m

in

° °°

°0

500 * *

Cou

nts/

31-6

0min

Fig. 5-6. The 60 min locomotor activity of the rats given repeated epibatidine (EPI, 0.6 or 3.0 µg/kg s.c.) or saline (SAL, 1 ml/kg s.c.). The insert shows the locomotor activities during the time period 31-60 min on test day 1. The drugs were administered repeatedly on five consecutive days and once more on challenge day 7 and the activity was measured on test days 1, 3 and 5 and on the challenge day. Data were analysed with ANOVA followed by Student Newman Keuls test: * P < 0.05 vs. SAL controls, O P < 0.05 vs. test day 1. Data are presented as means ± S.E.M. (n = 21-23, except on test day 5 and the challenge day SAL: n = 9).

5.2.4. Effects of nicotine and epibatidine on conditioned place preference (V)

The effects of nicotine (0.5 and 0.8 mg/kg s.c.) and epibatidine (0.1, 0.3 and 0.6 µg/kg s.c.) on

conditioned place preference are summarised in Fig. 5-7. Nicotine induced a significant place

preference at 0.5 (P < 0.01) and 0.8 mg/kg (P < 0.05). Epibatidine produced a significant

place preference at 0.1 µg/kg (P < 0.05), but not at the higher doses studied (0.3 or 0.6 µg/kg).

In all experiments the control rats given saline spent after the conditioning even less time in

the initially non-preferred white compartment with rod grid floor than during the habituation.

76

-150

-100

-50

0

50

100

150

SALNIC 0.5

NIC 0.8

** *

Cha

nge

in ti

me

(sec

) spe

ntin

dru

g-pa

ired

com

partm

ent

-150

-100

-50

0

50

100

150

SALEPI 0.1

EPI 0.3EPI 0.6

*

Fig. 5-7. The conditioned place preference of the rats given nicotine (NIC, 0.5 or 0.8 mg/kg s.c.), epibatidine (EPI, 0.01, 0.3 or 0.6 µg/kg s.c.) or saline (SAL, s.c.) in the drug-paired compartment on three consecutive days. Data were analysed with ANOVA followed by Student Newman Keuls: * P < 0.05, ** P < 0.01 vs. SAL controls. Data are presented as means ± S.E.M. (n = 18-24, except NIC 0.8: n = 9).

Furthermore, as shown in Table 5-6 nicotine increased the locomotor activity on conditioning

days 1 and 3 at 0.5 mg/kg [previously unpublished data; F(2,53) = 11.2, P < 0.0001, Student

Newman Keuls P < 0.01 on day 1, P < 0.001 on day 3] and on conditioning day 3 at 0.8

mg/kg (Student Newman Keuls P < 0.05), while epibatidine did not significantly alter the

locomotor activity during conditioning sessions.

Table 5-6. The effects of saline, nicotine (0.5 and 0.8 mg/kg) and epibatidine (0.1, 0.3 and 0.6 µg/kg) on the locomotor activity of rats in the drug-paired compartment during the first 20 min on the first and third conditioning day

Conditioning day 1 Conditioning day 3

Saline 236 ± 34 178 ± 30

Nicotine 0.5 mg/kg 474 ± 74 ** 550 ± 73 ***

E i

0.8 mg/kg 182 ± 59 416 ± 54 *

Epibatidine 0.1 µg/kg 361 ± 49 207 ± 35

0.3 µg/kg 284 ± 35 205 ± 29

0.6 µg/kg 256 ± 49 175 ± 24

Data were analysed with ANOVA followed by Student Newman Keuls test: * P < 0.05, ** P < 0.01, *** P < 0.001 vs. corresponding SAL controls. Data are presented as means ± S.E.M. (n = 9-24).

77

6. DISCUSSION

6.1. Microdialysis studies

6.1.1. Effects of nicotine and dopaminergic drugs on nigrostriatal dopamine (I)

In parkinsonian patients, smoking/nicotine is known to alleviate motor symptoms (Moll,

1926; Marshall and Schnieden, 1966; Ishikawa and Miyatake, 1993; Fagerström et al., 1994;

Kelton et al., 2000), suggesting that it stimulates dopaminergic systems in the brain. Indeed,

acute nicotine increases in vivo extracellular dopamine concentration in the CPu of rats when

given systemically (Damsma et al., 1988) or locally (Toth et al., 1992). When given

intranigrally, acute nicotine also produces contralateral rotations, a behaviour associated with

increased release of dopamine in the dorsal striatum (Kaakkola, 1980). In parkinsonian

monkeys, nicotine and some novel nAChR agonists have been found to enhance the effects of

levodopa (Schneider et al., 1998b; Domino et al., 1999). However, although acute nicotine

activates the nigrostriatal pathway, it fails to induce ipsilateral rotations in unilaterally 6-

OHDA lesioned rats when given systemically (Fuxe et al., 1977; Kaakkola, 1981). This might

be due to the particularly effective neuronal reuptake of dopamine in the CPu (Marshall et al.,

1990; Garris and Wightman, 1994; Hoffman and Gerhardt, 1998), which terminates the effect

of dopamine in the synapse. Benwell and Balfour (1992, 1997) have shown that nicotine

increases dopamine release in the dorsal striatum in the presence of the DAT inhibitor

nomifensine.

The present study is the first to collectively examine how the inhibition of either DAT,

COMT, MAO-B or presynaptic dopamine receptors, affects nicotine-induced dopamine

release in the nigrostriatal pathway of rats. Moreover, the effects of COMT inhibition on

nicotine-induced synaptic dopamine release have not previously been studied. When nicotine

was given acutely (s.c.) in the present study, it increased the extracellular level of dopamine in

the CPu, which corresponds with earlier reports (Imperato et al., 1986; Damsma et al., 1988).

The DAT inhibitor nomifensine, elevated the extracellular dopamine level to a greater extent

than nicotine alone, but in combination these two drugs produced an additive effect on the

elevation of the dopamine level. As discussed in detail below, this elevation was also reflected

78

by the ipsilateral rotational behaviour of 6-OHDA-lesioned rats, because nomifensine and

nicotine in combination induced significantly more ipsilateral rotations than either drug alone.

However, similar additive or synergistic effects on the extracellular dopamine level in the

CPu were not found when nicotine was administered in combination with the other drugs

studied. The COMT inhibitor tolcapone, or the dopamine receptor blocker haloperidol, did

not significantly increase the extracellular dopamine level or the nicotine-elevated dopamine

level in the CPu. Pretreatment with the MAO-B inhibitor selegiline, significantly increased

the basal extracellular dopamine concentration, but did not further enhance the nicotine-

elevated dopamine level. Furthermore, mecamylamine did not alter the effects of

nomifensine, tolcapone or haloperidol on the extracellular levels of dopamine, its metabolites

or 5-HIAA in the CPu (unpublished results). This differs from previous studies in which

mecamylamine inhibited the activity of dopaminergic neurons, for example, by reducing the

haloperidol-induced elevation of striatal HVA concentrations (Ahtee and Kaakkola, 1978;

Clarke et al., 1985a; Grenhoff et al., 1986; Haikala and Ahtee, 1988). To summarize, these

findings support the important role of dopamine reuptake in the termination of dopamine’s

effect in the synapse, while the role of COMT or MAO-B seems to be minimal under normal

metabolic conditions in the rat striatum (Huotari et al., 2002).

Nicotine significantly elevated the extracellular levels of DOPAC and HVA in the present

study. Previously, nicotine has been shown to slightly increase (Benwell and Balfour, 1997)

or fail to alter the levels of dopamine metabolites in the dorsal striatum (Imperato et al., 1986;

Damsma et al., 1988). Nomifensine did not significantly alter the levels of dopamine

metabolites, but it significantly attenuated the nicotine-induced increase in the DOPAC level.

Changes in the level of DOPAC are thought to reflect changes in the intracellular dopamine

metabolism, rather than changes in dopamine release (Roffler-Tarlov et al., 1971). Thus, it

seems that nomifensine, by inhibiting the reuptake of dopamine into the nerve terminal,

attenuates the nicotine-induced elevation in intracellular dopamine metabolism. Consistent

with its mechanism of action, inhibition of COMT, tolcapone increased the level of DOPAC

and decreased that of HVA in the CPu. This is in agreement with earlier reports (Kaakkola

and Wurtman, 1992). Tolcapone had an additive effect on the nicotine-induced increase in the

DOPAC level when compared with the elevation induced by either tolcapone or nicotine

alone. Thus, nicotine’s effect on dopamine metabolism is enhanced by inhibition of COMT.

However, neither inhibition of MAO-B (selegiline) nor blockade of presynaptic dopamine

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receptors (haloperidol), had such additive effects on the DOPAC level in combination with

nicotine. Thus, the nicotine-induced increase in dopamine metabolism is attenuated by

inhibition of DAT and enhanced by inhibition of COMT.

6.1.2. Effects of nicotine and epibatidine on dopaminergic transmission (II)

The aim of Study II was to clarify the effects of the nAChR agonist epibatidine, on

dopaminergic transmission in the CPu and NAc, and to compare those effects with that of

nicotine. (±)-Epibatidine has higher relative affinities at α4β2, α3β2, α3β4 and α7 nAChR

subtypes than nicotine (Table 2-2; Gerzanich et al., 1995; Xiao et al., 1998; Hahn et al.,

2003). Epibatidine also desensitizes α4β2 nAChRs at concentrations that do not activate the

receptor (Buisson et al., 2000). The α4β2 subtype is the most abundant nAChR subtype in the

brain (Whiting and Lindstrom, 1986; Wada et al., 1989; Seguela et al., 1993) and has an

important role in the regulation of dopamine release (Kulak et al., 1997; Kaiser et al., 1998;

Picciotto et al., 1998; Salminen et al., 2004). Epibatidine is considerably more potent in the

stimulation of dopamine release from rat striatal slices than (-)-nicotine (Sullivan et al., 1994).

In addition, epibatidine’s effects on dopamine overflow in the dorsal and ventral striatum

seem to differ from those of nicotine (Seppä and Ahtee, 2000). In the present study,

epibatidine was studied at a range of doses from 0.1 to 3.0 µg/kg and nicotine was studied at

the dose 0.5 mg/kg, at which it has been found to increase striatal dopamine overflow without

causing toxic side effects (Imperato et al., 1986; Benwell and Balfour, 1997; Ferger and

Kuschinsky, 1997; Seppä and Ahtee, 2000).

The dose-response curve of epibatidine-induced dopamine release in the CPu was bell-

shaped, because epibatidine increased dopamine overflow in the CPu at moderate doses (0.6

and 1.0 µg/kg), but not at higher doses (2.0 or 3.0 µg/kg). The low dose of epibatidine (0.3

µg/kg) modestly, but significantly decreased dopamine overflow in the CPu. A similar dose-

response curve has previously been reported for nicotine, with the maximal increase in

dopamine overflow in the CPu at the 0.4 mg/kg dose (Benwell and Balfour, 1997). The bell-

shaped dose-response curves of dopamine release in the CPu may result from dose-dependent

desensitization of nAChR subtypes, which mediate dopamine release (Haikala et al., 1986;

Haikala and Ahtee, 1988; Benwell et al., 1995). Moreover, these findings might further

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explain why nicotine at large doses fails to sufficiently stimulate the nigrostriatal pathway to

produce stereotypical behavioural responses, but instead produces catalepsy, a behaviour

associated with reduced activity of the nigrostriatal pathway (Zetler, 1968, 1971; Costall and

Naylor, 1973). The finding that very low or high doses of nAChR agonists do not stimulate

and probably desensitize nAChRs may explain the reportedly diverse effects of nicotine on

parkinsonian symptoms. The repeated effects of nicotine on these symptoms are

contradictory, ranging from a failure to improve motor deficits in MPTP-treated animals

(Fung et al., 1991) or even to worsen parkinsonian motor symptoms (Ebersbach et al., 1999),

to an improvement of motor symptoms in animals (Sershen et al., 1987) and parkinsonian

patients (Moll, 1926; Marshall and Schnieden, 1966; Ishikawa and Miyatake, 1993;

Fagerström et al., 1994; Kelton et al., 2000). The diversity of findings from epidemiological

studies that have investigated the beneficial effects of smoking on the occurrence of

Parkinson’s disease (reviewed by Allam et al., 2004), may also be explained by the bell-

shaped dose-response curve for nicotinic compounds on nigrostriatal dopamine release.

Epibatidine-induced dopamine overflow in the NAc differed from that in the CPu. Epibatidine

increased dopamine overflow in the NAc at the highest dose tested (3.0 µg/kg), but not at the

lower doses (0.1-2.0 µg/kg). This is consistent with Bednar et al. (2004), who recently

reported that epibatidine failed to increase accumbal dopamine overflow at the 2.5 µg/kg

dose. Thus, it seems that three to five times higher dose of epibatidine is required to elevate

dopamine release in the NAc, compared with the CPu. This might be related to structural

and/or physiological differences in the two brain nuclei. Furthermore, the mesolimbic

dopaminergic pathway may contain a different set of nAChR subtypes compared with the

nigrostriatal pathway. Indeed, α7* nAChRs are expressed at higher levels in the VTA than in

the SN (Wooltorton et al., 2003) and nicotine-induced dopamine release in the NAc can be

inhibited by MLA (a selective α7 nAChR antagonist) into the VTA (Schilström et al., 1998a).

This suggests a role for α7* receptors in the VTA, in the mediation of this effect. Although it

is probable that the dopamine release is regulated by more than one nAChR subtype, α7*

nAChRs are suggested to be preferentially involved in accumbal dopamine release, whereas

nAChRs containing α3/α6 subunit mediate effects on dopamine release in the CPu (Kulak et

al., 1997; Kaiser et al., 1998; Schilström et al., 1998a). According to Mansvelder et al. (2002),

long-lasting dopamine release in the mesolimbic system after acute nicotine administration

may be the result of differential sensitivity of nAChRs to desensitization. It is suggested that

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nAChRs mediating GABA release may be more prone to desensitization than those involved

in glutamate release. α4β2* and α7* nAChR subtypes are thought to mediate GABA release

(Wonnacott et al., 1989; Alkondon et al., 1997; Alkondon and Albuquerque, 2001), while

glutamate release is mediated via α7* nAChRs (Kaiser and Wonnacott, 2000). Epibatidine has

a higher relative affinity at α7 nAChRs than nicotine (Table 2-2; Gerzanich et al., 1995; Xiao

et al., 1998) and the effects of epibatidine on α4β2 nAChRs are characterized by slow offset,

possibly a consequence of longer-lasting binding of epibatidine to the receptor (Buisson et al.,

2000). The pronounced activation of nAChRs that mediate inhibitory GABAergic

transmission, may explain the present finding that the lower doses of epibatidine failed to

increase accumbal dopamine release. Increasing the dose of epibatidine to 3.0 µg/kg may

preferentially desensitize/inhibit these receptors and thus inhibit GABA release, while the

excitatory effects of glutamate on dopamine release remain.

Table 6-1 summarizes the effects of nicotine 0.5 mg/kg and epibatidine 0.6 µg/kg on

dopamine overflow in the CPu and NAc. These doses induce about similar effects on striatal

dopamine in rats without producing toxic side effects that higher doses, such as the 0.8 mg/kg

dose of nicotine and the 3.0 µg/kg dose of epibatidine produce. The present finding that

epibatidine enhances dopamine overflow in the CPu at lower doses than that in the NAc is the

reverse of the effects of nicotine, because nicotine enhances dopamine overflow from the

NAc at lower doses than from the CPu (Benwell and Balfour, 1997). Moreover, in the present

study nicotine increased dopamine overflow in both brain nuclei to a higher level than

epibatidine. These findings are probably due to the different affinities of epibatidine and

nicotine at various nAChR subtypes that are involved in the regulation of dopamine release,

as well as regional variations in the expression of these receptors. Indeed, as a result of its

higher affinity for nAChRs, epibatidine may desensitize/inactivate the nAChRs in the

mesolimbic dopaminergic pathway more easily than nicotine, which could result in a failure

by epibatidine to increase dopamine overflow in the NAc (Table 6-1). However, when

nicotine is applied, the nAChRs mediating the stimulatory effects on dopamine release may

remain functional. The modest stimulatory effect of epibatidine on the mesolimbic

dopaminergic pathway, compared to nicotine, may also explain why mice self-administer

nicotine, but do not self-administer epibatidine (Rasmussen and Swedberg, 1998).

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Table 6-1. A simplified summary of the effects of nicotine (0.5 mg/kg) and epibatidine (0.6 µg/kg) on the extracellular concentrations of dopamine in the caudate-putamen (CPu) and nucleus accumbens (NAc), the ipsilateral rotational behaviour of 6-OHDA-lesioned rats, the locomotor activity and the conditioned place preference

Parameters studied Nicotine 0.5 mg/kg Epibatidine 0.6 µg/kg

Dopamine (SAL pretreatment) CPu ↑ ↑

NAc ↑ Ø

Dopamine (NOM pretreatment) CPu ↑ ↑

NAc ↑ ↓

Rotations (SAL pretreatment) Acute Ø Ø

Repeated ↑ ↑

Rotations (NOM pretreatment) Acute ↑ Ø

Repeated Ø Ø

Locomotor activity Acute ↑ (↑)

Repeated ↑↑ (↑)

Conditioned place preference ↑ Ø

Acute = acute administration, Repeated = repeated administration, SAL = saline, NOM = nomifensine, ↑ = increased, ↑↑ = markedly increased, ↓ = decreased, Ø = unaltered.

Epibatidine (0.1-3.0 µg/kg) as well as nicotine 0.5 mg/kg elevated both DOPAC and HVA

levels in the NAc, but not in the CPu. It has been previously reported that epibatidine

modestly elevates the level of dopamine metabolites in the CPu (Seppä and Ahtee, 2000),

while nicotine either modestly increases (Study I; Seppä and Ahtee, 2000) or fails to alter the

level of dopamine metabolites in the CPu (Imperato et al., 1986; Damsma et al., 1988).

However, both nAChR agonists have repeatedly been found to elevate accumbal DOPAC and

HVA levels (Imperato et al., 1986; Damsma et al., 1989; Mirza et al., 1996; Seppä and Ahtee,

2000). Thus epibatidine, in a similar way to nicotine, elevates dopamine metabolism

preferentially in the NAc as compared with the CPu. Furthermore, its effects on dopamine

release seem to be separate from its effects on dopamine metabolism, which suggests that

these two effects are mediated by different nAChR subtypes.

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6.1.3. Effects of nicotine and epibatidine on dopamine in combination with nomifensine (III)

When the DAT inhibitor nomifensine is administered locally, it has been found to intensify

the effects of nicotine on striatal dopamine overflow (Benwell and Balfour, 1992, 1997). In

Study I, pretreatment with systemic nomifensine was found to enhance the effects of nicotine

on the extracellular dopamine level in the CPu. In the present study (III), nomifensine

increased the extracellular dopamine concentration preferentially in the NAc, which agrees

with previous findings (Carboni et al., 1989b; Cass et al., 1993). Nomifensine also decreased

the extracellular level of DOPAC in the CPu and to an even greater extent in the NAc. Indeed,

as nomifensine prevents the interaction of dopamine with intraneuronal MAO, the increased

level of extracellular dopamine and decreased level of DOPAC are expected. Both nicotine

and epibatidine elevated accumbal DOPAC level that was reduced by nomifensine, which

corresponds with their effects on accumbal dopamine metabolites shown in Studies I and II.

In the present study, nomifensine also increased 5-HIAA levels in the CPu and NAc, but this

effect was not altered by nicotine or epibatidine. It can be hypothesed that serotonergic

transmission might be altered as a result of increased extracellular dopamine level.

Consistent with the bell-shaped dose-response curve of dopamine overflow in the CPu found

in Study II, epibatidine at a moderate dose (0.6 µg/kg), but not at a higher dose (3.0 µg/kg)

elevated nomifensine-induced increase in extracellular dopamine in the CPu. Nicotine 0.5

mg/kg enhanced this nomifensine-elevated extracellular dopamine in the CPu. Thus, the

effects of nAChR agonists on dopamine overflow in the CPu appear to be similar in the

presence and absence of dopamine uptake inhibition (Table 6-1). Consistent with Study II and

with nicotine’s effects in the CPu, nicotine enhanced the increase in extracellular dopamine in

the NAc of nomifensine-pretreated rats. This finding agrees with studies by Benwell and

Balfour (1992, 1997) in which local nomifensine intensified nicotine’s effects on accumbal

dopaminergic transmission. It also agrees with previous reports that nicotine enhances the

elevation of accumbal dopamine induced by other DAT inhibitors (cocaine, methylphenidate)

(Zernig et al., 1997; Gerasimov et al., 2000). However, in contrast to nicotine, epibatidine at a

moderate dose (0.6 µg/kg) decreased (Table 6-1) and at a higher dose (3.0 µg/kg) did not

alter, the nomifensine-increased extracellular dopamine level. Thus, the different affinities of

epibatidine and nicotine for different nAChR subtypes (Table 2-2 on page 46), as well as

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regional variations in the expression of these receptors, probably explain our findings that

epibatidine’s effects on accumbal dopamine overflow differ from those of nicotine.

As discussed in the previous chapter, spatial and temporal differences in nAChR activity on

both excitatory and inhibitory neurons in the mesolimbic pathways might result in the

elevated release of mesolimbic dopamine after systemic administration of nicotine

(Mansvelder et al., 2002). Interestingly, smoking tobacco (10-50 ng/ml; Benowitz et al.,

1990) or subcutaneous administration of the 0.5 mg/kg dose of nicotine to rats (80 ng/ml;

Salminen et al., 1999), leads to plasma nicotine concentrations at which non-α7 nAChRs (e.g.

β2-subunit) are desensitized, while those containing the α7-subunit remain functional and are

likely to mediate the effects on accumbal dopamine release (Wooltorton et al., 2003).

However epibatidine, when compared to nicotine, has a higher relative affinity at α7 nAChRs

(Table 2-2; Gerzanich et al., 1995; Xiao et al., 1998) and at low concentration it desensitizes

nAChRs (e.g. α4β2*) more markedly than nicotine (Buisson et al., 2000). Thus, similar to the

findings in the saline-pretreated rats in Study II, the inhibitory effect of epibatidine at the

lower dose (0.6 µg/kg) on accumbal dopamine overflow in nomifensine-pretreated rats, could

also be due to desensitization of nAChR subtypes that mediate the effects on inhibitory

GABAergic transmission, at low epibatidine concentrations. In contrast, nicotine enhances

accumbal dopamine overflow in nomifensine-pretreated rats and in previous studies nicotine

has been found to elevate dopamine overflow at even lower doses (e.g. 0.1 mg/kg) when the

DAT is inhibited by intra-accumbal nomifensine (Benwell and Balfour, 1997).

The inhibitory effect of epibatidine in nomifensine-pretreated rats also differ from its effects

in saline-pretreated rats (Study II; Table 6-1). In saline-pretreated rats epibatidine did not alter

accumbal dopamine overflow at 0.6 µg/kg, but elevated it at 3.0 µg/kg. Thus, the effects of

the potent nAChR agonist epibatidine, on dopamine overflow in the NAc are different in the

presence of DAT inhibition by nomifensine, compared with epibatidine’s effects under

normal physiological conditions. Elevation of extracellular dopamine by nomifensine causes a

hyperpolarization of dopaminergic neurons and reduces their firing rate (Mercuri et al., 1992).

The present study has shown that the NAc is more sensitive to nomifensine than the CPu.

Recently, the effects of nicotine on dopamine release were shown to be dependent on the

firing rate of dopaminergic neurons. Desensitization of nAChRs inhibits dopamine release at

low firing rates, but accelerating the firing rate attenuates this effect (Rice and Cragg, 2004;

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Zhang and Sulzer, 2004). Thus, hyperpolarization induced by nomifensine might be expected

to shift the effects of nAChR agonists to low frequency effects, i.e. to a depression of

dopamine release. This might explain why the low dose of epibatidine, which probably

desensitized nicotinic receptors (Buisson et al., 2000), was found to decrease dopamine

overflow in the presence of nomifensine. Consistent with this, epibatidine was recently

reported to decrease accumbal dopamine overflow in Sprague-Dawley rats (Bednar et al.,

2004). The rats in the study by Bednar et al. recovered for 17-22 h after the surgery, while our

rats were tested 5-7 days after the implantation of the guide cannula. Hence, it is possible that

the extracellular dopamine level was elevated in the same manner as in our nomifensine-

pretreated rats, which elevation, in turn, could emphasize epibatidine’s effects on mechanisms

inhibiting the accumbal dopamine release.

6.2. Behavioural studies

6.2.1. Effects of acute nicotine on rotational behaviour in combination with

nomifensine or levodopa (I, unpublished results)

The rotational behaviour experiments were carried out to see if changes in the striatal

extracellular dopamine level correspond with rotational behaviour in unilaterally 6-OHDA-

lesioned rats. These rats represent a parkinsonian animal model, and their ipsilateral rotational

behaviour is thought to reflect an increase in dopamine release in the intact nigrostriatal

pathway (Ungerstedt, 1968; Ungerstedt and Arbuthnott, 1970; Ungerstedt, 1971a, b). In the

present study we investigated the effects of nicotine in combination with nomifensine,

because these two drugs produced the most substantial increase in extracellular dopamine

level in Study I. The modest increase in dopamine overflow induced by nicotine was

accompanied by few ipsilateral rotations, while the greater increase in dopamine induced by

nomifensine was accompanied by a greater number of ipsilateral rotations. However, the

combination of both nicotine and nomifensine had an additive effect on the number of

ipsilateral rotations, and that correlated with the elevated dopamine level in the CPu. The

combined effect of these drugs on rotations was inhibited by mecamylamine, suggesting that

it is mediated by neuronal nAChRs. Thus, even when acute nicotine releases dopamine in the

CPu, the released dopamine is inadequate to induce rotational behaviour, because the DAT

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termination of dopamine’s action in the synaptic cleft is so effective. Nicotine, or other

nAChR agonists, in combination with a DAT inhibitor could thus alleviate the motor

symptoms of the early stage of Parkinson’s disease when functional dopaminergic neurons

remain.

In the advanced stage of Parkinson’s disease, the most effective drug treatment is levodopa in

combination with peripheral inhibitors of dopamine metabolism (Olanow, 2004). Thus, we

carried out rotational experiments to see whether nicotine enhances the effects of levodopa on

contralateral rotational behaviour. Indeed, nicotine tended to potentiate the rotational

behaviour, although the response failed to reach significance, thought to be due to the

variability between animals. Differences are likely to have arisen in the concentrations of

carbidopa and tolcapone administered, because these drugs had to be given as suspension, due

to their poor solubility. Thus, the dose of levodopa crossing the blood-brain barrier probably

varied between animals. Although more experiments are needed to confirm these findings, the

data agrees with previous studies in which levodopa produced greater contraversive circling

in MPTP-treated monkeys in combination with nicotine than with saline (Domino et al.,

1999). In addition, novel nAChR agonists have been reported to potentiate levodopa’s effects

on motor symptoms in MPTP-treated monkeys (Schneider et al., 1998a, b), as well as to

reverse both cognitive and motor deficits in combination with levodopa and benserazide, in

reserpine-treated rats (Menzaghi et al., 1997a). Thus, the use of subtype-selective nAChR

agonist in combination with levodopa may allow a reduction in levodopa dosage in some

parkinsonian patients and thus reduce complications associated with long-term levodopa use.

6.2.2. Effects of repeated nicotine and epibatidine on rotational behaviour (IV)

The aim of Study IV was to investigate the acute and repeated effects of epibatidine and

nicotine on the rotational behaviour of 6-OHDA-lesioned rats. Although epibatidine

preferentially stimulates nigrostriatal dopaminergic transmission (Study II; Seppä and Ahtee,

2000) and therefore is likely to affect rotational behaviour, this has not previously been

investigated. However, the effect of systemic nicotine on rotational behaviour has previously

been studied following acute and chronic administration (Fuxe et al., 1977; Kaakkola, 1981;

Lapin et al., 1987; Domino et al., 1999), but a profound investigation on the development of

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sensitization of rotational behaviour after repeated nicotine administration has not. Acute

nomifensine administered intrastriatally, has been reported to produce contralateral rotations

in mice (Worms et al., 1986a) and when given systemically to produce ipsilateral rotations in

rodents with unilateral nigrostriatal lesions (Costall et al., 1975; Worms et al., 1986b; Nakachi

et al., 1995). To our knowledge, the effects of repeated nomifensine administration have not

previously been studied.

In the present study, acute epibatidine and nicotine produced only few ipsilateral rotations in

rats and the effect of nicotine was similar to that found in Study I (Table 6-1). Hence, it seems

that acute epibatidine, in a similar way to acute nicotine (Fuxe et al., 1977; Kaakkola, 1981),

fails to elevate synaptic dopamine concentration in the CPu sufficiently to produce rotational

behaviour. When the nAChR agonists were given repeatedly, the effect of nicotine on the

ipsilateral rotational behaviour was the most enhanced, that of epibatidine 0.6 µg/kg was

somewhat less enhanced (Table 6-1), and the effect of epibatidine 3.0 µg/kg was not

significantly enhanced. These findings are consistent with those from the microdialysis

studies (Study II). Study II showed that nicotine and epibatidine at 0.6 µg/kg, but not at 3.0

µg/kg, elevated extracellular dopamine in the CPu, which implies that the effects of nicotine

and epibatidine (0.6 µg/kg) on dopaminergic transmission in the nigrostriatal pathway were

sensitized by repeated administration of nAChR agonists.

In light of the results from Study I that nAChR agonists do not induce rotational behaviour,

due to the effective dopamine reuptake in the CPu, we studied the effects of either acute or

repeated epibatidine and nicotine in combination with nomifensine. Nomifensine alone

induced ipsilateral rotational behaviour and its effects were sensitized when administered

repeatedly. Indeed, acute systemic nomifensine has been reported to elevate extracellular

striatal dopamine level in rats (Carboni et al., 1989b). Acute nicotine enhanced the

nomifensine-induced rotational behaviour during the first 45 min, while acute epibatidine did

not alter this rotational behaviour (Table 6-1). In Study III, acute nicotine enhanced the

nomifensine-elevated dopamine level both in the CPu and NAc, while epibatidine enhanced

that in the CPu, but inhibited that in the NAc (Table 6-1). Hence, the more pronounced effect

of nicotine on rotational behaviour, compared with epibatidine, may be due to its elevating

effects in both dopaminergic nerve terminals. Furthermore, injection of 6-OHDA into the

MFB leads to an almost complete dopamine depletion in the nigrostriatal pathway, but also

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causes up to 90% depletion of the mesolimbic dopamine (Mikkola et al., 2002). These

findings thus suggest that the rotational behaviour is mainly mediated by the nigrostriatal

dopaminergic system, but the mesolimbic system probably modulates it (Pycock and

Marsden, 1978).

Repeated administration of nicotine and epibatidine (0.6 µg/kg) slightly enhanced the effect

of repeated nomifensine on rotational behaviour, but this effect was not significant (Table 6-

1). This might be due to a ceiling effect in the rotational behaviour induced by the elevated

endogenous dopamine in the synaptic cleft. In contrast to nicotine and the lower dose of

epibatidine (0.6 µg/kg), the 3.0 µg/kg dose of epibatidine tended to inhibit the sensitized

effect of repeated nomifensine on rotational behaviour, which may be due to desensitization

of nAChRs. Indeed, psychostimulant-induced sensitization evoked by amphetamine or

cocaine is associated with enhanced endogenous activation of nAChRs and blocking nAChRs

with antagonists inhibits the induction of behavioural sensitization (Schoffelmeer et al., 2002;

de Rover et al., 2004). Thus, it seems possible that a high dose of nAChR agonist that is likely

to desensitize at least some nAChR subtypes, could inhibit the development of sensitization in

the nomifensine-induced rotational behaviour. However, there may also be other indirect

mechanisms involved, such as release of neurotransmitters like GABA and glutamate that

modulate dopamine release. In fact, an irreversible GABA transaminase inhibitor has been

found to inhibit the sensitization of stereotypical behaviour and locomotor activity associated

with cocaine (Gardner et al., 2002). The sensitization of rotational behaviour of 6-OHDA-

lesioned rats has been associated with the potential of dopaminergic drugs to induce motor

complications, such as dyskinesia (Carey, 1991; Henry et al., 1998). Thus, the finding that the

3.0 µg/kg dose of epibatidine inhibited nomifensine-induced sensitization might be of

therapeutic interest.

6.2.2. Effects of nicotine and epibatidine on locomotor activity (V, unpublished results)

Nicotine is known to stimulate locomotor activity in habituated rats, an effect that becomes

sensitized after repeated nicotine administration (Morrison and Stephenson, 1972; Clarke and

Kumar, 1983a; Ksir et al., 1985; O'Neill et al., 1991; Benwell and Balfour, 1992). This

enhanced behavioural response to nicotine and other addictive drugs is thought to relate to

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neuronal adaptations, which lead to compulsive drug use. Drugs of abuse, such as nicotine are

known to stimulate cerebral dopaminergic transmission and both stimulation and sensitization

of the mesolimbic/ mesocortical dopaminergic pathway in particular is associated with the

locomotor stimulant actions (Clarke et al., 1988; Corrigall and Coen, 1991; Leikola-Pelho and

Jackson, 1992; Louis and Clarke, 1998). The effects of epibatidine on locomotor activity have

previously been studied after acute administration (Sacaan et al., 1996; Menzaghi et al.,

1997b; Reuben et al., 2000) and only one study has compared the acute effects epibatidine

with those of nicotine (Reuben et al., 2000). Epibatidine’s effects on dopamine release in the

dorsal and ventral striatum have been found to differ from those of nicotine in microdialysis

studies (Study II; Seppä and Ahtee, 2000), suggesting that also its effects on locomotor

activity might differ from those of nicotine.

At the ambient temperature 20-23°C, acute nicotine at 0.5 mg/kg significantly increased

locomotor activity of rats throughout the 2 h experiment. At the higher dose (0.8 mg/kg),

nicotine did not alter the locomotor activity during the first 30 min from the control level, but

increased thereafter the activity up to 2 h. When nicotine was given repeatedly, the acute

locomotor stimulant effects were enhanced at both doses (0.5 and 0.8 mg/kg). These findings

agree with several earlier studies in which repeated administration of nicotine enhanced its

stimulant effects (Ksir et al., 1985; O'Neill et al., 1991; Benwell and Balfour, 1992). These

sensitized effects of nicotine on locomotor activity are probably due to enhanced mesolimbic

dopaminergic transmission (Benwell and Balfour, 1992; Balfour et al., 1998).

The stimulatory effects of nicotine on striatal dopamine release and metabolism have

previously been found to be enhanced by an elevated ambient temperature of 30-33°C

(Haikala, 1986; Leikola-Pelho et al., 1990; Seppä et al., 2000). Therefore, we investigated

whether this enhancement of dopamine release is accompanied by enhanced locomotor

activity. At the ambient temperature of 30-33°C, the basal activity of the rats was significantly

higher than at 20-23°C and repeated exposure to the test boxes and saline injections did not

alter this effect. In earlier studies, the elevation of ambient temperature alone was not found to

alter basal extracellular concentrations of dopamine or its metabolites in the CPu or NAc

(Seppä et al., 2000; Seppä, 2001), suggesting that other mechanisms and/or neurotransmitters

may underlie this phenomenon. Furthermore, rats given either acute or repeated nicotine at

0.5 mg/kg were somewhat more active, while rats given nicotine at 0.8 mg/kg were somewhat

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less active, particularly on test day 5, at the elevated ambient temperature of 30-33°C,

compared to 20-23°C. However, the responses to acute or repeated nicotine were not

significantly different at 30-33°C from those at 20-23°C. Acute nicotine increased activity of

rats at 0.5 mg/kg immediately and at 0.8 mg/kg after a delay, and the stimulant response was

sensitized after repeated administration at both doses. This is in line with the findings that a

higher ambient temperature enhances nicotine-induced dopamine release only in the CPu, but

not in the NAc (Seppä et al., 2000; Seppä, 2001).

Acute epibatidine depressed locomotor activity at the highest dose (3.0 µg/kg) during the first

10 min, but activity was increased during 31-60 min at both 0.6 and 3.0 µg/kg. These findings

are consistent with earlier studies in which acute epibatidine at 3.0 µg/kg depressed the

activity of habituated rats for 10-15 min, but increased it between 30-40 min after

administration (Sacaan et al., 1996; Menzaghi et al., 1997b; Reuben et al., 2000). After

repeated administration of epibatidine, its locomotor depressant effects were attenuated and

its stimulant effects were enhanced, particularly at the higher dose (3.0 µg/kg). Sensitization

of locomotor activity was more pronounced at 3.0 µg/kg than at 0.6 µg/kg and this is

consistent with the findings in Study II that epibatidine elevates accumbal dopamine release at

3.0 µg/kg, but not at 0.6 µg/kg. In contrast, the 0.6 µg/kg dose of epibatidine, but not 3.0

µg/kg, elevates dopamine release in the CPu. Thus, stimulation of locomotor activity and its

sensitization appear to relate to increased dopaminergic transmission in the mesolimbic

terminal regions as suggested previously (Clarke et al., 1988; Corrigall and Coen, 1991;

Benwell and Balfour, 1992; Leikola-Pelho and Jackson, 1992; Louis and Clarke, 1998).

Comparatively, stimulation of locomotor activity by nicotine is greater than by epibatidine

(Table 6-1), which is consistent with a previous study (Reuben et al., 2000). Furthermore,

repeated epibatidine did not produce such a clear and rapid sensitization as nicotine (Table 6-

1). When compared with nicotine, the more modest stimulatory effect of epibatidine on

locomotor activity, is in line with its modest stimulatory effect on mesolimbic dopaminergic

transmission (Study II; Seppä and Ahtee, 2000). However, dopamine is unlikely to be the

only neurotransmitter mediating the locomotor effects studied here.

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6.2.3. Effects of nicotine and epibatidine on conditioned place preference (V)

Similarly to other drugs of abuse, nicotine has been found to effectively support drug-seeking

behaviour in rats, as demonstrated by the self-administration model (Corrigall and Coen,

1989; Corrigall et al., 1992; Donny et al., 1995, 1998; Corrigall, 1999; Donny et al., 1999)

and the conditioned place preference paradigm (Fudala et al., 1985; Fudala and Iwamoto,

1986; Acquas et al., 1989; Carboni et al., 1989a; Calcagnetti and Schechter, 1994; Vastola et

al., 2002; Le Foll and Goldberg, 2005). For both nicotine and other drugs of abuse, the

reinforcing and rewarding effects appear to depend upon the direct and/or indirect stimulation

of mesolimbic dopaminergic projections, because lesioning or pharmacological blockade of

these neurons attenuates self-administration (Singer et al., 1982; Corrigall and Coen, 1991;

Corrigall et al., 1992). In addition, the pedunculopontine nucleus that projects into the VTA

seems to be involved in the mediation of the rewarding effects of nicotine (Lança et al.,

2000b; Cousins et al., 2002). The modest effects of epibatidine on the mesolimbic

dopaminergic pathways, compared to those of nicotine (Study II; Seppä and Ahtee, 2000),

suggest that epibatidine might possess a low abuse potential. Indeed, mice did not self-

administer epibatidine (Rasmussen and Swedberg, 1998). Therefore, in the present study we

wanted to compare the effects of nicotine and epibatidine on the conditioned place preference.

Nicotine produced place preference in rats at 0.5 and 0.8 mg/kg and also increased locomotor

activity during the conditioning days. Correspondingly, nicotine has previously been reported

to elicit place preference in rats at 0.1-1.4 mg/kg under similar biased conditions (Fudala et

al., 1985; Fudala and Iwamoto, 1986; Acquas et al., 1989; Carboni et al., 1989a; Calcagnetti

and Schechter, 1994; Le Foll and Goldberg, 2005). In studies by Vastola et al. (2002) nicotine

induced place preference at 0.6 mg/kg in adolescent Sprague-Dawley rats (4-6 weeks old), but

not in adult rats (8-10 weeks). Epibatidine’s effects have not previously been studied using

the conditioned place preference, but Rasmussen et al. (1998) reported that mice did not self-

administer epibatidine infused i.v. at 0.25-1.25 µg/kg, although they did self-administer

nicotine (at doses up to 0.075 mg/kg per i.v. infusion). In the present study, epibatidine

induced place preference in rats at the lowest dose, 0.1 µg/kg, but not at 0.3 or 0.6 µg/kg.

When compared to nicotine, epibatidine’s effect on place preference was modest (Table 6-1),

although at 0.1 µg/kg it did elicit place preference. However, in contrast to nicotine,

epibatidine failed to significantly alter locomotor activity during the conditioning days when

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given acutely or repeatedly. Thus, the present findings imply that the actions of epibatidine on

the nAChR subtypes that regulate neurotransmitters involved in the mediation of locomotor

activity and motivation, differ from those of nicotine.

In Study II, epibatidine at the doses studied here (0.1, 0.3 or 0.6 µg/kg) did not alter accumbal

dopamine release, although it slightly, but significantly, elevated accumbal extracellular

concentration of dopamine metabolites and the serotonin metabolite 5-HIAA. The mesolimbic

dopaminergic pathway has been suggested to directly and/or indirectly mediate the

motivational effects of nicotine (Corrigall et al., 1992). However, other neurotransmitters,

such as endogenous opioids, may play a role in the acquisition of the reinforcing properties of

nAChR agonists (Pomerleau, 1998) and in the conditioning effects of 0.1 µg/kg of

epibatidine. The nAChR subtypes that mediate effects on these neurotransmitters may

desensitize at higher doses of epibatidine, which fail to elicit place preference. In conclusion,

epibatidine’s rewarding and reinforcing effects seem to occur transiently only at very low

doses. If the epibatidine dose is increased to levels found to alter striatal dopamine release

(Study II) and to stimulate locomotor activity (Study V) this effect is attenuated. Thus, these

findings support the idea that it is possible to develop subtype-selective nAChR agonists that

lack dependence-producing properties.

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7. SUMMARY AND CONCLUSIONS

In contrast to nicotine, which preferentially stimulated the mesolimbic dopaminergic pathway,

epibatidine increased dopamine overflow at lower doses in the nigrostriatal pathway than in

the mesolimbic pathway. This is probably due to the different affinities of nicotine and

epibatidine at various nAChR subtypes, as well as the fact that there are different nAChR

subtypes located on neurons in the nigrostriatal pathway compared to the mesolimbic

pathway. Indeed, α3/α6* nAChRs appear to mediate effects on dopamine release in the CPu,

while α7* nAChRs are preferentially involved in accumbal dopamine release.

Similar to nicotine, the dose-response curve of epibatidine-induced dopamine release in the

CPu was bell-shaped. Thus, high doses of nAChR agonists probably desensitize nAChR

subunits that mediate effects on dopamine release in the nigrostriatal terminal areas, while

those of the mesolimbic pathway remain functional. These findings may explain the

conflicting results regarding nicotine’s therapeutic effects in animals, as well as in

parkinsonian patients. The pronounced effects of nicotine on mesolimbic dopamine are likely

to be related to its ability to cause addiction.

Nicotine-induced dopamine release was enhanced by inhibition of dopamine reuptake by

nomifensine, but not by inhibition of COMT (tolcapone), MAO-B (selegiline) or presynaptic

dopamine receptors (haloperidol). Hence, effective dopamine reuptake mechanisms in the

CPu appear to terminate the actions of synaptic dopamine release stimulated by nicotine. This

may also explain why dopamine release induced by systemic nicotine fails to induce

ipsilateral rotations in unilaterally 6-OHDA lesioned rats. These findings imply that a DAT

inhibitor, such as nomifensine, could be used to potentiate the effects nicotine or other

nAChR agonists on nigrostriatal dopaminergic transmission.

When given in combination with nomifensine, both nicotine and epibatidine at a low dose

enhanced the nomifensine-induced increase in extracellular dopamine level in the CPu. In the

NAc, however, nicotine enhanced, while epibatidine inhibited the nomifensine-induced

elevation in dopamine level. Thus, nomifensine revealed the inhibitory effect of epibatidine

on accumbal dopamine release. This effect was characteristic for the NAc and was not found

in the CPu. This supports the idea that there are differences in nicotinic regulation of the

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mesolimbic and nigrostriatal dopaminergic pathways. In addition, drugs that substantially

elevate synaptic dopamine and thus induce hyperpolarization of dopaminergic neurons, may

modulate the net effects of nAChR agonists in the direction of inhibition.

The effects of nicotine and epibatidine on dopaminergic transmission of the nigrostriatal and

mesolimbic pathways corresponded with the rotational behaviour results. Both nicotine and

epibatidine induced ipsilateral rotational behaviour after repeated administration, but not

acutely, which suggests sensitization of dopaminergic transmission, particularly in the

nigrostriatal pathway.

Consistent with its modest effects on mesolimbic dopamine release, acute epibatidine

stimulated locomotor activity less than nicotine. When given repeatedly, epibatidine induced

sensitization of locomotor activity to a lesser extent than nicotine. Furthermore, contrary to

nicotine, epibatidine elicited transient conditioned place preference at a very low dose, but

failed to alter the preference of rats at doses that increase striatal dopamine release and

stimulate locomotor activity. These findings imply that epibatidine does not produce

dependence to the same degree as nicotine.

In conclusion, the present study shows that the effects of epibatidine on the nigrostriatal

dopaminergic pathway and the behaviours preferentially mediated by nigrostriatal dopamine,

resemble those of nicotine, while the effects on the mesolimbic dopaminergic pathways and

the behaviours mediated by mesolimbic dopamine, differ from those of nicotine. Thus, it

seems possible to stimulate the nigrostriatal dopaminergic pathway with nAChR agonists

without significantly affecting the mesolimbic dopaminergic pathway. This supports the

possibility that novel subtype-selective nAChR agonists that enhance dopaminergic

transmission, but lack addictive properties, could be developed. Such compounds may be

clinically useful for controlling tobacco and drug addiction, and improving brain function in

neurodegenerative brain diseases, such as Parkinson’s disease.

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8. ACKNOWLEDGEMENTS

This study was carried out in the Division of Pharmacology and Toxicology, Department/

Faculty of Pharmacy, University of Helsinki, during the years 2000-2005.

I wish to express my sincere gratitude to the following persons:

Professor Liisa Ahtee, M.D., my teacher and supervisor, for welcoming me to her lab and for

introducing the fascinating field of neuropharmacology to me. Her profound knowledge and

experience, as well as her endless encouragement, optimism and support have enabled me to

successfully complete this project. I will always appreciate her hospitality and kindness

during the preparation of the papers of this thesis.

Professor Raimo Tuominen, M.D., for his support and confidence in me during this thesis

project. I also wish to thank docent Seppo Kaakkola, M.D., for his contribution to the first

study and for valuable discussions during the preparation of its manuscript.

Professor Agneta Nordberg, M.D., Ph.D., and docent Pekka Rauhala, M.D., the reviewers of

this thesis, for devoting their time to review the manuscript, and for their valuable and

encouraging comments for its improvement. I am grateful to Miss Kate Hanrott, B.Sc.(Hons),

University of Bath, United Kingdom, for her skillful revision of the English language of the

manuscript.

All the co-authors of these studies. I am grateful to Anna Linnervuo, M.Sc.(Pharm), Paula

Mielikäinen, M.Sc.(Pharm), Päivi Paldánius, Lic.Med., and Minna Svensk, B.Sc.(Pharm) for

their excellent collaboration and contribution to the studies. I wish to thank Dr. Petteri

Piepponen for his support with the microdialysis studies. I am furthermore thankful to Marjo

Vaha and Kati Valkonen for their skillful technical assistance and friendship throughout this

project.

All my colleagues at the Division of Pharmacology and Toxicology for creating a pleasant

atmosphere to work in, and for all their help, support and both scientific and non-scientific

discussions during these years. Special thanks to the people in the nicotine group. I am

96

particularly grateful to Saara Nuutinen, M.Sc.(Pharm) for numerous inspiring and helpful

scientific discussions, for her constant encouragement and optimism on the days of both

success and problems, and for her valuable friendship, in and outside the lab. I wish to thank

Dr. Elina Ekokoski for all those scientific and non-scientific lunches between the

experiments, her strong knowledge and professionality is really something to look up to. I am

grateful to Anne Tammimäki, M.Sc.(Pharm), Mikko Airavaara, M.Sc.(Pharm), Jelena

Mijatovic, M.Sc.(Pharm), and Johanna Salimäki, Lic.Pharm., for their help, support and

friendship during these studies. I also wish to thank Dr. Ulla-Mari Parkkisenniemi-Kinnunen,

Dr. Helena Raattamaa and Dr. Tiina Seppä for their guidance, support and helpful advises

concerning both teaching and research.

All my dear friends. I particularly wish to thank Hanna Heikkilä, M.Sc.(Pharm), Elina Harju,

M.Sc.(Pharm), Sanna Heikkilä, M.Sc.(Pharm) and Laura Kasslin, M.Sc.(Pharm) for their

interest, encouragement and friendship, not to mention their endless confidence that I will pull

it through.

My warmest gratitude belongs to my family. I wish to thank my parents Sinikka and Tapio

Janhunen and my brother Olli Janhunen for their patience, support and love during my

studies. I am also deeply grateful to my grandparents, especially my grandmother Aune

Talvio, for her effortless support and encouragement throughout all my years at school, and

for teaching me the importance of education. Finally, I wish to thank Fernand for his love,

encouragement and positive thinking during my long period of writing, for all the happiness

that he has brought into my life, and for making me a cup of coffee when I needed it the most.

The financial support from the Academy of Finland, the Sigrid Jusélius Foundation, the

University of Helsinki’s Fund, the Graduate School in Pharmaceutical Research, the Finnish

Cultural Foundation (Elli Turunen Foundation), the Finnish Pharmaceutical Society, the

Finnish Parkinson Foundation and the Paulo Foundation is gratefully acknowledged.

Helsinki, May 2005

Sanna Janhunen

97

9. REFERENCES Abdulla FA, Calaminici M, Wonnacott S, Gray JA, Sinden JD, Stephenson JD (1995) Sensitivity of rat frontal cortical

neurones to nicotine is increased by chronic administration of nicotine and by lesions of the nucleus basalis magnocellularis: comparison with numbers of 3H nicotine binding sites. Synapse 21:281-288.

Abdulla FA, Bradbury E, Calaminici MR, Lippiello PM, Wonnacott S, Gray JA, Sinden JD (1996) Relationship between up-regulation of nicotine binding sites in rat brain and delayed cognitive enhancement observed after chronic or acute nicotinic receptor stimulation. Psychopharmacology 124:323-331.

Acquas E, Carboni E, Leone P, Di Chiara G (1989) SCH 23390 blocks drug-conditioned place-preference and place-aversion: anhedonia (lack of reward) or apathy (lack of motivation) after dopamine-receptor blockade? Psychopharmacology 99:151-155.

Adler LE, Olincy A, Waldo M, Harris JG, Griffith J, Stevens K, Flach K, Nagamoto H, Bickford P, Leonard S, Freedman R (1998) Schizophrenia, sensory gating, and nicotinic receptors. Schizophrenia Bull 24:189-202.

Agid Y (1991) Parkinson's disease: pathophysiology. Lancet 337:1321-1324. Ahtee L, Kaakkola S (1978) Effect of mecamylamine on the fate of dopamine in striatal and mesolimbic areas of rat brain;

interaction with morphine and haloperidol. Br J Pharmacol 62:213-218. Akaike A, Tamura Y, Yokota T, Shimohama S, Kimura J (1994) Nicotine-induced protection of cultured cortical neurons

against N-methyl-D-aspartate receptor-mediated glutamate cytotoxicity. Brain Res 644:181-187. Alkondon M, Albuquerque EX (1995) Diversity of nicotinic acetylcholine receptors in rat hippocampal neurons. III. Agonist

actions of the novel alkaloid epibatidine and analysis of type II current. J Pharmacol Exp Ther 274:771-782. Alkondon M, Albuquerque EX (2001) Nicotinic acetylcholine receptor alpha7 and alpha4beta2 subtypes differentially

control GABAergic input to CA1 neurons in rat hippocampus. J Neurophysiol 86:3043-3055. Alkondon M, Pereira EF, Albuquerque EX (1996a) Mapping the location of functional nicotinic and gamma-aminobutyric

acidA receptors on hippocampal neurons. J Pharmacol Exp Ther 279:1491-1506. Alkondon M, Pereira EF, Wonnacott S, Albuquerque EX (1992) Blockade of nicotinic currents in hippocampal neurons

defines methyllycaconitine as a potent and specific receptor antagonist. Mol Pharmacol 41:802-808. Alkondon M, Rocha ES, Maelicke A, Albuquerque EX (1996b) Diversity of nicotinic acetylcholine receptors in rat brain. V.

Alpha-Bungarotoxin-sensitive nicotinic receptors in olfactory bulb neurons and presynaptic modulation of glutamate release. J Pharmacol Exp Ther 278:1460-1471.

Alkondon M, Pereira EF, Barbosa CT, Albuquerque EX (1997) Neuronal nicotinic acetylcholine receptor activation modulates gamma-aminobutyric acid release from CA1 neurons of rat hippocampal slices. J Pharmacol Exp Ther 283:1396-1411.

Alkondon M, Pereira EF, Almeida LE, Randall WR, Albuquerque EX (2000) Nicotine at concentrations found in cigarette smokers activates and desensitizes nicotinic acetylcholine receptors in CA1 interneurons of rat hippocampus. Neuropharmacology 39:2726-2739.

Allam MF, Campbell MJ, Hofman A, Del Castillo AS, Fernandez-Crehuet Navajas R (2004) Smoking and Parkinson's disease: systematic review of prospective studies. Mov Disord 19:614-621.

Andén NE, Grabowska-Andén M, Lindgren S, Thornström U (1983) Synthesis rate of dopamine: difference between corpus striatum and limbic system as a possible explanation of variations in reactions to drugs. Naunyn-Schmiedebergs Arch Pharmacol 323:193-198.

Aosaki T, Kimura M, Graybiel AM (1995) Temporal and spatial characteristics of tonically active neurons of the primate's striatum. J Neurophysiol 73:1234-1252.

Arqueros L, Naquira D, Zunino E (1978) Nicotine-induced release of catecholamines from rat hippocampus and striatum. Biochem Pharmacol 27:2667-2674.

Asencio H, Bustos G, Gysling K, Labarca R (1991) N-Methyl-D-aspartate receptors and release of newly-synthesized 3H dopamine in nucleus accumbens slices and its relationship with neocortical afferences. Prog Neuro-Psychopharmacology Biol Psychiatry 15:663-676.

Aubert I, Araujo DM, Cecyre D, Robitaille Y, Gauthier S, Quirion R (1992) Comparative alterations of nicotinic and muscarinic binding sites in Alzheimer's and Parkinson's diseases. J Neurochem 58:529-541.

Avalos M, Parker MJ, Maddox FN, Carroll FI, Luetje CW (2002) Effects of pyridine ring substitutions on affinity, efficacy, and subtype selectivity of neuronal nicotinic receptor agonist epibatidine. J Pharmacol Exp Ther 302:1246-1252.

Azam L, Winzer-Serhan U, Leslie FM (2003) Co-expression of alpha7 and beta2 nicotinic acetylcholine receptor subunit mRNAs within rat brain cholinergic neurons. Neuroscience 119:965-977.

Azam L, Winzer-Serhan UH, Chen Y, Leslie FM (2002) Expression of neuronal nicotinic acetylcholine receptor subunit mRNAs within midbrain dopamine neurons. J Comp Neurol 444:260-274.

Badio B, Daly JW (1994) Epibatidine, a potent analgetic and nicotinic agonist. Mol Pharmacol 45:563-569. Baldo BA, Sadeghian K, Basso AM, Kelley AE (2002) Effects of selective dopamine D1 or D2 receptor blockade within

nucleus accumbens subregions on ingestive behavior and associated motor activity. Behav Brain Res 137:165-177. Balfour DJ (1994) Neural mechanisms underlying nicotine dependence. Addiction 89:1419-1423. Balfour DJ, Ridley DL (2000) The effects of nicotine on neural pathways implicated in depression: a factor in nicotine

addiction? Pharmacol Biochem Behav 66:79-85. Balfour DJ, Wright AE, Benwell ME, Birrell CE (2000) The putative role of extra-synaptic mesolimbic dopamine in the

neurobiology of nicotine dependence. Behav Brain Res 113:73-83. Balfour DJ, Benwell ME, Birrell CE, Kelly RJ, Al-Aloul M (1998) Sensitization of the mesoaccumbens dopamine response

to nicotine. Pharmacol Biochem Behav 59:1021-1030. Bancroft A, Levin ED (2000) Ventral hippocampal alpha4beta2 nicotinic receptors and chronic nicotine effects on memory.

Neuropharmacology 39:2770-2778.

98

Bannon AW, Gunther KL, Decker MW (1995) Is epibatidine really analgesic? Dissociation of the activity, temperature, and analgesic effects of (+/-)-epibatidine. Pharmacol Biochem Behav 51:693-698.

Baron JA (1986) Cigarette smoking and Parkinson's disease. Neurology 36:1490-1496. Bednar I, Friberg L, Nordberg A (2004) Modulation of dopamine release by the nicotinic agonist epibatidine in the frontal

cortex and the nucleus accumbens of naive and chronic nicotine treated rats. Neurochem Int 45:1049-1055. Behmand RA, Harik SI (1992) Nicotine enhances 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity. J Neurochem

58:776-779. Belluardo N, Mudo G, Blum M, Fuxe K (2000) Central nicotinic receptors, neurotrophic factors and neuroprotection. Behav

Brain Res 113:21-34. Bencherif M, Fowler K, Lukas RJ, Lippiello PM (1995) Mechanisms of up-regulation of neuronal nicotinic acetylcholine

receptors in clonal cell lines and primary cultures of fetal rat brain. J Pharmacol Exp Ther 275:987-994. Bencherif M, Bane AJ, Miller CH, Dull GM, Gatto GJ (2000) TC-2559: a novel orally active ligand selective at neuronal

acetylcholine receptors. Eur J Pharmacol 409:45-55. Bencherif M, Lovette ME, Fowler KW, Arrington S, Reeves L, Caldwell WS, Lippiello PM (1996) RJR-2403: a nicotinic

agonist with CNS selectivity I. In vitro characterization. J Pharmacol Exp Ther 279:1413-1421. Bencherif M, Schmitt JD, Bhatti BS, Crooks P, Caldwell WS, Lovette ME, Fowler K, Reeves L, Lippiello PM (1998) The

heterocyclic substituted pyridine derivative (+/-)-2-(-3-pyridinyl)-1-azabicyclo2.2.2 octane (RJR-2429): a selective ligand at nicotinic acetylcholine receptors. J Pharmacol Exp Ther 284:886-894.

Bennett BD, Wilson CJ (1999) Spontaneous activity of neostriatal cholinergic interneurons in vitro. J Neurosci 19:5586-5596.

Benowitz NL (1996) Pharmacology of nicotine: addiction and therapeutics. Annu Rev Pharmacol Toxicol 36:597-613. Benowitz NL, Porchet H, Jacob PI (1990) Pharmacokinetics, metabolism, and pharmacodynamics of nicotine. In: Nicotine

psychopharmacology: Molecular, cellular and behavioural aspects (Wonnacott S, Russell MAH, Stolerman IP, eds), pp 112-157. Oxford: Oxford University Press.

Benwell ME, Balfour DJ (1992) The effects of acute and repeated nicotine treatment on nucleus accumbens dopamine and locomotor activity. Br J Pharmacol 105:849-856.

Benwell ME, Balfour DJ (1997) Regional variation in the effects of nicotine on catecholamine overflow in rat brain. Eur J Pharmacol 325:13-20.

Benwell ME, Balfour DJ, Anderson JM (1988) Evidence that tobacco smoking increases the density of (-)3H nicotine binding sites in human brain. J Neurochem 50:1243-1247.

Benwell ME, Balfour DJ, Lucchi HM (1993) Influence of tetrodotoxin and calcium on changes in extracellular dopamine levels evoked by systemic nicotine. Psychopharmacology 112:467-474.

Benwell ME, Balfour DJ, Khadra LF (1994) Studies on the influence of nicotine infusions on mesolimbic dopamine and locomotor responses to nicotine. Clin Investigator 72:233-239.

Benwell ME, Balfour DJ, Birrell CE (1995) Desensitization of the nicotine-induced mesolimbic dopamine responses during constant infusion with nicotine. Br J Pharmacol 114:454-460.

Berke JD, Hyman SE (2000) Addiction, dopamine, and the molecular mechanisms of memory. Neuron 25:515-532. Berkovic S, Curtis L, Bertrand D (1999) Neuronal nicotinic receptor channel dysfunction in a human epilepsy syndrome. In:

Neuronal nicotinic receptors. Pharmacology and therapeutic opportunities (Arneric SP, Brioni JD, eds), 1st Edition, pp 287-306. New York: Wiley-Liss, Inc.

Bertolino M, Kellar KJ, Vicini S, Gillis RA (1997) Nicotinic receptor mediates spontaneous GABA release in the rat dorsal motor nucleus of the vagus. Neuroscience 79:671-681.

Bertrand D, Ballivet M, Rungger D (1990) Activation and blocking of neuronal nicotinic acetylcholine receptor reconstituted in Xenopus oocytes. Proc Natl Acad Sci U S A 87:1993-1997.

Bertrand D, Bertrand S, Ballivet M (1992) Pharmacological properties of the homomeric alpha7 receptor. Neurosci Lett 146:87-90.

Bertrand S, Weiland S, Berkovic SF, Steinlein OK, Bertrand D (1998) Properties of neuronal nicotinic acetylcholine receptor mutants from humans suffering from autosomal dominant nocturnal frontal lobe epilepsy. Br J Pharmacol 125:751-760.

Bertrand S, Patt JT, Spang JE, Westera G, Schubiger PA, Bertrand D (1999) Neuronal nAChR stereoselectivity to non-natural epibatidine derivatives. FEBS Lett 450:273-279.

Bezard E, Gross CE (1998) Compensatory mechanisms in experimental and human parkinsonism: towards a dynamic approach. Prog Neurobiol 55:93-116.

Bezard E, Bioulac B, Gross CE (1998) Glutamatergic compensatory mechanisms in experimental parkinsonism. Prog Neuro-Psychopharmacology Biol Psychiatry 22:609-623.

Bezard E, Boraud T, Bioulac B, Gross CE (1997) Compensatory effects of glutamatergic inputs to the substantia nigra pars compacta in experimental parkinsonism. Neuroscience 81:399-404.

Bezard E, Boraud T, Bioulac B, Gross CE (1999) Involvement of the subthalamic nucleus in glutamatergic compensatory mechanisms. Eur J Neurosci 11:2167-2170.

Bianchi C, Ferraro L, Tanganelli S, Morari M, Spalluto G, Simonato M, Beani L (1995) 5-Hydroxytryptamine-mediated effects of nicotine on endogenous GABA efflux from guinea-pig cortical slices. Br J Pharmacol 116:2724-2728.

Birkmeyer W, Riederer P (1985) Die Parkinson-Krankheit, 2nd Edition. Wien: Springer-Verlag. Birkmeyer W, Riederer P (1989) Understanding the Neurotransmitters: Key to the workings of the brain, 1st Edition. Wien:

Springer-Verlag. Blaha CD, Allen LF, Das S, Inglis WL, Latimer MP, Vincent SR, Winn P (1996) Modulation of dopamine efflux in the

nucleus accumbens after cholinergic stimulation of the ventral tegmental area in intact, pedunculopontine tegmental nucleus-lesioned, and laterodorsal tegmental nucleus-lesioned rats. J Neurosci 16:714-722.

99

Bolam JP, Smith Y (1990) The GABA and substance P input to dopaminergic neurones in the substantia nigra of the rat. Brain Res 529:57-78.

Bolam JP, Francis CM, Henderson Z (1991) Cholinergic input to dopaminergic neurons in the substantia nigra: a double immunocytochemical study. Neuroscience 41:483-494.

Bolam JP, Hanley JJ, Booth PA, Bevan MD (2000) Synaptic organisation of the basal ganglia. J Anat 196:527-542. Bonhaus DW, Bley KR, Broka CA, Fontana DJ, Leung E, Lewis R, Shieh A, Wong EH (1995) Characterization of the

electrophysiological, biochemical and behavioral actions of epibatidine. J Pharmacol Exp Ther 272:1199-1203. Bontempi B, Whelan KT, Risbrough VB, Lloyd GK, Menzaghi F (2003) Cognitive enhancing properties and tolerability of

cholinergic agents in mice: a comparative study of nicotine, donepezil, and SIB-1553A, a subtype-selective ligand for nicotinic acetylcholine receptors. Neuropsychopharmacology 28:1235-1246.

Boulter J, Connolly J, Deneris E, Goldman D, Heinemann S, Patrick J (1987) Functional expression of two neuronal nicotinic acetylcholine receptors from cDNA clones identifies a gene family. Proc Natl Acad Sci U S A 84:7763-7767.

Boulter J, Evans K, Goldman D, Martin G, Treco D, Heinemann S, Patrick J (1986) Isolation of a cDNA clone coding for a possible neural nicotinic acetylcholine receptor alpha-subunit. Nature 319:368-374.

Breese CR, Marks MJ, Logel J, Adams CE, Sullivan B, Collins AC, Leonard S (1997) Effect of smoking history on 3H nicotine binding in human postmortem brain. J Pharmacol Exp Ther 282:7-13.

Breese CR, Lee MJ, Adams CE, Sullivan B, Logel J, Gillen KM, Marks MJ, Collins AC, Leonard S (2000) Abnormal regulation of high affinity nicotinic receptors in subjects with schizophrenia. Neuropsychopharmacology 23:351-364.

Briggs CA, McKenna DG, Piattoni-Kaplan M (1995) Human alpha7 nicotinic acetylcholine receptor responses to novel ligands. Neuropharmacology 34:583-590.

Briggs CA, Anderson DJ, Brioni JD, Buccafusco JJ, Buckley MJ, Campbell JE, Decker MW, Donnelly-Roberts D, Elliott RL, Gopalakrishnan M, Holladay MW, Hui YH, Jackson WJ, Kim DJ, Marsh KC, O'Neill A, Prendergast MA, Ryther KB, Sullivan JP, Arneric SP (1997) Functional characterization of the novel neuronal nicotinic acetylcholine receptor ligand GTS-21 in vitro and in vivo. Pharmacol Biochem Behav 57:231-241.

Brioni JD, Arneric SP (1993) Nicotinic receptor agonists facilitate retention of avoidance training: participation of dopaminergic mechanisms. Behav Neural Biol 59:57-62.

Buccafusco JJ, Beach JW, Terry AV, Jr., Doad GS, Sood A, Arias E, Misawa H, Masai M, Fujii T, Kawashima K (2004) Novel analogs of choline as potential neuroprotective agents. J Alzheimers Dis 6(Suppl):S85-92.

Buisson B, Bertrand D (2001) Chronic exposure to nicotine upregulates the human alpha4beta2 nicotinic acetylcholine receptor function. J Neurosci 21:1819-1829.

Buisson B, Bertrand D (2002) Nicotine addiction: the possible role of functional upregulation. Trends Pharmacol Sci 23:130-136.

Buisson B, Vallejo YF, Green WN, Bertrand D (2000) The unusual nature of epibatidine responses at the alpha4beta2 nicotinic acetylcholine receptor. Neuropharmacology 39:2561-2569.

Buisson B, Gopalakrishnan M, Arneric SP, Sullivan JP, Bertrand D (1996) Human alpha4beta2 neuronal nicotinic acetylcholine receptor in HEK 293 cells: A patch-clamp study. J Neurosci 16:7880-7891.

Bättig K, Driscoll P, Schlatter J, Uster HJ (1976) Effects of nicotine on the exploratory locomotion patterns of female Roman high- and low-avoidance rats. Pharmacol Biochem Behav 4:435-439.

Cachelin AB, Jaggi R (1991) Beta subunits determine the time course of desensitization in rat alpha3 neuronal nicotinic acetylcholine receptors. Pflügers Arch Eur J Physiol 419:579-582.

Cadoni C, Di Chiara G (2000) Differential changes in accumbens shell and core dopamine in behavioral sensitization to nicotine. Eur J Pharmacol 387:R23-25.

Calabresi P, Lacey MG, North RA (1989) Nicotinic excitation of rat ventral tegmental neurones in vitro studied by intracellular recording. Br J Pharmacol 98:135-140.

Calcagnetti DJ, Schechter MD (1994) Nicotine place preference using the biased method of conditioning. Prog Neuro-Psychopharmacology Biol Psychiatry 18:925-933.

Carboni E, Acquas E, Leone P, Di Chiara G (1989a) 5HT3 receptor antagonists block morphine- and nicotine- but not amphetamine-induced reward. Psychopharmacologia 97:175-178.

Carboni E, Imperato A, Perezzani L, Di Chiara G (1989b) Amphetamine, cocaine, phencyclidine and nomifensine increase extracellular dopamine concentrations preferentially in the nucleus accumbens of freely moving rats. Neuroscience 28:653-661.

Carey RJ (1991) Chronic L-dopa treatment in the unilateral 6-OHDA rat: evidence for behavioral sensitization and biochemical tolerance. Brain Res 568:205-214.

Carlsson A (1959) The occurrence, distribution and physiological role of catecholamines in the nervous system. Pharmacol Rev 11:490-493.

Carlsson A, Lindqvist M, Magnusson T, Waldeck B (1958) On the presence of 3-hydroxytyramine in brain. Science 127:471. Carr LA, Rowell PP (1990) Attenuation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity by tobacco

smoke. Neuropharmacology 29:311-314. Cartier GE, Yoshikami D, Gray WR, Luo S, Olivera BM, McIntosh JM (1996) A new alpha-conotoxin which targets

alpha3beta2 nicotinic acetylcholine receptors. J Biol Chem 271:7522-7528. Cass WA, Zahniser NR, Flach KA, Gerhardt GA (1993) Clearance of exogenous dopamine in rat dorsal striatum and nucleus

accumbens: role of metabolism and effects of locally applied uptake inhibitors. J Neurochem 61:2269-2278. Champtiaux N, Han ZY, Bessis A, Rossi FM, Zoli M, Marubio L, McIntosh JM, Changeux JP (2002) Distribution and

pharmacology of alpha6-containing nicotinic acetylcholine receptors analyzed with mutant mice. J Neurosci 22:1208-1217.

100

Champtiaux N, Gotti C, Cordero-Erausquin M, David DJ, Przybylski C, Lena C, Clementi F, Moretti M, Rossi FM, Le Novere N, McIntosh JM, Gardier AM, Changeux JP (2003) Subunit composition of functional nicotinic receptors in dopaminergic neurons investigated with knock-out mice. J Neurosci 23:7820-7829.

Changeux JP, Edelstein SJ (1998) Allosteric receptors after 30 years. Neuron 21:959-980. Changeux JP, Benoit P, Bessis A, Cartaud J, Devillers-Thiery A, Fontaine B, Galzi JL, Klarsfeld A, Laufer R, Mulle C

(1990) The acetylcholine receptor: functional architecture and regulation. Adv Second Messenger Phosphoprotein Res 24:15-19.

Charléty PJ, Grenhoff J, Chergui K, De la Chapelle B, Buda M, Svensson TH, Chouvet G (1991) Burst firing of mesencephalic dopamine neurons is inhibited by somatodendritic application of kynurenate. Acta Physiol Scand 142:105-112.

Charpantier E, Barneoud P, Moser P, Besnard F, Sgard F (1998) Nicotinic acetylcholine subunit mRNA expression in dopaminergic neurons of the rat substantia nigra and ventral tegmental area. Neuroreport 9:3097-3101.

Chavez-Noriega LE, Gillespie A, Stauderman KA, Crona JH, Claeps BO, Elliott KJ, Reid RT, Rao TS, Velicelebi G, Harpold MM, Johnson EC, Corey-Naeve J (2000) Characterization of the recombinant human neuronal nicotinic acetylcholine receptors alpha3beta2 and alpha4beta2 stably expressed in HEK293 cells. Neuropharmacology 39:2543-2560.

Chergui K, Charléty PJ, Akaoka H, Saunier CF, Brunet JL, Buda M, Svensson TH, Chouvet G (1993) Tonic activation of NMDA receptors causes spontaneous burst discharge of rat midbrain dopamine neurons in vivo. Eur J Neurosci 5:137-144.

Chiodini FC, Tassonyi E, Hulo S, Bertrand D, Muller D (1999) Modulation of synaptic transmission by nicotine and nicotinic antagonists in hippocampus. Brain Res Bull 48:623-628.

Cimino M, Marini P, Fornasari D, Cattabeni F, Clementi F (1992) Distribution of nicotinic receptors in cynomolgus monkey brain and ganglia: localization of alpha3 subunit mRNA, alpha-bungarotoxin and nicotine binding sites. Neuroscience 51:77-86.

Cinciripini PM, Hecht SS, Henningfield JE, Manley MW, Kramer BS (1997) Tobacco addiction: implications for treatment and cancer prevention. J Natl Cancer Inst 89:1852-1867.

Clarke PB (1993a) Nicotine dependence - mechanisms and therapeutic strategies. Biochem Soc Symp 59:83-95. Clarke PB (1993b) Nicotinic receptors in mammalian brain: localization and relation to cholinergic innervation. Prog Brain

Res 98:77-83. Clarke PB, Kumar R (1983a) Characterization of the locomotor stimulant action of nicotine in tolerant rats. Br J Pharmacol

80:587-594. Clarke PB, Kumar R (1983b) The effects of nicotine on locomotor activity in non-tolerant and tolerant rats. Br J Pharmacol

78:329-337. Clarke PB, Pert A (1985) Autoradiographic evidence for nicotine receptors on nigrostriatal and mesolimbic dopaminergic

neurons. Brain Res 348:355-358. Clarke PB, Fibiger HC (1987) Apparent absence of nicotine-induced conditioned place preference in rats.

Psychopharmacology 92:84-88. Clarke PB, Hommer DW, Pert A, Skirboll LR (1985a) Electrophysiological actions of nicotine on substantia nigra single

units. Br J Pharmacol 85:827-835. Clarke PB, Hommer DW, Pert A, Skirboll LR (1987) Innervation of substantia nigra neurons by cholinergic afferents from

pedunculopontine nucleus in the rat: neuroanatomical and electrophysiological evidence. Neuroscience 23:1011-1019. Clarke PB, Fu DS, Jakubovic A, Fibiger HC (1988) Evidence that mesolimbic dopaminergic activation underlies the

locomotor stimulant action of nicotine in rats. J Pharmacol Exp Ther 246:701-708. Clarke PB, Schwartz RD, Paul SM, Pert CB, Pert A (1985b) Nicotinic binding in rat brain: autoradiographic comparison of

3Hacetylcholine, 3Hnicotine, and 125Ialpha-bungarotoxin. J Neurosci 5:1307-1315. Cobb WS, Abercrombie ED (2002) Distinct roles for nigral GABA and glutamate receptors in the regulation of dendritic

dopamine release under normal conditions and in response to systemic haloperidol. J Neurosci 22:1407-1413. Collins AC, Luo Y, Selvaag S, Marks MJ (1994) Sensitivity to nicotine and brain nicotinic receptors are altered by chronic

nicotine and mecamylamine infusion. J Pharmacol Exp Ther 271:125-133. Colquhoun LM, Patrick JW (1997) Pharmacology of neuronal nicotinic acetylcholine receptor subtypes. Adv Pharmacol

39:191-220. Cooper E, Couturier S, Ballivet M (1991) Pentameric structure and subunit stoichiometry of a neuronal nicotinic

acetylcholine receptor. Nature 350:235-238. Cooper JR, Bloom FE, Roth RH (2003) The biochemical basis of neuropharmacology, 8th Edition. New York: Oxford

University Press. Corrigall WA (1999) Nicotine self-administration in animals as a dependence model. Nicotine Tobacco Res 1:11-20. Corrigall WA, Coen KM (1989) Nicotine maintains robust self-administration in rats on a limited-access schedule.

Psychopharmacology 99:473-478. Corrigall WA, Coen KM (1991) Selective dopamine antagonists reduce nicotine self-administration. Psychopharmacology

104:171-176. Corrigall WA, Coen KM, Adamson KL (1994) Self-administered nicotine activates the mesolimbic dopamine system through

the ventral tegmental area. Brain Res 653:278-284. Corrigall WA, Franklin KB, Coen KM, Clarke PB (1992) The mesolimbic dopaminergic system is implicated in the

reinforcing effects of nicotine. Psychopharmacology 107:285-289. Corrigall WA, Coen KM, Zhang J, Adamson KL (2001) GABA mechanisms in the pedunculopontine tegmental nucleus

influence particular aspects of nicotine self-administration selectively in the rat. Psychopharmacology 158:190-197.

101

Corrigall WA, Coen KM, Adamson KL, Chow BL, Zhang J (2000) Response of nicotine self-administration in the rat to manipulations of mu-opioid and gamma-aminobutyric acid receptors in the ventral tegmental area. Psychopharmacologia 149:107-114.

Cosford ND, Bleicher L, Vernier JM, Chavez-Noriega L, Rao TS, Siegel RS, Suto C, Washburn M, Lloyd GK, McDonald IA (2000) Recombinant human receptors and functional assays in the discovery of altinicline (SIB-1508Y), a novel acetylcholine-gated ion channel (nAChR) agonist. Pharm Acta Helv 74:125-130.

Costa G, Abin-Carriquiry JA, Dajas F (2001) Nicotine prevents striatal dopamine loss produced by 6-hydroxydopamine lesion in the substantia nigra. Brain Res 888:336-342.

Costall B, Naylor RJ (1973) Neuroleptic and non-neuroleptic catalepsy. Arzneimittelforschung 23:674-683. Costall B, Kelly DM, Naylor RJ (1975) Nomifensine: a potent dopaminergic agonist of antiparkinson potential.

Psychopharmacologia 41:153-164. Court JA, Martin-Ruiz C, Graham A, Perry E (2000a) Nicotinic receptors in human brain: topography and pathology. J Chem

Neuroanat 20:281-298. Court JA, Piggott MA, Lloyd S, Cookson N, Ballard CG, McKeith IG, Perry RH, Perry EK (2000b) Nicotine binding in

human striatum: elevation in schizophrenia and reductions in dementia with Lewy bodies, Parkinson's disease and Alzheimer's disease and in relation to neuroleptic medication. Neuroscience 98:79-87.

Court JA, Lloyd S, Johnson M, Griffiths M, Birdsall NJ, Piggott MA, Oakley AE, Ince PG, Perry EK, Perry RH (1997) Nicotinic and muscarinic cholinergic receptor binding in the human hippocampal formation during development and aging. Brain Res Dev Brain Res 101:93-105.

Court JA, Lloyd S, Thomas N, Piggott MA, Marshall EF, Morris CM, Lamb H, Perry RH, Johnson M, Perry EK (1998) Dopamine and nicotinic receptor binding and the levels of dopamine and homovanillic acid in human brain related to tobacco use. Neuroscience 87:63-78.

Cousins MS, Roberts DC, de Wit H (2002) GABA(B) receptor agonists for the treatment of drug addiction: a review of recent findings. Drug Alcohol Depend 65:209-220.

Couturier S, Erkman L, Valera S, Rungger D, Bertrand S, Boulter J, Ballivet M, Bertrand D (1990a) Alpha5, alpha3, and non-alpha3. Three clustered avian genes encoding neuronal nicotinic acetylcholine receptor-related subunits. J Biol Chem 265:17560-17567.

Couturier S, Bertrand D, Matter JM, Hernandez MC, Bertrand S, Millar N, Valera S, Barkas T, Ballivet M (1990b) A neuronal nicotinic acetylcholine receptor subunit alpha7 is developmentally regulated and forms a homo-oligomeric channel blocked by alpha-BTX. Neuron 5:847-856.

Cui C, Booker TK, Allen RS, Grady SR, Whiteaker P, Marks MJ, Salminen O, Tritto T, Butt CM, Allen WR, Stitzel JA, McIntosh JM, Boulter J, Collins AC, Heinemann SF (2003) The beta3 nicotinic receptor subunit: a component of alpha-conotoxin MII-binding nicotinic acetylcholine receptors that modulate dopamine release and related behaviors. J Neurosci 23:11045-11053.

Cummings JL, Benson DF (1987) The role of the nucleus basalis of Meynert in dementia: review and reconsideration. Alzheimer Dis Assoc Dis 1:128-155.

Dahlström A, Fuxe K (1964) Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons. Acta Physiol Scand 62:1-55.

Dalack GW, Healy DJ, Meador-Woodruff JH (1998) Nicotine dependence in schizophrenia: clinical phenomena and laboratory findings. Am J Psychiatry 155:1490-1501.

Damaj MI, Creasy KR, Grove AD, Rosecrans JA, Martin BR (1994) Pharmacological effects of epibatidine optical enantiomers. Brain Res 664:34-40.

Damsma G, Day J, Fibiger HC (1989) Lack of tolerance to nicotine-induced dopamine release in the nucleus accumbens. Eur J Pharmacol 168:363-368.

Damsma G, Westerink BH, de Vries JB, Horn AS (1988) The effect of systemically applied cholinergic drugs on the striatal release of dopamine and its metabolites, as determined by automated brain dialysis in conscious rats. Neurosci Lett 89:349-354.

Dani JA (2001) Nicotinic receptor activity alters synaptic plasticity. ScientificWorldJournal 1:393-395. Davies AR, Hardick DJ, Blagbrough IS, Potter BV, Wolstenholme AJ, Wonnacott S (1999) Characterisation of the binding

of 3H methyllycaconitine: a new radioligand for labelling alpha7-type neuronal nicotinic acetylcholine receptors. Neuropharmacology 38:679-690.

de Fiebre CM, Meyer EM, Henry JC, Muraskin SI, Kem WR, Papke RL (1995) Characterization of a series of anabaseine-derived compounds reveals that the 3-(4)-dimethylaminocinnamylidine derivative is a selective agonist at neuronal nicotinic alpha7/125I-alpha-bungarotoxin receptor subtypes. Mol Pharmacol 47:164-171.

de Rover M, Mansvelder HD, Lodder JC, Wardeh G, Schoffelmeer AN, Brussaard AB (2004) Long-lasting nicotinic modulation of GABAergic synaptic transmission in the rat nucleus accumbens associated with behavioural sensitization to amphetamine. Eur J Neurosci 19:2859-2870.

Decker MW, Brioni JD, Bannon AW, Arneric SP (1995) Diversity of neuronal nicotinic acetylcholine receptors: lessons from behavior and implications for CNS therapeutics. Life Sci 56:545-570.

Deneris ES, Connolly J, Rogers SW, Duvoisin R (1991) Pharmacological and functional diversity of neuronal nicotinic acetylcholine receptors. Trends Pharmacol Sci 12:34-40.

Deneris ES, Boulter J, Swanson LW, Patrick J, Heinemann S (1989) Beta 3: a new member of nicotinic acetylcholine receptor gene family is expressed in brain. J Biol Chem 264:6268-6272.

Dewey SL, Brodie JD, Gerasimov M, Horan B, Gardner EL, Ashby CR, Jr. (1999) A pharmacologic strategy for the treatment of nicotine addiction. Synapse 31:76-86.

Di Chiara G (2000) Role of dopamine in the behavioural actions of nicotine related to addiction. Eur J Pharmacol 393:295-314.

102

Di Chiara G (2002) Nucleus accumbens shell and core dopamine: differential role in behavior and addiction. Behav Brain Res 137:75-114.

Di Chiara G, Imperato A (1988) Drugs abused by humans preferentially increase synaptic dopamine concentrations in the mesolimbic system of freely moving rats. Proc Natl Acad Sci U S A 85:5274-5278.

Dilger JP, Liu Y (1992) Desensitization of acetylcholine receptors in BC3H-1 cells. Pflügers Arch Eur J Physiol 420:479-485.

Dobelis P, Hutton S, Lu Y, Collins AC (2003) GABAergic systems modulate nicotinic receptor-mediated seizures in mice. J Pharmacol Exp Ther 306:1159-1166.

Domino EF, Ni L, Zhang H (1999) Nicotine alone and in combination with L-DOPA methyl ester or the D(2) agonist N-0923 in MPTP-induced chronic hemiparkinsonian monkeys. Exp Neurol 158:414-421.

Donny EC, Caggiula AR, Knopf S, Brown C (1995) Nicotine self-administration in rats. Psychopharmacology 122:390-394. Donny EC, Caggiula AR, Mielke MM, Jacobs KS, Rose C, Sved AF (1998) Acquisition of nicotine self-administration in

rats: the effects of dose, feeding schedule, and drug contingency. Psychopharmacology 136:83-90. Donny EC, Caggiula AR, Mielke MM, Booth S, Gharib MA, Hoffman A, Maldovan V, Shupenko C, McCallum SE (1999)

Nicotine self-administration in rats on a progressive ratio schedule of reinforcement. Psychopharmacology 147:135-142. Dowell C, Olivera BM, Garrett JE, Staheli ST, Watkins M, Kuryatov A, Yoshikami D, Lindstrom JM, McIntosh JM (2003)

Alpha-conotoxin PIA is selective for alpha6 subunit-containing nicotinic acetylcholine receptors. J Neurosci 23:8445-8452.

Dukat M, Glennon RA (2003) Epibatidine: impact on nicotinic receptor research. Cell Mol Neurobiol 23:365-378. Durany N, Zochling R, Boissl KW, Paulus W, Ransmayr G, Tatschner T, Danielczyk W, Jellinger K, Deckert J, Riederer P

(2000) Human post-mortem striatal alpha4beta2 nicotinic acetylcholine receptor density in schizophrenia and Parkinson's syndrome. Neurosci Lett 287:109-112.

Dursun SM, Kutcher S (1999) Smoking, nicotine and psychiatric disorders: evidence for therapeutic role, controversies and implications for future research. Med Hypotheses 52:101-109.

Ebersbach G, Stock M, Muller J, Wenning G, Wissel J, Poewe W (1999) Worsening of motor performance in patients with Parkinson's disease following transdermal nicotine administration. Mov Disord 14:1011-1013.

Elbaz A, Manubens-Bertran JM, Baldereschi M, Breteler MM, Grigoletto F, Lopez-Pousa S, Dartigues JF, Alperovitch A, Rocca WA, Tzourio C (2000) Parkinson's disease, smoking, and family history. EUROPARKINSON Study Group. J Neurol 247:793-798.

el-Bizri H, Clarke PB (1994a) Blockade of nicotinic receptor-mediated release of dopamine from striatal synaptosomes by chlorisondamine and other nicotinic antagonists administered in vitro. Br J Pharmacol 111:406-413.

el-Bizri H, Clarke PB (1994b) Regulation of nicotinic receptors in rat brain following quasi-irreversible nicotinic blockade by chlorisondamine and chronic treatment with nicotine. Br J Pharmacol 113:917-925.

Elgoyhen AB, Johnson DS, Boulter J, Vetter DE, Heinemann S (1994) Alpha9: an acetylcholine receptor with novel pharmacological properties expressed in rat cochlear hair cells. Cell 79:705-715.

Elgoyhen AB, Vetter DE, Katz E, Rothlin CV, Heinemann SF, Boulter J (2001) Alpha10: a determinant of nicotinic cholinergic receptor function in mammalian vestibular and cochlear mechanosensory hair cells. Proc Natl Acad Sci U S A 98:3501-3506.

Elliott KJ, Jones JM, Sacaan AI, Lloyd GK, Corey-Naeve J (1998) 6-Hydroxydopamine lesion of rat nigrostriatal dopaminergic neurons differentially affects nicotinic acetylcholine receptor subunit mRNA expression. J Mol Neurosci 10:251-260.

Elliott KJ, Ellis SB, Berckhan KJ, Urrutia A, Chavez-Noriega LE, Johnson EC, Velicelebi G, Harpold MM (1996) Comparative structure of human neuronal alpha2-alpha7 and beta2-beta4 nicotinic acetylcholine receptor subunits and functional expression of the alpha2, alpha3, alpha4, alpha7, beta2, and beta4 subunits. J Mol Neurosci 7:217-228.

Epping-Jordan MP, Picciotto MR, Changeux JP, Pich EM (1999) Assessment of nicotinic acetylcholine receptor subunit contributions to nicotine self-administration in mutant mice. Psychopharmacologia 147:25-26.

Erhardt S, Mathe JM, Chergui K, Engberg G, Svensson TH (2002) GABA(B) receptor-mediated modulation of the firing pattern of ventral tegmental area dopamine neurons in vivo. Naunyn-Schmiedebergs Arch Pharmacol 365:173-180.

Everhart D, Reiller E, Mirzoian A, McIntosh JM, Malhotra A, Luetje CW (2003) Identification of residues that confer alpha-conotoxin-PnIA sensitivity on the alpha 3 subunit of neuronal nicotinic acetylcholine receptors. J Pharmacol Exp Ther 306:664-670.

Everitt BJ, Wolf ME (2002) Psychomotor stimulant addiction: a neural systems perspective. J Neurosci 22:3312-3320. Fadda P, Scherma M, Fresu A, Collu M, Fratta W (2003) Baclofen antagonizes nicotine-, cocaine-, and morphine-induced

dopamine release in the nucleus accumbens of rat. Synapse 50:1-6. Fagerström KO, Pomerleau O, Giordani B, Stelson F (1994) Nicotine may relieve symptoms of Parkinson's disease.

Psychopharmacology 116:117-119. Falk L, Nordberg A, Seiger A, Kjaeldgaard A, Hellström-Lindahl E (2005) Smoking during early pregnancy affects the

expression pattern of both nicotinic and muscarinic acetylcholine receptors in human first trimester brainstem and cerebellum. Neuroscience 132:389-397.

Fattore L, Cossu G, Martellotta MC, Fratta W (2002) Baclofen antagonizes intravenous self-administration of nicotine in mice and rats. Alcohol Alcoholism 37:495-498.

Feldman RS, Meyer JS, Quenzer LF (1997) Principles in Neuropsychopharmacology. Massachusetts: Sinauer Associates, Inc.

Fenster CP, Rains MF, Noerager B, Quick MW, Lester RA (1997) Influence of subunit composition on desensitization of neuronal acetylcholine receptors at low concentrations of nicotine. J Neurosci 17:5747-5759.

Fenster CP, Whitworth TL, Sheffield EB, Quick MW, Lester RA (1999a) Upregulation of surface alpha4beta2 nicotinic receptors is initiated by receptor desensitization after chronic exposure to nicotine. J Neurosci 19:4804-4814.

103

Fenster CP, Hicks JH, Beckman ML, Covernton PJ, Quick MW, Lester RA (1999b) Desensitization of nicotinic receptors in the central nervous system. Ann NY Acad Sci 868:620-623.

Ferger B, Kuschinsky K (1997) Biochemical studies support the assumption that dopamine plays a minor role in the EEG effects of nicotine. Psychopharmacology 129:192-196.

Ferrari R, Le Novere N, Picciotto MR, Changeux JP, Zoli M (2002) Acute and long-term changes in the mesolimbic dopamine pathway after systemic or local single nicotine injections. Eur J Neurosci 15:1810-1818.

Fisher JL, Dani JA (2000) Nicotinic receptors on hippocampal cultures can increase synaptic glutamate currents while decreasing the NMDA-receptor component. Neuropharmacology 39:2756-2769.

Fisher JL, Pidoplichko VI, Dani JA (1998) Nicotine modifies the activity of ventral tegmental area dopaminergic neurons and hippocampal GABAergic neurons. J Physiol (Paris) 92:209-213.

Flores CM, Davila-Garcia MI, Ulrich YM, Kellar KJ (1997) Differential regulation of neuronal nicotinic receptor binding sites following chronic nicotine administration. J Neurochem 69:2216-2219.

Flores CM, Rogers SW, Pabreza LA, Wolfe BB, Kellar KJ (1992) A subtype of nicotinic cholinergic receptor in rat brain is composed of alpha4 and beta2 subunits and is up-regulated by chronic nicotine treatment. Mol Pharmacol 41:31-37.

Fonck C, Nashmi R, Deshpande P, Damaj MI, Marks MJ, Riedel A, Schwarz J, Collins AC, Labarca C, Lester HA (2003) Increased sensitivity to agonist-induced seizures, straub tail, and hippocampal theta rhythm in knock-in mice carrying hypersensitive alpha4 nicotinic receptors. J Neurosci 23:2582-2590.

Fowler JS, Logan J, Wang GJ, Volkow ND (2003) Monoamine oxidase and cigarette smoking. Neurotoxicology 24:75-82. Fowler JS, Volkow ND, Wang GJ, Pappas N, Logan J, MacGregor R, Alexoff D, Shea C, Schlyer D, Wolf AP, Warner D,

Zezulkova I, Cilento R (1996) Inhibition of monoamine oxidase B in the brains of smokers. Nature 379:733-736. Fratiglioni L, Wang HX (2000) Smoking and Parkinson's and Alzheimer's disease: review of the epidemiological studies.

Behav Brain Res 113:117-120. Free RB, Bryant DL, McKay SB, Kaser DJ, McKay DB (2002) 3H Epibatidine binding to bovine adrenal medulla: evidence

for alpha3beta4* nicotinic receptors. Neurosci Lett 318:98-102. Freedman R, Hall M, Adler LE, Leonard S (1995) Evidence in postmortem brain tissue for decreased numbers of

hippocampal nicotinic receptors in schizophrenia. Biol Psychiatry 38:22-33. Fu Y, Matta SG, Gao W, Sharp BM (2000a) Local alpha-bungarotoxin-sensitive nicotinic receptors in the nucleus accumbens

modulate nicotine-stimulated dopamine secretion in vivo. Neuroscience 101:369-375. Fu Y, Matta SG, Gao W, Brower VG, Sharp BM (2000b) Systemic nicotine stimulates dopamine release in nucleus

accumbens: re-evaluation of the role of N-methyl-D-aspartate receptors in the ventral tegmental area. J Pharmacol Exp Ther 294:458-465.

Fucile S, Matter JM, Erkman L, Ragozzino D, Barabino B, Grassi F, Alema S, Ballivet M, Eusebi F (1998) The neuronal alpha6 subunit forms functional heteromeric acetylcholine receptors in human transfected cells. Eur J Neurosci 10:172-178.

Fudala PJ, Iwamoto ET (1986) Further studies on nicotine-induced conditioned place preference in the rat. Pharmacol Biochem Behav 25:1041-1049.

Fudala PJ, Iwamoto ET (1987) Conditioned aversion after delay place conditioning with nicotine. Psychopharmacology 92:376-381.

Fudala PJ, Teoh KW, Iwamoto ET (1985) Pharmacologic characterization of nicotine-induced conditioned place preference. Pharmacol Biochem Behav 22:237-241.

Fung YK (1989) Effects of chronic nicotine pretreatment on (+)-amphetamine and nicotine-induced synthesis and release of 3H dopamine from 3H tyrosine in rat nucleus accumbens. J Pharm Pharmacol 41:66-68.

Fung YK, Fiske LA, Lau YS (1991) Chronic administration of nicotine fails to alter the MPTP-induced neurotoxicity in mice. Gen Pharmacol 22:669-672.

Futami T, Takakusaki K, Kitai ST (1995) Glutamatergic and cholinergic inputs from the pedunculopontine tegmental nucleus to dopamine neurons in the substantia nigra pars compacta. Neurosci Res 21:331-342.

Fuxe K, Janson AM, Jansson A, Andersson K, Eneroth P, Agnati LF (1990) Chronic nicotine treatment increases dopamine levels and reduces dopamine utilization in substantia nigra and in surviving forebrain dopamine nerve terminal systems after a partial di-mesencephalic hemitransection. Naunyn-Schmiedebergs Arch Pharmacol 341:171-181.

Fuxe K, Agnati L, Eneroth P, Gustafsson JA, Hökfelt T, Lofström A, Skett B, Skett P (1977) The effect of nicotine on central catecholamine neurons and gonadotropin secretion. I. Studies in the male rat. Med Biol 55:148-157.

Gainetdinov RR, Jones SR, Caron MG (1999) Functional hyperdopaminergia in dopamine transporter knock-out mice. Biol Psychiatry 46:303-311.

Gardner EL, Schiffer WK, Horan BA, Highfield D, Dewey SL, Brodie JD, Ashby CR, Jr. (2002) Gamma-vinyl GABA, an irreversible inhibitor of GABA transaminase, alters the acquisition and expression of cocaine-induced sensitization in male rats. Synapse 46:240-250.

Gariano RF, Groves PM (1988) Burst firing induced in midbrain dopamine neurons by stimulation of the medial prefrontal and anterior cingulate cortices. Brain Res 462:194-198.

Garris PA, Wightman RM (1994) Different kinetics govern dopaminergic transmission in the amygdala, prefrontal cortex, and striatum: an in vivo voltammetric study. J Neurosci 14:442-450.

Gatto GJ, Bohme GA, Caldwell WS, Letchworth SR, Traina VM, Obinu MC, Laville M, Reibaud M, Pradier L, Dunbar G, Bencherif M (2004) TC-1734: an orally active neuronal nicotinic acetylcholine receptor modulator with antidepressant, neuroprotective and long-lasting cognitive effects. CNS Drug Rev 10:147-166.

Gentry CL, Wilkins LH, Jr., Lukas RJ (2003) Effects of prolonged nicotinic ligand exposure on function of heterologously expressed, human alpha4beta2- and alpha4beta4-nicotinic acetylcholine receptors. J Pharmacol Exp Ther 304:206-216.

Gerasimov MR, Franceschi M, Volkow ND, Rice O, Schiffer WK, Dewey SL (2000) Synergistic interactions between nicotine and cocaine or methylphenidate depend on the dose of dopamine transporter inhibitor. Synapse 38:432-437.

104

Gerdeman GL, Partridge JG, Lupica CR, Lovinger DM (2003) It could be habit forming: drugs of abuse and striatal synaptic plasticity. Trends Neurosci 26:184-192.

Gerzanich V, Anand R, Lindstrom J (1994) Homomers of alpha8 and alpha7 subunits of nicotinic receptors exhibit similar channel but contrasting binding site properties. Mol Pharmacol 45:212-220.

Gerzanich V, Kuryatov A, Anand R, Lindstrom J (1997) "Orphan" alpha6 nicotinic AChR subunit can form a functional heteromeric acetylcholine receptor. Mol Pharmacol 51:320-327.

Gerzanich V, Wang F, Kuryatov A, Lindstrom J (1998) Alpha5 subunit alters desensitization, pharmacology, Ca++ permeability and Ca++ modulation of human neuronal alpha3 nicotinic receptors. J Pharmacol Exp Ther 286:311-320.

Gerzanich V, Peng X, Wang F, Wells G, Anand R, Fletcher S, Lindstrom J (1995) Comparative pharmacology of epibatidine: a potent agonist for neuronal nicotinic acetylcholine receptors. Mol Pharmacol 48:774-782.

Gilbert DG (1979) Paradoxical tranquilizing and emotion-reducing effects of nicotine. Psychol Bull 86:643-661. Giorguieff-Chesselet MF, Kemel ML, Wandscheer D, Glowinski J (1979) Regulation of dopamine release by presynaptic

nicotinic receptors in rat striatal slices: effect of nicotine in a low concentration. Life Sci 25:1257-1262. Girault JA, Spampinato U, Savaki HE, Glowinski J, Besson MJ (1986) In vivo release of 3H gamma-aminobutyric acid in the

rat neostriatum I. Characterization and topographical heterogeneity of the effects of dopaminergic and cholinergic agents. Neuroscience 19:1101-1108.

Girod R, Crabtree G, Ernstrom G, Ramirez-Latorre J, McGehee D, Turner J, Role L (1999) Heteromeric complexes of alpha5 and/or alpha7 subunits. Effects of calcium and potential role in nicotine-induced presynaptic facilitation. Ann NY Acad Sci 868:578-590.

Glassman AH (1993) Cigarette smoking: implications for psychiatric illness. Am J Psychiatry 150:546-553. Goldberg SR, Spealman RD, Goldberg DM (1981) Persistent behavior at high rates maintained by intravenous self-

administration of nicotine. Science 214:573-575. Goodman FR (1974) Effects of nicotine on distribution and release of 14C-norepinephrine and 14C-dopamine in rat brain

striatum and hypothalamus slices. Neuropharmacology 13:1025-1032. Gopalakrishnan M, Monteggia LM, Anderson DJ, Molinari EJ, Piattoni-Kaplan M, Donnelly-Roberts D, Arneric SP,

Sullivan JP (1996) Stable expression, pharmacologic properties and regulation of the human neuronal nicotinic acetylcholine alpha4beta2 receptor. J Pharmacol Exp Ther 276:289-297.

Gopalakrishnan M, Buisson B, Touma E, Giordano T, Campbell JE, Hu IC, Donnelly-Roberts D, Arneric SP, Bertrand D, Sullivan JP (1995) Stable expression and pharmacological properties of the human alpha7 nicotinic acetylcholine receptor. Eur J Pharmacol 290:237-246.

Gorell JM, Rybicki BA, Johnson CC, Peterson EL (1999) Smoking and Parkinson's disease: a dose-response relationship. Neurology 52:115-119.

Gotham AM, Brown RG, Marsden CD (1988) 'Frontal' cognitive function in patients with Parkinson's disease 'on' and 'off' levodopa. Brain 111:299-321.

Gotti C, Fornasari D, Clementi F (1997a) Human neuronal nicotinic receptors. Prog Neurobiol 53:199-237. Gotti C, Hanke W, Maury K, Moretti M, Ballivet M, Clementi F, Bertrand D (1994) Pharmacology and biophysical

properties of alpha7 and alpha7-alpha8 alpha-bungarotoxin receptor subtypes immunopurified from the chick optic lobe. Eur J Neurosci 6:1281-1291.

Gotti C, Moretti M, Maggi R, Longhi R, Hanke W, Klinke N, Clementi F (1997b) Alpha7 and alpha8 nicotinic receptor subtypes immunopurified from chick retina have different immunological, pharmacological and functional properties. Eur J Neurosci 9:1201-1211.

Gould E, Woolf NJ, Butcher LL (1989) Cholinergic projections to the substantia nigra from the pedunculopontine and laterodorsal tegmental nuclei. Neuroscience 28:611-623.

Grace AA, Bunney BS (1984a) The control of firing pattern in nigral dopamine neurons: burst firing. J Neurosci 4:2877-2890.

Grace AA, Bunney BS (1984b) The control of firing pattern in nigral dopamine neurons: single spike firing. J Neurosci 4:2866-2876.

Grady S, Marks MJ, Wonnacott S, Collins AC (1992) Characterization of nicotinic receptor-mediated 3H dopamine release from synaptosomes prepared from mouse striatum. J Neurochem 59:848-856.

Grady SR, Marks MJ, Collins AC (1994) Desensitization of nicotine-stimulated 3H dopamine release from mouse striatal synaptosomes. J Neurochem 62:1390-1398.

Grady SR, Meinerz NM, Cao J, Reynolds AM, Picciotto MR, Changeux JP, McIntosh JM, Marks MJ, Collins AC (2001) Nicotinic agonists stimulate acetylcholine release from mouse interpeduncular nucleus: a function mediated by a different nAChR than dopamine release from striatum. J Neurochem 76:258-268.

Graybiel AM (1990) Neurotransmitters and neuromodulators in the basal ganglia. Trends Neurosci 13:244-254. Grenhoff J, Svensson TH (1988) Selective stimulation of limbic dopamine activity by nicotine. Acta Physiol Scand 133:595-

596. Grenhoff J, Svensson TH (1992) Nicotinic and muscarinic components of rat brain dopamine synthesis stimulation induced

by physostigmine. Naunyn-Schmiedebergs Arch Pharmacol 346:395-398. Grenhoff J, Aston-Jones G, Svensson TH (1986) Nicotinic effects on the firing pattern of midbrain dopamine neurons. Acta

Physiol Scand 128:351-358. Grenhoff J, Tung CS, Svensson TH (1988) The excitatory amino acid antagonist kynurenate induces pacemaker-like firing of

dopamine neurons in rat ventral tegmental area in vivo. Acta Physiol Scand 134:567-568. Grillner P, Svensson TH (2000) Nicotine-induced excitation of midbrain dopamine neurons in vitro involves ionotropic

glutamate receptor activation. Synapse 38:1-9. Grilly DM, Simon BB, Levin ED (2000) Nicotine enhances stimulus detection performance of middle- and old-aged rats: a

longitudinal study. Pharmacol Biochem Behav 65:665-670.

105

Groot-Kormelink PJ, Luyten WH, Colquhoun D, Sivilotti LG (1998) A reporter mutation approach shows incorporation of the "orphan" subunit beta3 into a functional nicotinic receptor. J Biol Chem 273:15317-15320.

Grottick AJ, Trube G, Corrigall WA, Huwyler J, Malherbe P, Wyler R, Higgins GA (2000) Evidence that nicotinic alpha7 receptors are not involved in the hyperlocomotor and rewarding effects of nicotine. J Pharmacol Exp Ther 294:1112-1119.

Guan ZZ, Zhang X, Blennow K, Nordberg A (1999) Decreased protein level of nicotinic receptor alpha7 subunit in the frontal cortex from schizophrenic brain. Neuroreport 10:1779-1782.

Guan ZZ, Zhang X, Ravid R, Nordberg A (2000) Decreased protein levels of nicotinic receptor subunits in the hippocampus and temporal cortex of patients with Alzheimer's disease. J Neurochem 74:237-243.

Guan ZZ, Nordberg A, Mousavi M, Rinne JO, Hellström-Lindahl E (2002) Selective changes in the levels of nicotinic acetylcholine receptor protein and of corresponding mRNA species in the brains of patients with Parkinson's disease. Brain Res 956:358-366.

Guo JZ, Tredway TL, Chiappinelli VA (1998) Glutamate and GABA release are enhanced by different subtypes of presynaptic nicotinic receptors in the lateral geniculate nucleus. J Neurosci 18:1963-1969.

Göldner FM, Dineley KT, Patrick JW (1997) Immunohistochemical localization of the nicotinic acetylcholine receptor subunit alpha6 to dopaminergic neurons in the substantia nigra and ventral tegmental area. Neuroreport 8:2739-2742.

Hadjiconstantinou M, Hubble JP, Wemlinger TA, Neff NH (1994) Enhanced MPTP neurotoxicity after treatment with isoflurophate or cholinergic agonists. J Pharmacol Exp Ther 270:639-644.

Hahn B, Sharples CG, Wonnacott S, Shoaib M, Stolerman IP (2003) Attentional effects of nicotinic agonists in rats. Neuropharmacology 44:1054-1067.

Haikala H (1986) Different changes in striatal dopamine metabolism induced by nicotine in mice kept at different ambient temperatures. Evidence for partly separate metabolic routes of dopamine derived from separate compartmentations. Naunyn-Schmiedebergs Arch Pharmacol 334:373-376.

Haikala H (1987) Use of a novel type of rotating disc electrode and a flow cell with laminar flow pattern for the electrochemical detection of biogenic monoamines and their metabolites after Sephadex gel chromatographic purification and high-performance liquid chromatographic isolation from rat brain. J Neurochem 49:1033-1041.

Haikala H, Ahtee L (1988) Antagonism of the nicotine-induced changes of the striatal dopamine metabolism in mice by mecamylamine and pempidine. Naunyn-Schmiedebergs Arch Pharmacol 338:169-173.

Haikala H, Karmalahti T, Ahtee L (1986) The nicotine-induced changes in striatal dopamine metabolism of mice depend on body temperature. Brain Res 375:313-319.

Hamani C, Lozano AM (2003) Physiology and pathophysiology of Parkinson's disease. Ann NY Acad Sci 991:15-21. Han Z-Y, Zoli M, Cardona A, Bourgeois J-P, Changeux J-P, Le Novere N (2003) Localization of 3H nicotine, 3H cytisine,

3H epibatidine, and 125I bungarotoxin binding sites in the brain of macaca mulatta. J Comp Neurol 461:49-60. Harsing LG, Sershen H, Vizi SE, Lajtha A (1992) N-type calcium channels are involved in the dopamine releasing effect of

nicotine. Neurochem Res 17:729-734. Hauber W (1998) Involvement of basal ganglia transmitter systems in movement initiation. Prog Neurobiol 56:507-540. Heimer L, Zahm DS, Churchill L, Kalivas PW, Wohltmann C (1991) Specificity in the projection patterns of accumbal core

and shell in the rat. Neuroscience 41:89-125. Hellström-Lindahl E, Mousavi M, Ravid R, Nordberg A (2004a) Reduced levels of Abeta 40 and Abeta 42 in brains of

smoking controls and Alzheimer's patients. Neurobiol Dis 15:351-360. Hellström-Lindahl E, Mousavi M, Zhang X, Ravid R, Nordberg A (1999) Regional distribution of nicotinic receptor subunit

mRNAs in human brain: comparison between Alzheimer and normal brain. Brain Res Mol Brain Res 66:94-103. Hellström-Lindahl E, Court J, Keverne J, Svedberg M, Lee M, Marutle A, Thomas A, Perry E, Bednar I, Nordberg A (2004b)

Nicotine reduces Abeta in the brain and cerebral vessels of APPsw mice. Eur J Neurosci 19:2703-2710. Henningfield JE, Goldberg SR (1983) Control of behavior by intravenous nicotine injections in human subjects. Pharmacol

Biochem Behav 19:1021-1026. Henry B, Crossman AR, Brotchie JM (1998) Characterization of enhanced behavioral responses to L-DOPA following

repeated administration in the 6-hydroxydopamine-lesioned rat model of Parkinson's disease. Exp Neurol 151:334-342. Herning RI, Jones RT, Benowitz NL, Mines AH (1983) How a cigarette is smoked determines blood nicotine levels. Clin

Pharmacol Ther 33:84-90. Hill JA, Zoli M, Bourgeois JP, Changeux JP (1993) Immunocytochemical localization of a neuronal nicotinic receptor: the

beta2-subunit. J Neurosci 13:1551-1568. Hoffman AF, Gerhardt GA (1998) In vivo electrochemical studies of dopamine clearance in the rat substantia nigra: effects

of locally applied uptake inhibitors and unilateral 6-hydroxydopamine lesions. J Neurochem 70:179-189. Hogg RC, Bertrand D (2004) Nicotinic acetylcholine receptors as drug targets. Curr Drug Target CNS Neurol Disord 3:123-

130. Holladay MW, Dart MJ, Lynch JK (1997) Neuronal nicotinic acetylcholine receptors as targets for drug discovery. J Med

Chem 40:4169-4194. Huotari M, Santha M, Lucas LR, Karayiorgou M, Gogos JA, Mannisto PT (2002) Effect of dopamine uptake inhibition on

brain catecholamine levels and locomotion in catechol-O-methyltransferase-disrupted mice. J Pharmacol Exp Ther 303:1309-1316.

Imperato A, Mulas A, Di Chiara G (1986) Nicotine preferentially stimulates dopamine release in the limbic system of freely moving rats. Eur J Pharmacol 132:337-338.

Ishikawa A, Miyatake T (1993) Effects of smoking in patients with early-onset Parkinson's disease. J Neurol Sci 117:28-32. Ito R, Robbins TW, Everitt BJ (2004) Differential control over cocaine-seeking behavior by nucleus accumbens core and

shell. Nat Neuroscience 7:389-397.

106

Iwamoto ET (1990) Nicotine conditions place preferences after intracerebral administration in rats. Psychopharmacology 100:251-257.

Jacobs I, Anderson DJ, Surowy CS, Puttfarcken PS (2002) Differential regulation of nicotinic receptor-mediated neurotransmitter release following chronic (-)-nicotine administration. Neuropharmacology 43:847-856.

James JR, Villanueva HF, Johnson JH, Arezo S, Rosecrans JA (1994) Evidence that nicotine can acutely desensitize central nicotinic acetylcholinergic receptors. Psychopharmacology 114:456-462.

Janson AM, Moller A (1993) Chronic nicotine treatment counteracts nigral cell loss induced by a partial mesodiencephalic hemitransection: an analysis of the total number and mean volume of neurons and glia in substantia nigra of the male rat. Neuroscience 57:931-941.

Janson AM, Fuxe K, Goldstein M (1992) Differential effects of acute and chronic nicotine treatment on MPTP-(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) induced degeneration of nigrostriatal dopamine neurons in the black mouse. Clin Investigator 70:232-238.

Janson AM, Meana JJ, Goiny M, Herrera-Marschitz M (1991) Chronic nicotine treatment counteracts the decrease in extracellular neostriatal dopamine induced by a unilateral transection at the mesodiencephalic junction in rats: a microdialysis study. Neurosci Lett 134:88-92.

Janson AM, Hedlund PB, Fuxe K, von Euler G (1994) Chronic nicotine treatment counteracts dopamine D2 receptor upregulation induced by a partial meso-diencephalic hemitransection in the rat. Brain Res 655:25-32.

Janson AM, Fuxe K, Agnati LF, Kitayama I, Harfstrand A, Andersson K, Goldstein M (1988) Chronic nicotine treatment counteracts the disappearance of tyrosine-hydroxylase-immunoreactive nerve cell bodies, dendrites and terminals in the mesostriatal dopamine system of the male rat after partial hemitransection. Brain Res 455:332-345.

Jellinger KA (1997) Morphological substrates of dementia in parkinsonism. A critical update. J Neural Transm Suppl 51:57-82.

Jellinger KA (1999) Post mortem studies in Parkinson's disease--is it possible to detect brain areas for specific symptoms? J Neural Transm Suppl 56:1-29.

Jeyarasasingam G, Tompkins L, Quik M (2002) Stimulation of non-alpha7 nicotinic receptors partially protects dopaminergic neurons from 1-methyl-4-phenylpyridinium-induced toxicity in culture. Neuroscience 109:275-285.

Johnson DS, Martinez J, Elgoyhen AB, Heinemann SF, McIntosh JM (1995) Alpha-conotoxin ImI exhibits subtype-specific nicotinic acetylcholine receptor blockade: preferential inhibition of homomeric alpha7 and alpha9 receptors. Mol Pharmacol 48:194-199.

Jones GM, Sahakian BJ, Levy R, Warburton DM, Gray JA (1992) Effects of acute subcutaneous nicotine on attention, information processing and short-term memory in Alzheimer's disease. Psychopharmacology 108:485-494.

Jorenby DE, Steinpreis RE, Sherman JE, Baker TB (1990) Aversion instead of preference learning indicated by nicotine place conditioning in rats. Psychopharmacology 101:533-538.

Kaakkola S (1980) Contralateral circling behaviour induced by intranigral injection of morphine and enkephalin analogue FK 33-824 in rats. Acta Pharmacol Toxicol 47:385-393.

Kaakkola S (1981) Effect of nicotinic and muscarinic drugs on amphetamine- and apomorphine-induced circling behaviour in rats. Acta Pharmacol Toxicol 48:162-167.

Kaakkola S, Wurtman RJ (1992) Effects of COMT inhibitors on striatal dopamine metabolism: a microdialysis study. Brain Res 587:241-249.

Kaakkola S, Männistö PT, Nissinen E (1987) Striatal membrane-bound and soluble catechol-O-methyl-transferase after selective neuronal lesions in the rat. J Neural Transm Gen Sect 69:221-228.

Kaiser S, Wonnacott S (2000) Alpha-bungarotoxin-sensitive nicotinic receptors indirectly modulate 3H dopamine release in rat striatal slices via glutamate release. Mol Pharmacol 58:312-318.

Kaiser SA, Soliakov L, Harvey SC, Luetje CW, Wonnacott S (1998) Differential inhibition by alpha-conotoxin-MII of the nicotinic stimulation of 3H-dopamine release from rat striatal synaptosomes and slices. J Neurochem 70:1069-1076.

Kaneko S, Maeda T, Kume T, Kochiyama H, Akaike A, Shimohama S, Kimura J (1997) Nicotine protects cultured cortical neurons against glutamate-induced cytotoxicity via alpha7-neuronal receptors and neuronal CNS receptors. Brain Res 765:135-140.

Karlin A (1993) Structure of nicotinic acetylcholine receptors. Curr Opin Neurobiol 3:299-309. Karlin A, Akabas MH (1995) Toward a structural basis for the function of nicotinic acetylcholine receptors and their cousins.

Neuron 15:1231-1244. Karreman M, Westerink BH, Moghaddam B (1996) Excitatory amino acid receptors in the ventral tegmental area regulate

dopamine release in the ventral striatum. J Neurochem 67:601-607. Katz B, Thessleff S (1957) A study of desensitization produced by acetylcholine at the motor end-plate. J Physiol (Lond)

138:63-80. Kawai H, Berg DK (2001) Nicotinic acetylcholine receptors containing alpha7 subunits on rat cortical neurons do not

undergo long-lasting inactivation even when up-regulated by chronic nicotine exposure. J Neurochem 78:1367-1378. Kayadjanian N, Retaux S, Menetrey A, Besson MJ (1994) Stimulation by nicotine of the spontaneous release of 3H gamma-

aminobutyric acid in the substantia nigra and in the globus pallidus of the rat. Brain Res 649:129-135. Ke L, Eisenhour CM, Bencherif M, Lukas RJ (1998) Effects of chronic nicotine treatment on expression of diverse nicotinic

acetylcholine receptor subtypes. I. Dose- and time-dependent effects of nicotine treatment. J Pharmacol Exp Ther 286:825-840.

Kehr W (1976) 3-Methoxytyramine as an indicator of impulse-induced dopamine release in rat brain in vivo. Naunyn-Schmiedebergs Arch Pharmacol 293:209-215.

Kelton MC, Kahn HJ, Conrath CL, Newhouse PA (2000) The effects of nicotine on Parkinson's disease. Brain Cogn 43:274-282.

107

Kem WR, Mahnir VM, Papke RL, Lingle CJ (1997) Anabaseine is a potent agonist on muscle and neuronal alpha-bungarotoxin-sensitive nicotinic receptors. J Pharmacol Exp Ther 283:979-992.

Kita H, Kitai ST (1987) Efferent projections of the subthalamic nucleus in the rat: light and electron microscopic analysis with the PHA-L method. J Comp Neurol 260:435-452.

Klink R, de Kerchove d'Exaerde A, Zoli M, Changeux JP (2001) Molecular and physiological diversity of nicotinic acetylcholine receptors in the midbrain dopaminergic nuclei. J Neurosci 21:1452-1463.

Koob GF (1996) Drug addiction: the yin and yang of hedonic homeostasis. Neuron 16:893-896. Koob GF, Le Moal M (2001) Drug addiction, dysregulation of reward, and allostasis. Neuropsychopharmacology 24:97-129. Koós T, Tepper JM (1999) Inhibitory control of neostriatal projection neurons by GABAergic interneurons. Nat

Neuroscience 2:467-472. Ksir C, Hakan R, Hall DP, Jr., Kellar KJ (1985) Exposure to nicotine enhances the behavioral stimulant effect of nicotine and

increases binding of 3H acetylcholine to nicotinic receptors. Neuropharmacology 24:527-531. Kulak JM, Nguyen TA, Olivera BM, McIntosh JM (1997) Alpha-conotoxin MII blocks nicotine-stimulated dopamine release

in rat striatal synaptosomes. J Neurosci 17:5263-5270. Kuryatov A, Gerzanich V, Nelson M, Olale F, Lindstrom J (1997) Mutation causing autosomal dominant nocturnal frontal

lobe epilepsy alters Ca2+ permeability, conductance, and gating of human alpha4beta2 nicotinic acetylcholine receptors. J Neurosci 17:9035-9047.

Kuryatov A, Olale F, Cooper J, Choi C, Lindstrom J (2000) Human alpha6 AChR subtypes: subunit composition, assembly, and pharmacological responses. Neuropharmacology 39:2570-2590.

Lança AJ, Adamson KL, Coen KM, Chow BL, Corrigall WA (2000a) The pedunculopontine tegmental nucleus and the role of cholinergic neurons in nicotine self-administration in the rat: a correlative neuroanatomical and behavioral study. Neuroscience 96:735-742.

Lança JA, Sanelli TR, Corrigall WA (2000b) Nicotine-induced fos expression in the pedunculopontine mesencephalic tegmentum in the rat. Neuropharmacology 39:2808-2817.

Lange KW, Wells FR, Jenner P, Marsden CD (1993) Altered muscarinic and nicotinic receptor densities in cortical and subcortical brain regions in Parkinson's disease. J Neurochem 60:197-203.

Lange KW, Paul GM, Naumann M, Gsell W (1995) Dopaminergic effects on cognitive performance in patients with Parkinson's disease. J Neural Transm Suppl 46:423-432.

Lapin EP, Maker HS, Sershen H, Hurd Y, Lajtha A (1987) Dopamine-like action of nicotine: lack of tolerance and reverse tolerance. Brain Res 407:351-363.

Le Foll B, Goldberg SR (2005) Nicotine induces conditioned place preferences over a large range of doses in rats. Psychopharmacology 178:481-492.

Le Novere N, Changeux JP (1995) Molecular evolution of the nicotinic acetylcholine receptor: an example of multigene family in excitable cells. J Mol Evol 40:155-172.

Le Novere N, Zoli M, Changeux JP (1996) Neuronal nicotinic receptor alpha6 subunit mRNA is selectively concentrated in catecholaminergic nuclei of the rat brain. Eur J Neurosci 8:2428-2439.

Le Novere N, Zoli M, Lena C, Ferrari R, Picciotto MR, Merlo-Pich E, Changeux JP (1999) Involvement of alpha6 nicotinic receptor subunit in nicotine-elicited locomotion, demonstrated by in vivo antisense oligonucleotide infusion. Neuroreport 10:2497-2501.

Lecca D, Shim I, Costa E, Javaid JI (2000) Striatal application of nicotine, but not of lobeline, attenuates dopamine release in freely moving rats. Neuropharmacology 39:88-98.

Lee M, Martin-Ruiz C, Graham A, Court J, Jaros E, Perry R, Iversen P, Bauman M, Perry E (2002) Nicotinic receptor abnormalities in the cerebellar cortex in autism. Brain 125:1483-1495.

Leikola-Pelho T, Jackson DM (1992) Preferential stimulation of locomotor activity by ventral tegmental microinjections of (-)-nicotine. Pharmacol Toxicol 70:50-52.

Leikola-Pelho T, Heinämäki J, Laakso I, Ahtee L (1990) Chronic nicotine treatment changes differentially the effects of acute nicotine on the three main dopamine metabolites in mouse striatum. Naunyn-Schmiedebergs Arch Pharmacol 342:400-406.

Lena C, Changeux JP (1993) Allosteric modulations of the nicotinic acetylcholine receptor. Trends Neurosci 16:181-186. Lena C, Changeux JP (1997a) Pathological mutations of nicotinic receptors and nicotine-based therapies for brain disorders.

Curr Opin Neurobiol 7:674-682. Lena C, Changeux JP (1997b) Role of Ca2+ ions in nicotinic facilitation of GABA release in mouse thalamus. J Neurosci

17:576-585. Lena C, Changeux JP (1998) Allosteric nicotinic receptors, human pathologies. J Physiol (Paris) 92:63-74. Lena C, Changeux JP, Mulle C (1993) Evidence for "preterminal" nicotinic receptors on GABAergic axons in the rat

interpeduncular nucleus. J Neurosci 13:2680-2688. Leonard S, Gault J, Hopkins J, Logel J, Vianzon R, Short M, Drebing C, Berger R, Venn D, Sirota P, Zerbe G, Olincy A,

Ross RG, Adler LE, Freedman R (2002) Association of promoter variants in the alpha7 nicotinic acetylcholine receptor subunit gene with an inhibitory deficit found in schizophrenia. Arch Gen Psychiatry 59:1085-1096.

Lerman C, Caporaso N, Main D, Audrain J, Boyd NR, Bowman ED, Shields PG (1998) Depression and self-medication with nicotine: the modifying influence of the dopamine D4 receptor gene. Health Psychol 17:56-62.

Levin ED, Torry D (1996) Acute and chronic nicotine effects on working memory in aged rats. Psychopharmacology 123:88-97.

Levin ED, Simon BB (1998) Nicotinic acetylcholine involvement in cognitive function in animals. Psychopharmacologia 138:217-230.

Levin ED, Rezvani AH (2000) Development of nicotinic drug therapy for cognitive disorders. Eur J Pharmacol 393:141-146.

108

Levin ED, Rezvani AH (2002) Nicotinic treatment for cognitive dysfunction. Curr Drug Target CNS Neurol Disord 1:423-431.

Levin ED, Conners CK, Silva D, Canu W, March J (2001) Effects of chronic nicotine and methylphenidate in adults with attention deficit/hyperactivity disorder. Exp Clin Psychopharmacol 9:83-90.

Levin ED, Conners CK, Silva D, Hinton SC, Meck WH, March J, Rose JE (1998) Transdermal nicotine effects on attention. Psychopharmacology 140:135-141.

Li Y, Papke RL, He YJ, Millard WJ, Meyer EM (1999) Characterization of the neuroprotective and toxic effects of alpha7 nicotinic receptor activation in PC12 cells. Brain Res 830:218-225.

Lichtensteiger W, Felix D, Lienhart R, Hefti F (1976) A quantitative correlation between single unit activity and fluorescence intensity of dopamine neurones in zona compacta of substantia nigra, as demonstrated under the influence of nicotine and physostigmine. Brain Res 117:85-103.

Lichtensteiger W, Hefti F, Felix D, Huwyler T, Melamed E, Schlumpf M (1982) Stimulation of nigrostriatal dopamine neurones by nicotine. Neuropharmacology 21:963-968.

Limberger N, Spath L, Starke K (1986) A search for receptors modulating the release of gamma3H aminobutyric acid in rabbit caudate nucleus slices. J Neurochem 46:1109-1117.

Lindstrom J, Anand R, Peng X, Gerzanich V, Wang F, Li Y (1995) Neuronal nicotinic receptor subtypes. Ann NY Acad Sci 757:100-116.

Lippiello PM, Bencherif M, Gray JA, Peters S, Grigoryan G, Hodges H, Collins AC (1996) RJR-2403: a nicotinic agonist with CNS selectivity II. In vivo characterization. J Pharmacol Exp Ther 279:1422-1429.

Lloyd GK, Williams M (2000) Neuronal nicotinic acetylcholine receptors as novel drug targets. J Pharmacol Exp Ther 292:461-467.

Lloyd GK, Menzaghi F, Bontempi B, Suto C, Siegel R, Akong M, Stauderman K, Velicelebi G, Johnson E, Harpold MM, Rao TS, Sacaan AI, Chavez-Noriega LE, Washburn MS, Vernier JM, Cosford ND, McDonald LA (1998) The potential of subtype-selective neuronal nicotinic acetylcholine receptor agonists as therapeutic agents. Life Sci 62:1601-1606.

Louis M, Clarke PB (1998) Effect of ventral tegmental 6-hydroxydopamine lesions on the locomotor stimulant action of nicotine in rats. Neuropharmacology 37:1503-1513.

Lu Y, Marks MJ, Collins AC (1999) Desensitization of nicotinic agonist-induced 3H gamma-aminobutyric acid release from mouse brain synaptosomes is produced by subactivating concentrations of agonists. J Pharmacol Exp Ther 291:1127-1134.

Lu Y, Grady S, Marks MJ, Picciotto M, Changeux JP, Collins AC (1998) Pharmacological characterization of nicotinic receptor-stimulated GABA release from mouse brain synaptosomes. J Pharmacol Exp Ther 287:648-657.

Luetje CW (2004) Getting past the asterisk: the subunit composition of presynaptic nicotinic receptors that modulate striatal dopamine release. Mol Pharmacol 65:1333-1335.

Luetje CW, Patrick J (1991) Both alpha- and beta-subunits contribute to the agonist sensitivity of neuronal nicotinic acetylcholine receptors. J Neurosci 11:837-845.

Luetje CW, Wada K, Rogers S, Abramson SN, Tsuji K, Heinemann S, Patrick J (1990) Neurotoxins distinguish between different neuronal nicotinic acetylcholine receptor subunit combinations. J Neurochem 55:632-640.

Lukas RJ (1995) Diversity and patterns of regulation of nicotinic receptor subtypes. Ann NY Acad Sci 757:153-168. Lukas RJ, Changeux JP, Le Novere N, Albuquerque EX, Balfour DJ, Berg DK, Bertrand D, Chiappinelli VA, Clarke PB,

Collins AC, Dani JA, Grady SR, Kellar KJ, Lindstrom JM, Marks MJ, Quik M, Taylor PW, Wonnacott S (1999) International Union of Pharmacology. XX. Current status of the nomenclature for nicotinic acetylcholine receptors and their subunits. Pharmacol Rev 51:397-401.

Luo S, Kulak JM, Cartier GE, Jacobsen RB, Yoshikami D, Olivera BM, McIntosh JM (1998) Alpha-conotoxin AuIB selectively blocks alpha3 beta4 nicotinic acetylcholine receptors and nicotine-evoked norepinephrine release. J Neurosci 18:8571-8579.

Maggio R, Riva M, Vaglini F, Fornai F, Molteni R, Armogida M, Racagni G, Corsini GU (1998) Nicotine prevents experimental parkinsonism in rodents and induces striatal increase of neurotrophic factors. J Neurochem 71:2439-2446.

Maldonado-Irizarry CS, Kelley AE (1995) Excitotoxic lesions of the core and shell subregions of the nucleus accumbens differentially disrupt body weight regulation and motor activity in rat. Brain Res Bull 38:551-559.

Mansvelder HD, McGehee DS (2000) Long-term potentiation of excitatory inputs to brain reward areas by nicotine. Neuron 27:349-357.

Mansvelder HD, Keath JR, McGehee DS (2002) Synaptic mechanisms underlie nicotine-induced excitability of brain reward areas. Neuron 33:905-919.

Mansvelder HD, De Rover M, McGehee DS, Brussaard AB (2003) Cholinergic modulation of dopaminergic reward areas: upstream and downstream targets of nicotine addiction. Eur J Pharmacol 480:117-123.

Marchi M, Risso F, Viola C, Cavazzani P, Raiteri M (2002) Direct evidence that release-stimulating alpha7* nicotinic cholinergic receptors are localized on human and rat brain glutamatergic axon terminals. J Neurochem 80:1071-1078.

Marin P, Maus M, Desagher S, Glowinski J, Premont J (1994) Nicotine protects cultured striatal neurones against N-methyl-D-aspartate receptor-mediated neurotoxicity. Neuroreport 5:1977-1980.

Markou A, Paterson NE, Semenova S (2004) Role of gamma-aminobutyric acid (GABA) and metabotropic glutamate receptors in nicotine reinforcement: potential pharmacotherapies for smoking cessation. Ann NY Acad Sci 1025:491-503.

Marks MJ, Burch JB, Collins AC (1983) Effects of chronic nicotine infusion on tolerance development and nicotinic receptors. J Pharmacol Exp Ther 226:817-825.

Marks MJ, Stitzel JA, Collins AC (1985) Time course study of the effects of chronic nicotine infusion on drug response and brain receptors. J Pharmacol Exp Ther 235:619-628.

109

Marks MJ, Grady SR, Collins AC (1993) Downregulation of nicotinic receptor function after chronic nicotine infusion. J Pharmacol Exp Ther 266:1268-1276.

Marks MJ, Smith KW, Collins AC (1998) Differential agonist inhibition identifies multiple epibatidine binding sites in mouse brain. J Pharmacol Exp Ther 285:377-386.

Marks MJ, Stitzel JA, Romm E, Wehner JM, Collins AC (1986) Nicotinic binding sites in rat and mouse brain: comparison of acetylcholine, nicotine, and alpha-bungarotoxin. Mol Pharmacol 30:427-436.

Marks MJ, Pauly JR, Gross SD, Deneris ES, Hermans-Borgmeyer I, Heinemann SF, Collins AC (1992) Nicotine binding and nicotinic receptor subunit RNA after chronic nicotine treatment. J Neurosci 12:2765-2784.

Marrero MB, Papke RL, Bhatti BS, Shaw S, Bencherif M (2004) The neuroprotective effect of 2-(3-pyridyl)-1-azabicyclo3.2.2 nonane (TC-1698), a novel alpha7 ligand, is prevented through angiotensin II activation of a tyrosine phosphatase. J Pharmacol Exp Ther 309:16-27.

Marshall D, Soliakov L, Redfern P, Wonnacott S (1996) Tetrodotoxin-sensitivity of nicotine-evoked dopamine release from rat striatum. Neuropharmacology 35:1531-1536.

Marshall DL, Redfern PH, 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. J Neurochem 68:1511-1519.

Marshall J, Schnieden H (1966) Effect of adrenaline, noradrenaline, atropine and nicotine on some types of human tremor. J Neurol Neurosurg Psychiatry 29.

Marshall JF, O'Dell SJ, Navarrete R, Rosenstein AJ (1990) Dopamine high-affinity transport site topography in rat brain: major differences between dorsal and ventral striatum. Neuroscience 37:11-21.

Martin LF, Kem WR, Freedman R (2004) Alpha-7 nicotinic receptor agonists: potential new candidates for the treatment of schizophrenia. Psychopharmacology 174:54-64.

Martin-Ruiz CM, Lee M, Perry RH, Baumann M, Court JA, Perry EK (2004) Molecular analysis of nicotinic receptor expression in autism. Brain Res Mol Brain Res 123:81-90.

Martin-Ruiz CM, Piggott M, Gotti C, Lindstrom J, Mendelow AD, Siddique MS, Perry RH, Perry EK, Court JA (2000) Alpha and beta nicotinic acetylcholine receptors subunits and synaptophysin in putamen from Parkinson's disease. Neuropharmacology 39:2830-2839.

Martin-Ruiz CM, Court JA, Molnar E, Lee M, Gotti C, Mamalaki A, Tsouloufis T, Tzartos S, Ballard C, Perry RH, Perry EK (1999) Alpha4 but not alpha3 and alpha7 nicotinic acetylcholine receptor subunits are lost from the temporal cortex in Alzheimer's disease. J Neurochem 73:1635-1640.

Marubio LM, del Mar Arroyo-Jimenez M, Cordero-Erausquin M, Lena C, Le Novere N, de Kerchove d'Exaerde A, Huchet M, Damaj MI, Changeux JP (1999) Reduced antinociception in mice lacking neuronal nicotinic receptor subunits. Nature 398:805-810.

Marubio LM, Gardier AM, Durier S, David D, Klink R, Arroyo-Jimenez MM, McIntosh JM, Rossi F, Champtiaux N, Zoli M, Changeux JP (2003) Effects of nicotine in the dopaminergic system of mice lacking the alpha4 subunit of neuronal nicotinic acetylcholine receptors. Eur J Neurosci 17:1329-1337.

Marutle A, Warpman U, Bogdanovic N, Nordberg A (1998) Regional distribution of subtypes of nicotinic receptors in human brain and effect of aging studied by (+/-) 3H epibatidine. Brain Res 801:143-149.

Maurice T, Lockhart BP, Privat A (1996) Amnesia induced in mice by centrally administered beta-amyloid peptides involves cholinergic dysfunction. Brain Res 706:181-193.

McEvoy JP, Allen TB (2002) The importance of nicotinic acetylcholine receptors in schizophrenia, bipolar disorder and Tourette's syndrome. Curr Drug Target CNS Neurol Disord 1:433-442.

McGehee DS, Role LW (1995) Physiological diversity of nicotinic acetylcholine receptors expressed by vertebrate neurons. Annu Rev Physiol 57:521-546.

Mendoza J, Angeles-Castellanos M, Escobar C (2005) Differential role of the accumbens shell and core subterritories in food-entrained rhythms of rats. Behav Brain Res 158:133-142.

Menzaghi F, Whelan KT, Risbrough VB, Rao TS, Lloyd GK (1997a) Interactions between a novel cholinergic ion channel agonist, SIB-1765F and L-DOPA in the reserpine model of Parkinson's disease in rats. J Pharmacol Exp Ther 280:393-401.

Menzaghi F, Whelan KT, Risbrough VB, Rao TS, Lloyd GK (1997b) Effects of a novel cholinergic ion channel agonist SIB-1765F on locomotor activity in rats. J Pharmacol Exp Ther 280:384-392.

Mercuri NB, Calabresi P, Bernardi G (1992) The electrophysiological actions of dopamine and dopaminergic drugs on neurons of the substantia nigra pars compacta and ventral tegmental area. Life Sci 51:711-718.

Mereu G, Yoon KW, Boi V, Gessa GL, Naes L, Westfall TC (1987) Preferential stimulation of ventral tegmental area dopaminergic neurons by nicotine. Eur J Pharmacol 141:395-399.

Meyer EM, Kuryatov A, Gerzanich V, Lindstrom J, Papke RL (1998) Analysis of 3-(4-hydroxy, 2-methoxybenzylidene)anabaseine selectivity and activity at human and rat alpha-7 nicotinic receptors. J Pharmacol Exp Ther 287:918-925.

Meyer EM, Tay ET, Papke RL, Meyers C, Huang GL, de Fiebre CM (1997) 32,4-Dimethoxybenzylidene anabaseine (DMXB) selectively activates rat alpha7 receptors and improves memory-related behaviors in a mecamylamine-sensitive manner. Brain Res 768:49-56.

Mihailescu S, Drucker-Colin R (2000) Nicotine, brain nicotinic receptors, and neuropsychiatric disorders. Arch Med Res 31:131-144.

Mikkola JA, Janhunen S, Hyytiä P, Kiianmaa K, Ahtee L (2002) Rotational behaviour in AA and ANA rats after repeated administration of morphine and cocaine. Pharmacol Biochem Behav 73:697-702.

110

Minana MD, Montoliu C, Llansola M, Grisolia S, Felipo V (1998) Nicotine prevents glutamate-induced proteolysis of the microtubule-associated protein MAP-2 and glutamate neurotoxicity in primary cultures of cerebellar neurons. Neuropharmacology 37:847-857.

Mirza NR, Pei Q, Stolerman IP, Zetterström TS (1996) The nicotinic receptor agonists (-)-nicotine and isoarecolone differ in their effects on dopamine release in the nucleus accumbens. Eur J Pharmacol 295:207-210.

Mogg AJ, Whiteaker P, McIntosh JM, Marks M, Collins AC, Wonnacott S (2002) Methyllycaconitine is a potent antagonist of alpha-conotoxin-MII-sensitive presynaptic nicotinic acetylcholine receptors in rat striatum. J Pharmacol Exp Ther 302:197-204.

Molinari EJ, Delbono O, Messi ML, Renganathan M, Arneric SP, Sullivan JP, Gopalakrishnan M (1998) Up-regulation of human alpha7 nicotinic receptors by chronic treatment with activator and antagonist ligands. Eur J Pharmacol 347:131-139.

Moll H (1926) The treatment of post-encephalitic parkinsonism by nicotine. Br Med J 1:1079-1081. Moore RY (2003) Organization of midbrain dopamine systems and the pathophysiology of Parkinson's disease. Parkinsonism

Rel Disord 9 Suppl 2:S65-71. Morens DM, Grandinetti A, Reed D, White LR, Ross GW (1995) Cigarette smoking and protection from Parkinson's disease:

false association or etiologic clue? Neurology 45:1041-1051. Morrison CF, Stephenson JA (1972) The occurrence of tolerance to a central depressant effect of nicotine. Br J Pharmacol

46:151-156. Museo E, Wise RA (1990) Microinjections of a nicotinic agonist into dopamine terminal fields: effects on locomotion.

Pharmacol Biochem Behav 37:113-116. Männistö PT, Kaakkola S (1999) Catechol-O-methyltransferase (COMT): biochemistry, molecular biology, pharmacology,

and clinical efficacy of the new selective COMT inhibitors. Pharmacol Rev 51:593-628. Nakachi N, Kiuchi Y, Inagaki M, Inazu M, Yamazaki Y, Oguchi K (1995) Effects of various dopamine uptake inhibitors on

striatal extracellular dopamine levels and behaviours in rats. Eur J Pharmacol 281:195-203. Neal MJ, Cunningham JR, Matthews KL (2001) Activation of nicotinic receptors on GABAergic amacrine cells in the rabbit

retina indirectly stimulates dopamine release. Vis Neurosci 18:55-64. Newhouse PA, Potter A, Singh A (2004) Effects of nicotinic stimulation on cognitive performance. Curr Opin Pharmacol

4:36-46. Nisell M, Nomikos GG, Svensson TH (1994a) Infusion of nicotine in the ventral tegmental area or the nucleus accumbens of

the rat differentially affects accumbal dopamine release. Pharmacol Toxicol 75:348-352. Nisell M, Nomikos GG, Svensson TH (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. Nisell M, Marcus M, Nomikos GG, Svensson TH (1997) Differential effects of acute and chronic nicotine on dopamine

output in the core and shell of the rat nucleus accumbens. J Neural Transm (Budapest) 104:1-10. Nisell M, Nomikos GG, Hertel P, Panagis G, Svensson TH (1996) Condition-independent sensitization of locomotor

stimulation and mesocortical dopamine release following chronic nicotine treatment in the rat. Synapse 22:369-381. Nitsch C, Riesenberg R (1988) Immunocytochemical demonstration of GABAergic synaptic connections in rat substantia

nigra after different lesions of the striatonigral projection. Brain Res 461:127-142. Nitsch C, Mews K, Wagner A, Hassler R (1984) Effects of frontal motor cortex ablation on the ultrastructure of cat

substantia nigra. Acta Anat 119:193-202. Nomikos GG, Schilström B, Hildebrand BE, Panagis G, Grenhoff J, Svensson TH (2000) Role of alpha7 nicotinic receptors

in nicotine dependence and implications for psychiatric illness. Behav Brain Res 113:97-103. Nordberg A (1994) Human nicotinic receptors - their role in aging and dementia. Neurochem Int 25:93-97. Nordberg A, Nyberg P, Windblad B (1985) Topographic distribution of choline acetyltransferase activity and muscarinic and

nicotinic receptors in Parkinson brains. Neurochem Pathol 3:223-236. Nordberg A, Lundqvist H, Hartvig P, Andersson J, Johansson M, Hellström-Lindahi E, Langström B (1997) Imaging of

nicotinic and muscarinic receptors in Alzheimer's disease: effect of tacrine treatment. Dement Geriatr Cogn Disord 8:78-84.

Nordberg A, Hellström-Lindahl E, Lee M, Johnson M, Mousavi M, Hall R, Perry E, Bednar I, Court J (2002) Chronic nicotine treatment reduces beta-amyloidosis in the brain of a mouse model of Alzheimer's disease (APPsw). J Neurochem 81:655-658.

Nordberg A, Lilja A, Lundqvist H, Hartvig P, Amberla K, Viitanen M, Warpman U, Johansson M, Hellström-Lindahl E, Bjurling P, et al. (1992) Tacrine restores cholinergic nicotinic receptors and glucose metabolism in Alzheimer patients as visualized by positron emission tomography. Neurobiol Aging 13:747-758.

Nose T, Takemoto H (1974) Effect of oxotremorine on homovanillic acid concentration in the striatum of the rat. Eur J Pharmacol 25:51-55.

Nuutinen S, Ahtee L, Tuominen RK (2005) Time and brain region specific up-regulation of low affinity neuronal nicotinic receptors during chronic nicotine administration in mice. Eur J Pharmacol, in press

Obeso JA, Olanow CW, Nutt JG (2000) Levodopa motor complications in Parkinson's disease. Trends Neurosci 23:S2-7. Ochoa EL (1994) Nicotine-related brain disorders: the neurobiological basis of nicotine dependence. Cell Mol Neurobiol

14:195-225. Ochoa EL, Li L, McNamee MG (1990) Desensitization of central cholinergic mechanisms and neuroadaptation to nicotine.

Mol Neurobiol 4:251-287. Ogden DC, Colquhoun D (1985) Ion channel block by acetylcholine, carbachol and suberyldicholine at the frog

neuromuscular junction. Proc R Soc Lond B Biol Sci 225:329-355. Olanow CW (2004) The scientific basis for the current treatment of Parkinson's disease. Ann Rev Med 55:41-60.

111

O'Neill MF, Dourish CT, Iversen SD (1991) Evidence for an involvement of D1 and D2 dopamine receptors in mediating nicotine-induced hyperactivity in rats. Psychopharmacology 104:343-350.

Orr-Urtreger A, Göldner FM, Saeki M, Lorenzo I, Goldberg L, De Biasi M, Dani JA, Patrick JW, Beaudet AL (1997) Mice deficient in the alpha7 neuronal nicotinic acetylcholine receptor lack alpha-bungarotoxin binding sites and hippocampal fast nicotinic currents. J Neurosci 17:9165-9171.

Owman C, Fuxe K, Janson AM, Kahrstrom J (1989) Studies of protective actions of nicotine on neuronal and vascular functions in the brain of rats: comparison between sympathetic noradrenergic and mesostriatal dopaminergic fiber systems, and the effect of a dopamine agonist. Prog Brain Res 79:267-276.

Pagliusi SR, Tessari M, DeVevey S, Chiamulera C, Pich EM (1996) The reinforcing properties of nicotine are associated with a specific patterning of c-fos expression in the rat brain. Eur J Neurosci 8:2247-2256.

Panagis G, Nisell M, Nomikos GG, Chergui K, Svensson TH (1996) Nicotine injections into the ventral tegmental area increase locomotion and Fos-like immunoreactivity in the nucleus accumbens of the rat. Brain Res 730:133-142.

Papke RL, Heinemann SF (1994) Partial agonist properties of cytisine on neuronal nicotinic receptors containing the beta2 subunit. Mol Pharmacol 45:142-149.

Papke RL, Thinschmidt JS, Moulton BA, Meyer EM, Poirier A (1997) Activation and inhibition of rat neuronal nicotinic receptors by ABT-418. Br J Pharmacol 120:429-438.

Parain K, Hapdey C, Rousselet E, Marchand V, Dumery B, Hirsch EC (2003) Cigarette smoke and nicotine protect dopaminergic neurons against the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine Parkinsonian toxin. Brain Res 984:224-232.

Parent A (1990) Extrinsic connections of the basal ganglia. Trends Neurosci 13:254-258. Parent A, Sato F, Wu Y, Gauthier J, Levesque M, Parent M (2000) Organization of the basal ganglia: the importance of

axonal collateralization. Trends Neurosci 23:S20-27. Parker MJ, Beck A, Luetje CW (1998) Neuronal nicotinic receptor beta2 and beta4 subunits confer large differences in

agonist binding affinity. Mol Pharmacol 54:1132-1139. Parkinson J (1817) An Essay on the Shaking Palsy. London: Sherwood. Paterson D, Nordberg A (2000) Neuronal nicotinic receptors in the human brain. Prog Neurobiol 61:75-111. Pauly JR, Marks MJ, Gross SD, Collins AC (1991) An autoradiographic analysis of cholinergic receptors in mouse brain

after chronic nicotine treatment. J Pharmacol Exp Ther 258:1127-1136. Pauly JR, Marks MJ, Robinson SF, van de Kamp JL, Collins AC (1996) Chronic nicotine and mecamylamine treatment

increase brain nicotinic receptor binding without changing alpha4 or beta2 mRNA levels. J Pharmacol Exp Ther 278:361-369.

Paxinos G, Watson C (1986) The Rat Brain in Stereotaxic Coordinates, 2nd Edition. San Diego, CA: Academic Press. Peng H, Ferris RL, Matthews T, Hiel H, Lopez-Albaitero A, Lustig LR (2004) Characterization of the human nicotinic

acetylcholine receptor subunit alpha9 (CHRNA9) and alpha10 (CHRNA10) in lymphocytes. Life Sci 76:263-280. Peng X, Gerzanich V, Anand R, Whiting PJ, Lindstrom J (1994a) Nicotine-induced increase in neuronal nicotinic receptors

results from a decrease in the rate of receptor turnover. Mol Pharmacol 46:523-530. Peng X, Katz M, Gerzanich V, Anand R, Lindstrom J (1994b) Human alpha7 acetylcholine receptor: cloning of the alpha7

subunit from the SH-SY5Y cell line and determination of pharmacological properties of native receptors and functional alpha7 homomers expressed in Xenopus oocytes. Mol Pharmacol 45:546-554.

Peng X, Gerzanich V, Anand R, Wang F, Lindstrom J (1997) Chronic nicotine treatment up-regulates alpha3 and alpha7 acetylcholine receptor subtypes expressed by the human neuroblastoma cell line SH-SY5Y. Mol Pharmacol 51:776-784.

Pereira EF, Alkondon M, McIntosh JM, Albuquerque EX (1996) Alpha-conotoxin-ImI: a competitive antagonist at alpha-bungarotoxin-sensitive neuronal nicotinic receptors in hippocampal neurons. J Pharmacol Exp Ther 278:1472-1483.

Perkins KA (2002) Chronic tolerance to nicotine in humans and its relationship to tobacco dependence. Nicotine Tobacco Res 4:405-422.

Perry DC, Davila-Garcia MI, Stockmeier CA, Kellar KJ (1999) Increased nicotinic receptors in brains from smokers: membrane binding and autoradiography studies. J Pharmacol Exp Ther 289:1545-1552.

Perry DC, Xiao Y, Nguyen HN, Musachio JL, Davila-Garcia MI, Kellar KJ (2002) Measuring nicotinic receptors with characteristics of alpha4beta2, alpha3beta2 and alpha3beta4 subtypes in rat tissues by autoradiography. J Neurochem 82:468-481.

Perry EK, Perry RH (1995) Acetylcholine and hallucinations: disease-related compared to drug-induced alterations in human consciousness. Brain Cogn 28:240-258.

Perry EK, Morris CM, Court JA, Cheng A, Fairbairn AF, McKeith IG, Irving D, Brown A, Perry RH (1995) Alteration in nicotine binding sites in Parkinson's disease, Lewy body dementia and Alzheimer's disease: possible index of early neuropathology. Neuroscience 64:385-395.

Perry EK, Irving D, Kerwin JM, McKeith IG, Thompson P, Collerton D, Fairbairn AF, Ince PG, Morris CM, Cheng AV, et al. (1993) Cholinergic transmitter and neurotrophic activities in Lewy body dementia: similarity to Parkinson's and distinction from Alzheimer disease. Alzheimer Dis Assoc Disord 7:69-79.

Perry EK, Lee ML, Martin-Ruiz CM, Court JA, Volsen SG, Merrit J, Folly E, Iversen PE, Bauman ML, Perry RH, Wenk GL (2001) Cholinergic activity in autism: abnormalities in the cerebral cortex and basal forebrain. Am J Psychiatry 158:1058-1066.

Peto R, Lopez AD, Boreham J, Thun M, Heath C, Jr., Doll R (1996) Mortality from smoking worldwide. Br Med Bull 52:12-21.

Picciotto MR, Zoli M (2002) Nicotinic receptors in aging and dementia. J Neurobiol 53:641-655. Picciotto MR, Zoli M, Rimondini R, Lena C, Marubio LM, Pich EM, Fuxe K, Changeux JP (1998) Acetylcholine receptors

containing the beta2 subunit are involved in the reinforcing properties of nicotine. Nature 391:173-177.

112

Pich EM, Pagliusi SR, Tessari M, Talabot-Ayer D, Hooft van Huijsduijnen R, Chiamulera C (1997) Common neural substrates for the addictive properties of nicotine and cocaine. Science 275:83-86.

Pidoplichko VI, Noguchi J, Areola OO, Liang Y, Peterson J, Zhang T, Dani JA (2004) Nicotinic cholinergic synaptic mechanisms in the ventral tegmental area contribute to nicotine addiction. Learn Mem 11:60-69.

Pietilä K, Lähde T, Attila M, Ahtee L, Nordberg A (1998) Regulation of nicotinic receptors in the brain of mice withdrawn from chronic oral nicotine treatment. Naunyn-Schmiedebergs Arch Pharmacol 357:176-182.

Piggott MA, Marshall EF, Thomas N, Lloyd S, Court JA, Jaros E, Burn D, Johnson M, Perry RH, McKeith IG, Ballard C, Perry EK (1999) Striatal dopaminergic markers in dementia with Lewy bodies, Alzheimer's and Parkinson's diseases: rostrocaudal distribution. Brain 122:1449-1468.

Pillon B, Dubois B, Bonnet AM, Esteguy M, Guimaraes J, Vigouret JM, Lhermitte F, Agid Y (1989) Cognitive slowing in Parkinson's disease fails to respond to levodopa treatment: the 15-objects test. Neurology 39:762-768.

Pomerleau OF (1998) Endogenous opioids and smoking: a review of progress and problems. Psychoneuroendocrinology 23:115-130.

Pontieri FE, Tanda G, Orzi F, Di Chiara G (1996) Effects of nicotine on the nucleus accumbens and similarity to those of addictive drugs. Nature 382:255-257.

Prince RJ, Fernandes KG, Gregory JC, Martyn ID, Lippiello PM (1996) Modulation of nicotine-evoked 3H dopamine release from rat striatal synaptosomes by voltage-sensitive calcium channel ligands. Biochem Pharmacol 52:613-618.

Puttfarcken PS, Jacobs I, Faltynek CR (2000) Characterization of nicotinic acetylcholine receptor-mediated 3H-dopamine release from rat cortex and striatum. Neuropharmacology 39:2673-2680.

Pycock CJ, Marsden CD (1978) The rotating rodent: a two component system? Eur J Pharmacol 47:167-175. Pycock CJ, Horton RW (1979) Dopamine-dependent hyperactivity in the rat following manipulation of GABA mechanisms

in the region of the nucleus accumbens. J Neural Transm Gen Sect 45:17-33. Qian C, Li T, Shen TY, Libertine-Garahan L, Eckman J, Biftu T, Ip S (1993) Epibatidine is a nicotinic analgesic. Eur J

Pharmacol 250:R13-14. Quik M, Jeyarasasingam G (2000) Nicotinic receptors and Parkinson's disease. Eur J Pharmacol 393:223-230. Quik M, Kulak JM (2002) Nicotine and nicotinic receptors; relevance to Parkinson's disease. Neurotoxicology 23:581-594. Quik M, Philie J, Choremis J (1997) Modulation of alpha7 nicotinic receptor-mediated calcium influx by nicotinic agonists.

Mol Pharmacol 51:499-506. Quik M, Polonskaya Y, Kulak JM, McIntosh JM (2001) Vulnerability of 125I-alpha-conotoxin MII binding sites to

nigrostriatal damage in monkey. J Neurosci 21:5494-5500. Quik M, Polonskaya Y, McIntosh JM, Kulak JM (2002) Differential nicotinic receptor expression in monkey basal ganglia:

effects of nigrostriatal damage. Neuroscience 112:619-630. Quik M, Bordia T, Forno L, McIntosh JM (2004) Loss of alpha-conotoxinMII- and A85380-sensitive nicotinic receptors in

Parkinson's disease striatum. J Neurochem 88:668-679. Quik M, Polonskaya Y, Gillespie A, G KL, Langston JW (2000) Differential alterations in nicotinic receptor alpha6 and

beta3 subunit messenger RNAs in monkey substantia nigra after nigrostriatal degeneration. Neuroscience 100:63-72. Quik M, Bordia T, Okihara M, Fan H, Marks MJ, McIntosh JM, Whiteaker P (2003) L-DOPA treatment modulates nicotinic

receptors in monkey striatum. Mol Pharmacol 64:619-628. Quik M, Vailati S, Bordia T, Kulak JM, Fan H, McIntosh JM, Clementi F, Gotti C (2005) Subunit composition of nicotinic

receptors in monkey striatum: effect of treatments with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine or L-DOPA. Mol Pharmacol 67:32-41.

Ramirez-Latorre J, Yu CR, Qu X, Perin F, Karlin A, Role L (1996) Functional contributions of alpha5 subunit to neuronal acetylcholine receptor channels. Nature 380:347-351.

Rang HP, Dale MM, Ritter JM (1999) Pharmacology, 4th Edition. Edinburgh: Churchill Livingstone. Rao TS, Adams PB, Correa LD, Santori EM, Sacaan AI, Reid RT, Suto CM, Vernier JM (2003) In vitro pharmacological

characterization of (+/-)-42-(1-methyl-2-pyrrolidinyl)ethyl thio]phenol hydrochloride (SIB-1553A), a nicotinic acetylcholine receptor ligand. Brain Res 981:85-98.

Rapier C, Lunt GG, Wonnacott S (1988) Stereoselective nicotine-induced release of dopamine from striatal synaptosomes: concentration dependence and repetitive stimulation. J Neurochem 50:1123-1130.

Rapier C, Lunt GG, Wonnacott S (1990) Nicotinic modulation of 3H dopamine release from striatal synaptosomes: pharmacological characterisation. J Neurochem 54:937-945.

Rasmussen T, Swedberg MD (1998) Reinforcing effects of nicotinic compounds: intravenous self-administration in drug-naive mice. Pharmacol Biochem Behav 60:567-573.

Reavill C, Stolerman IP (1990) Locomotor activity in rats after administration of nicotinic agonists intracerebrally. Br J Pharmacol 99:273-278.

Reid MS, Ho LB, Berger SP (1996) Effects of environmental conditioning on the development of nicotine sensitization: behavioral and neurochemical analysis. Psychopharmacology 126:301-310.

Reuben M, Boye S, Clarke PB (2000) Nicotinic receptors modulating somatodendritic and terminal dopamine release differ pharmacologically. Eur J Pharmacol 393:39-49.

Revah F, Bertrand D, Galzi JL, Devillers-Thiery A, Mulle C, Hussy N, Bertrand S, Ballivet M, Changeux JP (1991) Mutations in the channel domain alter desensitization of a neuronal nicotinic receptor. Nature 353:846-849.

Rice ME, Cragg SJ (2004) Nicotine amplifies reward-related dopamine signals in striatum. Nat Neuroscience 7:583-584. Rinne JO, Myllykylä T, Lonnberg P, Marjamäki P (1991) A postmortem study of brain nicotinic receptors in Parkinson's and

Alzheimer's disease. Brain Res 547:167-170. Rivett AJ, Francis A, Roth JA (1983) Localization of membrane-bound catechol-O-methyltransferase. J Neurochem 40:1494-

1496.

113

Robinson TE, Berridge KC (1993) The neural basis of drug craving: an incentive-sensitization theory of addiction. Brain Res Brain Res Rev 18:247-291.

Roceri M, Molteni R, Fumagalli F, Racagni G, Gennarelli M, Corsini G, Maggio R, Riva M (2001) Stimulatory role of dopamine on fibroblast growth factor-2 expression in rat striatum. J Neurochem 76:990-997.

Roffler-Tarlov S, Sharman DF, Tegerdine P (1971) 3,4-Dihydroxyphenylacetic acid and 4-hydroxy-3-methoxyphenylacetic acid in the mouse striatum: a reflection of intra- and extra-neuronal metabolism of dopamine? Br J Pharmacol 42:343-351.

Role LW (1992) Diversity in primary structure and function of neuronal nicotinic acetylcholine receptor channels. Curr Opin Neurobiol 2:254-262.

Role LW, Berg DK (1996) Nicotinic receptors in the development and modulation of CNS synapses. Neuron 16:1077-1085. Romanelli L, Ohman B, Adem A, Nordberg A (1988) Subchronic treatment of rats with nicotine: interconversion of nicotinic

receptor subtypes in brain. Eur J Pharmacol 148:289-291. Rose JE, Corrigall WA (1997) Nicotine self-administration in animals and humans: similarities and differences.

Psychopharmacology 130:28-40. Rosecrans JA, Meltzer LT (1981) Central sites and mechanisms of action of nicotine. Neurosci Biobehav Rev 5:497-501. Roth KA, McIntire SL, Barchas JD (1982) Nicotinic-catecholaminergic interactions in rat brain: evidence for cholinergic

nicotinic and muscarinic interactions with hypothalamic epinephrine. J Pharmacol Exp Ther 221:416-420. Rowell PP (1995) Nanomolar concentrations of nicotine increase the release of 3H dopamine from rat striatal synaptosomes.

Neurosci Lett 189:171-175. Rowell PP (2002) Effects of nicotine on dopaminergic neurotransmission. In: Nicotinic receptors in the nervous system

(Levin ED, ed), pp 51-80. New York: CRC Press. Rowell PP, Hillebrand JA (1994) Characterization of nicotine-induced desensitization of evoked dopamine release from rat

striatal synaptosomes. J Neurochem 63:561-569. Rowell PP, Li M (1997) Dose-response relationship for nicotine-induced up-regulation of rat brain nicotinic receptors. J

Neurochem 68:1982-1989. Rowell PP, Carr LA, Garner AC (1987) Stimulation of 3H dopamine release by nicotine in rat nucleus accumbens. J

Neurochem 49:1449-1454. Russell MA, Raw M, Taylor C, Feyerabend C, Saloojee Y (1978) Blood nicotine and carboxyhemoglobin levels after rapid-

smoking aversion therapy. J Consult Clin Psychol 46:1423-1431. Rusted JM, Newhouse PA, Levin ED (2000) Nicotinic treatment for degenerative neuropsychiatric disorders such as

Alzheimer's disease and Parkinson's disease. Behav Brain Res 113:121-129. Ryan RE, Ross SA, Drago J, Loiacono RE (2001) Dose-related neuroprotective effects of chronic nicotine in 6-

hydroxydopamine treated rats, and loss of neuroprotection in alpha4 nicotinic receptor subunit knockout mice. Br J Pharmacol 132:1650-1656.

Sabbagh MN, Lukas RJ, Sparks DL, Reid RT (2002) The nicotinic acetylcholine receptor, smoking, and Alzheimer's disease. J Alzheimers Dis 4:317-325.

Sacaan AI, Dunlop JL, Lloyd GK (1995) Pharmacological characterization of neuronal acetylcholine gated ion channel receptor-mediated hippocampal norepinephrine and striatal dopamine release from rat brain slices. J Pharmacol Exp Ther 274:224-230.

Sacaan AI, Menzaghi F, Dunlop JL, Correa LD, Whelan KT, Lloyd GK (1996) Epibatidine: a nicotinic acetylcholine receptor agonist releases monoaminergic neurotransmitters: in vitro and in vivo evidence in rats. J Pharmacol Exp Ther 276:509-515.

Sacaan AI, Reid RT, Santori EM, Adams P, Correa LD, Mahaffy LS, Bleicher L, Cosford ND, Stauderman KA, McDonald IA, Rao TS, Lloyd GK (1997) Pharmacological characterization of SIB-1765F: a novel cholinergic ion channel agonist. J Pharmacol Exp Ther 280:373-383.

Sakurai Y, Takano Y, Kohjimoto Y, Honda K, Kamiya HO (1982) Enhancement of 3H dopamine release and its 3H metabolites in rat striatum by nicotinic drugs. Brain Res 242:99-106.

Sallette J, Bohler S, Benoit P, Soudant M, Pons S, Le Novere N, Changeux JP, Corringer PJ (2004) An extracellular protein microdomain controls up-regulation of neuronal nicotinic acetylcholine receptors by nicotine. J Biol Chem 279:18767-18775.

Salminen O, Seppä T, Gäddnäs H, Ahtee L (1999) The effects of acute nicotine on the metabolism of dopamine and the expression of Fos protein in striatal and limbic brain areas of rats during chronic nicotine infusion and its withdrawal. J Neurosci 19:8145-8151.

Salminen O, Murphy KL, McIntosh JM, Drago J, Marks MJ, Collins AC, Grady SR (2004) Subunit composition and pharmacology of two classes of striatal presynaptic nicotinic acetylcholine receptors mediating dopamine release in mice. Mol Pharmacol 65:1526-1535.

Salokangas RK, Vilkman H, Ilonen T, Taiminen T, Bergman J, Haaparanta M, Solin O, Alanen A, Syvälahti E, Hietala J (2000) High levels of dopamine activity in the basal ganglia of cigarette smokers. Am J Psychiatry 157:632-634.

Santiago M, Westerink BH (1992) The role of GABA receptors in the control of nigrostriatal dopaminergic neurons: dual-probe microdialysis study in awake rats. Eur J Pharmacol 219:175-181.

Sargent PB (1993) The diversity of neuronal nicotinic acetylcholine receptors. Ann Rev Neurosci 16:403-443. Satoh K, Fibiger HC (1986) Cholinergic neurons of the laterodorsal tegmental nucleus: efferent and afferent connections. J

Comp Neurol 253:277-302. Schiffer WK, Gerasimov MR, Bermel RA, Brodie JD, Dewey SL (2000) Stereoselective inhibition of dopaminergic activity

by gamma vinyl-GABA following a nicotine or cocaine challenge: a PET/microdialysis study. Life Sci 66:L169-173.

114

Schilström B, Svensson HM, Svensson TH, Nomikos GG (1998a) Nicotine and food induced dopamine release in the nucleus accumbens of the rat: putative role of alpha7 nicotinic receptors in the ventral tegmental area. Neuroscience 85:1005-1009.

Schilström B, Nomikos GG, Nisell M, Hertel P, Svensson TH (1998b) N-methyl-D-aspartate receptor antagonism in the ventral tegmental area diminishes the systemic nicotine-induced dopamine release in the nucleus accumbens. Neuroscience 82:781-789.

Schneider JS, Van Velson M, Menzaghi F, Lloyd GK (1998a) Effects of the nicotinic acetylcholine receptor agonist SIB-1508Y on object retrieval performance in MPTP-treated monkeys: comparison with levodopa treatment. Ann Neurol 43:311-317.

Schneider JS, Tinker JP, Menzaghi F, Lloyd GK (2003) The subtype-selective nicotinic acetylcholine receptor agonist SIB-1553A improves both attention and memory components of a spatial working memory task in chronic low dose 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated monkeys. J Pharmacol Exp Ther 306:401-406.

Schneider JS, Pope-Coleman A, Van Velson M, Menzaghi F, Lloyd GK (1998b) Effects of SIB-1508Y, a novel neuronal nicotinic acetylcholine receptor agonist, on motor behavior in parkinsonian monkeys. Mov Disord 13:637-642.

Schneider JS, Tinker JP, Van Velson M, Menzaghi F, Lloyd GK (1999) Nicotinic acetylcholine receptor agonist SIB-1508Y improves cognitive functioning in chronic low-dose MPTP-treated monkeys. J Pharmacol Exp Ther 290:731-739.

Schoepfer R, Conroy WG, Whiting P, Gore M, Lindstrom J (1990) Brain alpha-bungarotoxin binding protein cDNAs and MAbs reveal subtypes of this branch of the ligand-gated ion channel gene superfamily. Neuron 5:35-48.

Schoffelmeer AN, De Vries TJ, Wardeh G, van de Ven HW, Vanderschuren LJ (2002) Psychostimulant-induced behavioral sensitization depends on nicotinic receptor activation. J Neurosci 22:3269-3276.

Schwartz RD, Kellar KJ (1983) Nicotinic cholinergic receptor binding sites in the brain: regulation in vivo. Science 220:214-216.

Schwartz RD, Kellar KJ (1985) In vivo regulation of 3H acetylcholine recognition sites in brain by nicotinic cholinergic drugs. J Neurochem 45:427-433.

Schwartz RD, Lehmann J, Kellar KJ (1984) Presynaptic nicotinic cholinergic receptors labeled by 3H acetylcholine on catecholamine and serotonin axons in brain. J Neurochem 42:1495-1498.

Scott WK, Zhang F, Stajich JM, Scott BL, Stacy MA, Vance JM (2005) Family-based case-control study of cigarette smoking and Parkinson disease. Neurology 64:442-447.

Seguela P, Wadiche J, Dineley-Miller K, Dani JA, Patrick JW (1993) Molecular cloning, functional properties, and distribution of rat brain alpha7: a nicotinic cation channel highly permeable to calcium. J Neurosci 13:596-604.

Seppä T (2001) Role of nicotinic acetylcholine receptors in crérebral dopaminergic transmission and expression of Fos protein. Helsinki: University Press.

Seppä T, Ahtee L (2000) Comparison of the effects of epibatidine and nicotine on the output of dopamine in the dorsal and ventral striatum of freely-moving rats. Naunyn-Schmiedebergs Arch Pharmacol 362:444-447.

Seppä T, Ruotsalainen M, Laakso I, Tuominen R, Ahtee L (2000) Effect of acute nicotine administration on striatal dopamine output and metabolism in rats kept at different ambient temperatures. Br J Pharmacol 130:1147-1155.

Sershen H, Hashim A, Lajtha A (1987) Behavioral and biochemical effects of nicotine in an MPTP-induced mouse model of Parkinson's disease. Pharmacol Biochem Behav 28:299-303.

Sgard F, Charpantier E, Barneoud P, Besnard F (1999) Nicotinic receptor subunit mRNA expression in dopaminergic neurons of the rat brain. Ann NY Acad Sci 868:633-635.

Sgard F, Charpantier E, Bertrand S, Walker N, Caput D, Graham D, Bertrand D, Besnard F (2002) A novel human nicotinic receptor subunit, alpha10, that confers functionality to the alpha9-subunit. Mol Pharmacol 61:150-159.

Sharples CG, Kaiser S, Soliakov L, Marks MJ, Collins AC, Washburn M, Wright E, Spencer JA, Gallagher T, Whiteaker P, Wonnacott S (2000) UB-165: a novel nicotinic agonist with subtype selectivity implicates the alpha4beta2* subtype in the modulation of dopamine release from rat striatal synaptosomes. J Neurosci 20:2783-2791.

Shim I, Javaid JI, Wirtshafter D, Jang SY, Shin KH, Lee HJ, Chung YC, Chun BG (2001) Nicotine-induced behavioral sensitization is associated with extracellular dopamine release and expression of c-Fos in the striatum and nucleus accumbens of the rat. Behav Brain Res 121:137-147.

Shimohama S, Akaike A, Kimura J (1996) Nicotine-induced protection against glutamate cytotoxicity. Nicotinic cholinergic receptor-mediated inhibition of nitric oxide formation. Ann NY Acad Sci 777:356-361.

Shoaib M, Schindler CW, Goldberg SR, Pauly JR (1997) Behavioural and biochemical adaptations to nicotine in rats: influence of MK801, an NMDA receptor antagonist. Psychopharmacology 134:121-130.

Shoaib M, Benwell ME, Akbar MT, Stolerman IP, Balfour DJ (1994) Behavioural and neurochemical adaptations to nicotine in rats: influence of NMDA antagonists. Br J Pharmacol 111:1073-1080.

Singer G, Wallace M, Hall R (1982) Effects of dopaminergic nucleus accumbens lesions on the acquisition of schedule induced self injection of nicotine in the rat. Pharmacol Biochem Behav 17:579-581.

Smith Y, Parent A (1988) Neurons of the subthalamic nucleus in primates display glutamate but not GABA immunoreactivity. Brain Res 453:353-356.

Smith Y, Bolam JP (1990) The output neurones and the dopaminergic neurones of the substantia nigra receive a GABA-containing input from the globus pallidus in the rat. J Comp Neurol 296:47-64.

Smith Y, Kieval JZ (2000) Anatomy of the dopamine system in the basal ganglia. Trends Neurosci 23:S28-33. Soliakov L, Wonnacott S (1996) Voltage-sensitive Ca2+ channels involved in nicotinic receptor-mediated 3H dopamine

release from rat striatal synaptosomes. J Neurochem 67:163-170. Spande TF, Garraffo HM, Edwards MW, Yeh HJC, Pannell L, Daly JW (1992) Epibatidine: A novel

(Chloropyridyl)azabicycloheptane with potent analgesic activity from an Equadoran poison frog. J Am Chem Soc 114:3475-3478.

115

Steinlein OK, Mulley JC, Propping P, Wallace RH, Phillips HA, Sutherland GR, Scheffer IE, Berkovic SF (1995) A missense mutation in the neuronal nicotinic acetylcholine receptor alpha 4 subunit is associated with autosomal dominant nocturnal frontal lobe epilepsy. Nat Genetics 11:201-203.

Stevens KE, Kem WR, Mahnir VM, Freedman R (1998) Selective alpha7-nicotinic agonists normalize inhibition of auditory response in DBA mice. Psychopharmacology 136:320-327.

Stevens KE, Freedman R, Collins AC, Hall M, Leonard S, Marks MJ, Rose GM (1996) Genetic correlation of inhibitory gating of hippocampal auditory evoked response and alpha-bungarotoxin-binding nicotinic cholinergic receptors in inbred mouse strains. Neuropsychopharmacology 15:152-162.

Stolerman IP, Shoaib M (1991) The neurobiology of tobacco addiction. Trends Pharmacol Sci 12:467-473. Stolerman IP, Fink R, Jarvik ME (1973) Acute and chronic tolerance to nicotine measured by activity in rats.

Psychopharmacologia 30:329-342. Stolerman IP, Chandler CJ, Garcha HS, Newton JM (1997) Selective antagonism of behavioural effects of nicotine by

dihydro-beta-erythroidine in rats. Psychopharmacology 129:390-397. Stratford TR, Kelley AE (1997) GABA in the nucleus accumbens shell participates in the central regulation of feeding

behavior. J Neurosci 17:4434-4440. Stratford TR, Kelley AE (1999) Evidence of a functional relationship between the nucleus accumbens shell and lateral

hypothalamus subserving the control of feeding behavior. J Neurosci 19:11040-11048. Suaud-Chagny MF, Chérgui K, Chouvet G, Gonon F (1992) Relationship between dopamine release in the rat nucleus

accumbens and the discharge activity of dopaminergic neurons during local in vivo application of amino acids in the ventral tegmental area. Neuroscience 49:63-72.

Sugaya K, Giacobini E, Chiappinelli VA (1990) Nicotinic acetylcholine receptor subtypes in human frontal cortex: changes in Alzheimer's disease. J Neurosci Res 27:349-359.

Sullivan JP, Decker MW, Brioni JD, Donnelly-Roberts D, Anderson DJ, Bannon AW, Kang CH, Adams P, Piattoni-Kaplan M, Buckley MJ, et al. (1994) (+/-)-Epibatidine elicits a diversity of in vitro and in vivo effects mediated by nicotinic acetylcholine receptors. J Pharmacol Exp Ther 271:624-631.

Sullivan JP, Donnelly-Roberts D, Briggs CA, Anderson DJ, Gopalakrishnan M, Xue IC, Piattoni-Kaplan M, Molinari E, Campbell JE, McKenna DG, Gunn DE, Lin NH, Ryther KB, He Y, Holladay MW, Wonnacott S, Williams M, Arneric SP (1997) ABT-089 2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine: I. A potent and selective cholinergic channel modulator with neuroprotective properties. J Pharmacol Exp Ther 283:235-246.

Summers KL, Giacobini E (1995) Effects of local and repeated systemic administration of (-)nicotine on extracellular levels of acetylcholine, norepinephrine, dopamine, and serotonin in rat cortex. Neurochem Res 20:753-759.

Svensson TH, Tung CS (1989) Local cooling of pre-frontal cortex induces pacemaker-like firing of dopamine neurons in rat ventral tegmental area in vivo. Acta Physiol Scand 136:135-136.

Svensson TH, Mathe JM, Nomikos GG, Schilström B (1998) Role of excitatory amino acids in the ventral tegmental area for central actions of non-competitive NMDA-receptor antagonists and nicotine. Amino Acids 14:51-56.

Teaktong T, Graham AJ, Johnson M, Court JA, Perry EK (2004) Selective changes in nicotinic acetylcholine receptor subtypes related to tobacco smoking: an immunohistochemical study. Neuropathol Appl Neurobiol 30:243-254.

Teng L, Crooks PA, Sonsalla PK, Dwoskin LP (1997) Lobeline and nicotine evoke 3H overflow from rat striatal slices preloaded with 3H dopamine: differential inhibition of synaptosomal and vesicular 3H dopamine uptake. J Pharmacol Exp Ther 280:1432-1444.

Tepper JM, Paladini CA, Celada P (1998) GABAergic control of the firing pattern of substantia nigra dopaminergic neurons. Adv Pharmacol 42:694-699.

Toth E, Vizi ES, Lajtha A (1993) Effect of nicotine on levels of extracellular amino acids in regions of the rat brain in vivo. Neuropharmacology 32:827-832.

Toth E, Sershen H, Hashim A, Vizi ES, Lajtha A (1992) Effect of nicotine on extracellular levels of neurotransmitters assessed by microdialysis in various brain regions: role of glutamic acid. Neurochem Res 17:265-271.

Tzschentke TM (1998) Measuring reward with the conditioned place preference paradigm: a comprehensive review of drug effects, recent progress and new issues. Prog Neurobiol 56:613-672.

Ungerstedt U (1968) 6-Hydroxydopamine induced degeneration of central monoamine neurons. Eur J Pharmacol 5:107-110. Ungerstedt U (1971a) Postsynaptic supersensitivity after 6-hydroxydopamine induced degeneration of the nigrostriatal

dopamine system. Acta Physiol Scand Suppl 367:69-93. Ungerstedt U (1971b) Striatal dopamine release after amphetamine or nerve degeneration revealed by rotational behaviour.

Acta Physiol Scand Suppl 367:49-68. Ungerstedt U (1971c) Stereotaxic mapping of the monoamine pathways in the rat brain. Acta Physiol Scand Suppl 367:1-48. Ungerstedt U, Arbuthnott GW (1970) Quantitative recording of rotational behavior in rats after 6-hydroxydopamine lesions

of the nigrostriatal dopamine system. Brain Res 24:485-493. US Department of Health and Human Sciences (1988) The health consequences of smoking: nicotine addiction. A report of

the Surgeon General Maryland: Office on Smoking and health. Usunoff KG, Romansky KV, Malinov GB, Ivanov DP, Blagov ZA, Galabov GP (1982) Electron microscopic evidence for

the existence of a corticonigral tract in the cat. J Hirnforschung 23:23-29. Vastola BJ, Douglas LA, Varlinskaya EI, Spear LP (2002) Nicotine-induced conditioned place preference in adolescent and

adult rats. Physiol Behav 77:107-114. Vernier JM, Holsenback H, Cosford ND, Whitten JP, Menzaghi F, Reid R, Rao TS, Sacaan AI, Lloyd GK, Suto CM,

Chavez-Noriega LE, Washburn MS, Urrutia A, McDonald IA (1998) Conformationally restricted analogues of nicotine and anabasine. Bioorganic Med Chem Lett 8:2173-2178.

Vizi ES, Lendvai B (1999) Modulatory role of presynaptic nicotinic receptors in synaptic and non-synaptic chemical communication in the central nervous system. Brain Res Brain Res Rev 30:219-235.

116

Wachtel H, Andén NE (1978) Motor activity of rats following intracerebral injections of drugs influencing GABA mechanisms. Naunyn-Schmiedebergs Arch Pharmacol 302:133-139.

Wada E, McKinnon D, Heinemann S, Patrick J, Swanson LW (1990) The distribution of mRNA encoded by a new member of the neuronal nicotinic acetylcholine receptor gene family (alpha5) in the rat central nervous system. Brain Res 526:45-53.

Wada E, Wada K, Boulter J, Deneris E, Heinemann S, Patrick J, Swanson LW (1989) Distribution of alpha2, alpha3, alpha4, and beta2 neuronal nicotinic receptor subunit mRNAs in the central nervous system: a hybridization histochemical study in the rat. J Comp Neurol 284:314-335.

Wang F, Gerzanich V, Wells GB, Anand R, Peng X, Keyser K, Lindstrom J (1996) Assembly of human neuronal nicotinic receptor alpha5 subunits with alpha3, beta2, and beta4 subunits. J Biol Chem 271:17656-17665.

Wang F, Nelson ME, Kuryatov A, Olale F, Cooper J, Keyser K, Lindstrom J (1998) Chronic nicotine treatment up-regulates human alpha3 beta2 but not alpha3 beta4 acetylcholine receptors stably transfected in human embryonic kidney cells. J Biol Chem 273:28721-28732.

Warburton DD, Rusted JJ, Muller CC (1992) Patterns of facilitation of memory by nicotine. Behav Pharmacol 3:375-378. Warpman U, Nordberg A (1995) Epibatidine and ABT 418 reveal selective losses of alpha4 beta2 nicotinic receptors in

Alzheimer brains. Neuroreport 6:2419-2423. Weiland S, Witzemann V, Villarroel A, Propping P, Steinlein O (1996) An amino acid exchange in the second

transmembrane segment of a neuronal nicotinic receptor causes partial epilepsy by altering its desensitization kinetics. FEBS Lett 398:91-96.

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. Pharmacol Biochem Behav 37:743-746.

Westerink BH, Spaan SJ (1982a) Estimation of the turnover of 3-methoxytyramine in the rat striatum by HPLC with electrochemical detection: implications for the sequence in the cerebral metabolism of dopamine. J Neurochem 38:342-347.

Westerink BH, Spaan SJ (1982b) On the significance of endogenous 3-methoxytyramine for the effects of centrally acting drugs on dopamine release in the rat brain. J Neurochem 38:680-686.

Westerink BH, Santiago M, De Vries JB (1992a) In vivo evidence for a concordant response of terminal and dendritic dopamine release during intranigral infusion of drugs. Naunyn-Schmiedebergs Arch Pharmacol 346:637-643.

Westerink BH, Santiago M, De Vries JB (1992b) The release of dopamine from nerve terminals and dendrites of nigrostriatal neurons induced by excitatory amino acids in the conscious rat. Naunyn-Schmiedebergs Arch Pharmacol 345:523-529.

Westerink BH, Kwint HF, deVries JB (1996) The pharmacology of mesolimbic dopamine neurons: a dual-probe microdialysis study in the ventral tegmental area and nucleus accumbens of the rat brain. J Neurosci 16:2605-2611.

Westfall TC (1974) Effect of nicotine and other drugs on the release of 3H-norepinephrine and 3H-dopamine from rat brain slices. Neuropharmacology 13:693-700.

Westfall TC, Mereu G, Vickery L, Perry H, Naes L, Yoon KW (1989) Regulation by nicotine of midbrain dopamine neurons. Prog Brain Res 79:173-185.

Whiteaker P, Sharples CG, Wonnacott S (1998) Agonist-induced up-regulation of alpha4beta2 nicotinic acetylcholine receptors in M10 cells: pharmacological and spatial definition. Mol Pharmacol 53:950-962.

Whiteaker P, Garcha HS, Wonnacott S, Stolerman IP (1995) Locomotor activation and dopamine release produced by nicotine and isoarecolone in rats. Br J Pharmacol 116:2097-2105.

Whiteaker P, Marks MJ, Grady SR, Lu Y, Picciotto MR, Changeux JP, Collins AC (2000) Pharmacological and null mutation approaches reveal nicotinic receptor diversity. Eur J Pharmacol 393:123-135.

Whiteaker P, Peterson CG, Xu W, McIntosh JM, Paylor R, Beaudet AL, Collins AC, Marks MJ (2002) Involvement of the alpha3 subunit in central nicotinic binding populations. J Neurosci 22:2522-2529.

Whiting P, Lindstrom J (1986) Pharmacological properties of immuno-isolated neuronal nicotinic receptors. J Neurosci 6:3061-3069.

WHO (2003) World Health Report 2003: Shaping the future. Geneva: World Health Organization. Wirdefeldt K, Gatz M, Pawitan Y, Pedersen NL (2005) Risk and protective factors for Parkinson's disease: a study in

Swedish twins. Ann Neurol 57:27-33. Wirtshafter D, Stratford TR, Asin KE (1987) Evidence that serotonergic projections to the substantia nigra in the rat arise in

the dorsal, but not the median, raphe nucleus. Neurosci Lett 77:261-266. Wise RA, Bozarth MA (1987) A psychomotor stimulant theory of addiction. Psychol Rev 94:469-492. Wonnacott S (1990) The paradox of nicotinic acetylcholine receptor upregulation by nicotine. Trends Pharmacol Sci 11:216-

219. Wonnacott S (1997) Presynaptic nicotinic ACh receptors. Trends Neurosci 20:92-98. Wonnacott S, Sidhpura N, Balfour DJ (2005) Nicotine: from molecular mechanisms to behaviour. Curr Opin Pharmacol

5:53-59. Wonnacott S, Drasdo A, Sanderson E, Rowell P (1990) Presynaptic nicotinic receptors and the modulation of transmitter

release. Ciba Found Symp 152:87-101; discussion 102-105. Wonnacott S, Irons J, Rapier C, Thorne B, Lunt GG (1989) Presynaptic modulation of transmitter release by nicotinic

receptors. Prog Brain Res 79:157-163. Wonnacott S, Kaiser S, Mogg A, Soliakov L, Jones IW (2000) Presynaptic nicotinic receptors modulating dopamine release

in the rat striatum. Eur J Pharmacol 393:51-58. Woolf NJ (1991) Cholinergic systems in mammalian brain and spinal cord. Prog Neurobiol 37:475-524. Woolf NJ, Butcher LL (1981) Cholinergic neurons in the caudate-putamen complex proper are intrinsically organized: a

combined Evans blue and acetylcholinesterase analysis. Brain Res Bull 7:487-507.

117

Wooltorton JR, Pidoplichko VI, Broide RS, Dani JA (2003) Differential desensitization and distribution of nicotinic acetylcholine receptor subtypes in midbrain dopamine areas. J Neurosci 23:3176-3185.

Worms P, Gueudet C, Biziere K (1986a) Induction of turning by direct intrastriatal injection of dopaminomimetic drugs in mice: pharmacological analysis of a simple screening model. Life Sci 39:2199-2208.

Worms P, Kan JP, Wermuth CG, Biziere K (1986b) Dopamine-like activities of an aminopyridazine derivative, CM 30366: a behavioural study. Naunyn-Schmiedebergs Arch Pharmacol 334:246-252.

Xiao Y, Meyer EL, Thompson JM, Surin A, Wroblewski J, Kellar KJ (1998) Rat alpha3/beta4 subtype of neuronal nicotinic acetylcholine receptor stably expressed in a transfected cell line: pharmacology of ligand binding and function. Mol Pharmacol 54:322-333.

Yang X, Criswell HE, Breese GR (1996) Nicotine-induced inhibition in medial septum involves activation of presynaptic nicotinic cholinergic receptors on gamma-aminobutyric acid-containing neurons. J Pharmacol Exp Ther 276:482-489.

Yin R, French ED (2000) A comparison of the effects of nicotine on dopamine and non-dopamine neurons in the rat ventral tegmental area: an in vitro electrophysiological study. Brain Res Bull 51:507-514.

Yong VW, Perry TL (1986) Monoamine oxidase B, smoking, and Parkinson's disease. J Neurol Sci 72:265-272. Yoshida M, Yokoo H, Tanaka T, Mizoguchi K, Emoto H, Ishii H, Tanaka M (1993) Facilitatory modulation of mesolimbic

dopamine neuronal activity by a mu-opioid agonist and nicotine as examined with in vivo microdialysis. Brain Res 624:277-280.

Yu CR, Role LW (1998) Functional contribution of the alpha7 subunit to multiple subtypes of nicotinic receptors in embryonic chick sympathetic neurones. J Physiol 509:651-665.

Yu ZJ, Wecker L (1994) Chronic nicotine administration differentially affects neurotransmitter release from rat striatal slices. J Neurochem 63:186-194.

Zarrindast MR, Sadegh M, Shafaghi B (1996) Effects of nicotine on memory retrieval in mice. Eur J Pharmacol 295:1-6. Zernig G, O'Laughlin IA, Fibiger HC (1997) Nicotine and heroin augment cocaine-induced dopamine overflow in nucleus

accumbens. Eur J Pharmacol 337:1-10. Zetler G (1968) Cataleptic state and hypothermia in mice, caused by central cholinergic stimulation and antagonized by

anticholinergic and antidepressant drugs. Intl J Neuropharmacol 7:325-335. Zetler G (1971) Pharmacological differentiation of "nicotinic" and "muscarinic" catalepsy. Neuropharmacology 10:289-296. Zhang H, Sulzer D (2004) Frequency-dependent modulation of dopamine release by nicotine. Nat Neuroscience 7:581-582. Zhang X, Gong ZH, Hellström-Lindahl E, Nordberg A (1995) Regulation of alpha4 beta2 nicotinic acetylcholine receptors in

M10 cells following treatment with nicotinic agents. Neuroreport 6:313-317. Zhou FM, Liang Y, Dani JA (2001) Endogenous nicotinic cholinergic activity regulates dopamine release in the striatum. Nat

Neuroscience 4:1224-1229. Zigmond MJ, Abercrombie ED, Berger TW, Grace AA, Stricker EM (1990) Compensations after lesions of central

dopaminergic neurons: some clinical and basic implications. Trends Neurosci 13:290-296. Zoli M, Lena C, Picciotto MR, Changeux JP (1998) Identification of four classes of brain nicotinic receptors using beta2

mutant mice. J Neurosci 18:4461-4472. Zoli M, Moretti M, Zanardi A, McIntosh JM, Clementi F, Gotti C (2002) Identification of the nicotinic receptor subtypes

expressed on dopaminergic terminals in the rat striatum. J Neurosci 22:8785-8789.