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

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CONTENTS

ABSTRACT ...... 6 LIST OF ABBREVIATIONS ...... 7 LIST OF ORIGINAL PUBLICATIONS ...... 8 1. INTRODUCTION...... 9 2. REVIEW OF THE LITERATURE ...... 11 2.1. Ascending pathways ...... 11 2.1.1. Functional implications of nigrostriatal and mesolimbic pathways ...... 13 2.2. Synthesis and release of ...... 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 receptors (nAChRs) ...... 22 2.6.1. Structure and classification of nAChRs ...... 23 2.6.2. Distribution of nAChRs in the ...... 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

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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 . Nicotine, the main psychoactive ingredient in tobacco, acts at neuronal nicotinic acetylcholine receptors (nAChRs) to induce release of dopamine in the caudate- and , 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 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 , 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 .

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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, 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 SNc substantia nigra SNr substantia nigra pars reticulata TPP tegmental pedunculopontine nucleus TTX tetrodotoxin VTA

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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 . 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.

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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 , 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 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 , 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, , 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 , 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 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), , , septum and , while the other, the , 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 , 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 . 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 (Heimer et al., 1991). The shell is involved in integration and expression of emotions and the projections between the shell and 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 of the hypothalamus to the and . 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 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 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

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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 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, 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 , integrate them with each other and with inputs from the limbic areas and focus the integrated outputs to regions of the frontal lobes and 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 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 , dopaminergic inputs from

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Cortex TPP/LTD (a) Glu ACh

GABA Glu GABA

DA Glu Thalamus SNc/VTA Glu

GABA

ACh GP GABA ACh GABA SNr

GABA Dorsal GABA striatum

Endopeduncular Glu GABA nucleus STN

PFC (b) VTA TPP/LTD

Glu GABA GABA Glu

Glu

GABA

ACh DA NAc

GABA DA ACh Interneuron GABA Projection neuron AGABA 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 = , 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.

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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 .

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 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 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 ↓↓↓ Dopamine-β-hydroxylase ↓(↓) HVA ↓↓ Phenylethanolamine-N- Tyrosine hydroxylase ↓↓↓ methyltransferase ↓ Biopterin ↓↓ 5-HT (serotonin) ↓(↓) Aromatic amino acid 5-HT decarboxylase ↓ = decarboxylase ↓(↓) = 5-HIAA ↓(↓) MAO = 5-HT receptors ↓ = COMT = GABA ↓(↓) cAMP-dependent protein Glutamate dehydrogenase ↓(↓) kinase = GABA receptors ↓(↓) D1 dopamine receptors ↑↓ = Substance P ↓(↓) D2 dopamine receptors ↑↓ = Leu-enkephalin binding ↓ = ↑ Noradrenaline ↓(↑) 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, 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 . 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 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 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 terminals and , 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 (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 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

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(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).

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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 Functional potency References Ki (nM) EC50 (µM) nAChR subtype ACh NIC EPI ACh NIC EPI α4β2 0.014- a,b,c,e,f,g,k,l,n,p, 8.6-34 1.7-8.1 0.01-0.09 3-270 0.59-14 0.043 q,u,v,x,y,z,,ac

α7 4000- 400- d,h,l,m,o,p, 20-233 79-322 40-83 0.17-1.3 19.000 14.000 q,s,t,w,y,z

α3β2 0.014- g,h,l,r,t, 29 16 26-126 6.6-7.7 0.1-0.19 0.035 u,v,x,aa α6β2 8.0 2.5-3.8 0.034 n.d. n.d. n.d. f,ac

α3β4 0.07- a,l,p,t,u,v, 560-881 183-475 0.15-0.59 160-217 7.5-110 0.128 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;

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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).

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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 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;

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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).

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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 , 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 . 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 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;

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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).

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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

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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.

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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).

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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%).

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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).

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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 Dopamine DOPAC HVA 5-HIAA Treatment (fmol/20 µl) (pmol/20 µl) (pmol/20 µl) (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).

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Caudate-putamen Nucleus accumbens

175 SAL 175 SAL NIC ↑↑↑ NIC ↑↑↑ 150 150 EPI 0.6 ↑↑↑ EPI 0.6

125 125

100 100

DA% of the baseline 75 75

-60 0 60 120 180 240 -60 0 60 120 180 240 175 SAL 175 SAL EPI 1.0 ↑↑↑ EPI 1.0 150 150 EPI 2.0 EPI 2.0

125 EPI 3.0 125 EPI 3.0 ↑↑↑

100 100

DA% of the baseline 75 75

-60 0 60 120 180 240 -60 0 60 120 180 240

175 SAL 175 SAL EPI 0.1 EPI 0.1 150 150 EPI 0.3 ↓↓↓ EPI 0.3 125 125

100 100

DA% of the baseline 75 75

-30 0 30 60 90 120 150 -30 0 30 60 90 120 150 Time (min) Time (min)

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%).

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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%).

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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).

Caudate-putamen Nucleus accumbens NOM + SAL ••• NOM + SAL ••• NOM + NIC ↑↑↑ NOM + NIC ↑↑ 600 NOM + EPI 0.6 ↑↑↑ 600 NOM + EPI 0.6 ↓↓ NOM + EPI 2.0 NOM + EPI 2.0 500 500 NOM + EPI 3.0 NOM + EPI 3.0 400 SAL + SAL 400 SAL+ SAL

300 300

200 200

DA% ofDA% the baseline 100 100

-60 0 60 120 180 240 -60 0 60 120 180 240

Time (min) Time (min)

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%).

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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%).

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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.

A NOM + NIC * # B SAL + NOM + NIC 50 50 MEC 2 + NOM + NIC NOM + SAL MEC 5 + NOM + NIC ↓ SAL + NIC 40 40 MEC 2 + SAL + SAL MEC 5 + SAL + SAL 30 30

20 20

10 10 Ipsilateral rotations/ 15 min 0 0

-30 0 30 60 90 120 150 180 -30 0 30 60 90 120 150 180 Time (min) Time (min)

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.

Test day 1 Test day 3 Test day 5 Challenge day 120 # A 100 °° # ** 80 ° * * 60

40 * **

Ipsilateral rotations/ 3 h 20

0 SAL + SAL SAL + NIC SAL + EPI 0.6 SAL + EPI 3.0

1200 B ### °° 1000 °°° ° # ° ° 800 ° ° ° 600

400

Ipsilateral rotations/ 3 h 200

0 NOM + SAL NOM + NIC NOM + EPI 0.6 NOM + EPI 3.0

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

2000 4000 Test day 1 •• 1000 °

3000 20-23 C 2 h Counts/ 0 30-33°C 20-23°C 30-33°C 2000 counts # * • *** 1000 * * Locomotor activity 0 0-60 61-120 0-60 61-120 0-60 61-120 Saline NIC 0.5 mg/kg NIC 0.8 mg/kg

4000 Test day 3 # ° 3000 °°°*** °°° *** ***°°° 2000 ** ** counts *** 1000 •• ** Locomotor activity 0 0-60 61-120 0-60 61-120 0-60 61-120 Saline NIC 0.5 mg/kg NIC 0.8 mg/kg

°°° % 4000 Test day 5 ** °°° *** 3000 °°° *** °°°• 2000 ° counts * * 1000 Locomotor activity 0 0-60 61-120 0-60 61-120 0-60 61-120 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

3500 SAL EPI 0.6 3000 500 * * EPI 3.0 2500

Counts/ 31-60min ° 0 * 2000 ° ° * 1500 ° *

1000

500 Locomotor activity counts/ 60 min 0 Saline day Test day 1 Test day 3 Test day 5 Challenge

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

SAL NIC 0.8 SAL EPI 0.3 150 NIC 0.5 150 EPI 0.1 EPI 0.6

100 100 * * ** 50 50

0 0

-50 -50

-100 -100 Change intime (sec) spent in drug-paired compartment -150 -150

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 *** 0.8 mg/kg 182 ± 59 416 ± 54 * Ei 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).

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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

79 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

80 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

81 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

84 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

86 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

87 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 88 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

89 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

90 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

92 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

94 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

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