KUOPION YLIOPISTON JULKAISUJA A. FARMASEUTTISET TIETEET 113 KUOPIO UNIVERSITY PUBLICATIONS A. PHARMACEUTICAL SCIENCES 113

TIINA KÄÄRIÄINEN Interaction of the and Systems in Rat Brain

Studies in Parkinsonian Models and Brain Microdialysis

Doctoral dissertation

To be presented by permission of the Faculty of Pharmacy of the University of Kuopio for public examination in Mediteknia Auditorium, Mediteknia building, University of Kuopio, on Saturday 13th December 2008, at 1 p.m.

Department of Pharmacology and Toxicology Faculty of Pharmacy University of Kuopio

JOKA KUOPIO 2008 Distributor: Kuopio University Library P.O. Box 1627 FI-70211 KUOPIO FINLAND Tel. +358 40 355 3430 Fax +358 17 163 410 http://www.uku.fi/kirjasto/julkaisutoiminta/julkmyyn.html

Series Editor: Docent Pekka Jarho, Ph.D. Department of Pharmaceutical Chemistry

Author’s address: Department of Pharmacology and Toxicology University of Kuopio P.O. Box 1627 FI-70211 KUOPIO Tel. +358 40 355 3776 Fax +358 17 162 424 E-mail: [email protected]

Supervisors: Professor Pekka T. Männistö, M.D., Ph.D. Division of Pharmacology and Toxicology Faculty of Pharmacy University of Helsinki Senior assistant Anne Lecklin, Ph.D. Department of Pharmacology and Toxicology University of Kuopio

Reviewers: Docent Pekka Rauhala, M.D., Ph.D. Institute of Biomedicine University of Helsinki Docent Seppo Kaakkola, M.D., Ph.D. Department of Neurology Helsinki University Central Hospital

Opponent: Professor Raimo K. Tuominen, M.D., Ph.D. Division of Pharmacology and Toxicology Faculty of Pharmacy University of Helsinki

ISBN 978-951-27-0851-2 ISBN 978-951-27-1144-4 (PDF) ISSN 1235-0478 Kopijyvä Kuopio 2008 Finland 3

Kääriäinen, Tiina. Interaction of the dopaminergic and serotonergic systems in rat brain: Studies in parkinsonian models and brain microdialysis. Kuopio University Publications A. Pharmaceutical Sciences 113. 2008. 125 p. ISBN 978-951-27-0851-2 ISBN 978-951-27-1144-4 (PDF) ISSN 1235-0478

ABSTRACT

Dopamine and (5-hydroxytryptamine; 5-HT) are involved in several essential brain functions as well as in various neurological and mental disorders. Anatomical, electrophysiological and biochemical data point to an important interplay of the serotonergic and dopaminergic pathways. In particular, the serotonergic system has a modulatory role of on dopaminergic neurotransmission in the central nervous system (CNS). Various 5-HT subtypes modulate release both directly by 5-HT receptors located on dopaminergic neurons, and indirectly via 5-HT heteroreceptors present on GABAergic neurons. Brain 5-HT/dopamine interactions are also putative targets for drug therapies in disorders related to alterations in dopaminergic or serotonergic neurotransmission, such as Parkinson’s disease (PD), schizophrenia and depression. However, the mechanisms to explain the interactions between 5-HT and dopamine in these brain dysfunctions are not well defined. The objectives of this study were 1) to explore the role of striatal 5-HT in the unilateral rat model of PD, and 2) to assess the ability of 5-HT2A/ and 5-HT1A receptor ligands to modulate accumbal and striatal dopamine release and in normal rats. Also 3) the advantages and disadvantages of two types of 6-hydroxydopamine (6-OHDA)-lesion models were evaluated in neuroprotection studies with a flavonoid, quercetin. It was shown that acute intrastriatal L-dopa infusion decreased contralateral rotation of unilaterally 6-OHDA-lesioned rats, evidently by desensitization of ipsilateral dopamine D2 receptors. In an acute study, the decrease in the amount of rotations and imbalance of striatal 5-HT levels between intact and lesion side were abolished by inhibition of 5-HT synthesis. In a long-term study, continuous 2- week intrastriatal L-dopa infusion in 6-OHDA-rats also decreased the number of contralateral rotations induced by peripheral L-dopa and induced marked alterations in the ipsilateral serotonergic system (such as a long-term increase in 5-HT synthesizing hydroxylase) as assessed on day 70 post- infusion. In normal rats, the 5-HT2A/2C antagonist, , had an antidopaminergic effect similar to that of neuroleptics or the anxiolytic 5-HT1A at a high dose. At least a 5-fold margin was found between the anxiolytic and neuroleptic doses of deramciclane in the rat. Striatal 5-HT content was differently affected by the two types of unilateral 6-OHDA-lesion models. Since it is known that striatal 5-HT is reduced in PD patients, it seems that the 6-OHDA medial forebrain bundle- lesioned rat models this situation unilaterally rather well. In these models, quercetin had no effect indicative of neuroprotection against nigrostriatal dopaminergic or serotonergic 6-OHDA-induced neuron damage in vivo. In conclusion, these results highlight the role of 5-HT neurons in the striatal dopaminergic imbalance that is responsible for the rotational behavior seen in the unilateral 6-OHDA rat. This study provides new information about the serotonergic effects after local exogenous L-dopa in acute and long-term administrations into the striatum of dopamine depleted rat. The results may help to further clarify the role of the serotonergic system in the action of L-dopa under dopamine deficiency. Additionally, the clear unilateral 5-HT damage in this widely used rat model should be taken into account in further studies. This study also confirms earlier reports of dopamine modulating properties of 5-HT1A and 5-HT2A/2C ligands, particularly in the mesolimbic system, and provides further evidence that combined affinity of 5-HT/ dopamine D2 receptors may be beneficial in drug actions modulating dopaminergic tone in the CNS. The lack of effect of quercetin in 6-OHDA-rats casts serious doubt on the neuroprotective effects of this bioflavonoid in experimental PD in vivo.

National Library of Medicine Classification: WL 359, WL 307, QY 58, QV 126, WK 725 Medical Subject Headings: Parkinson Disease; Disease Models, Animal; Brain; Corpus Striatum; Agents; Serotonin; Receptors, Serotonin; Dopamine; Receptors, Dopamine; Ligands; Levodopa; Behavior, Animal; Rotation; Tryptophan Hydroxylase; Flavonoids; Quercetin; ; Microdialysis; Rats 4 5

“If the human brain were so simple that we could understand it, we would be so simple that we couldn’t.” Emerson Pugh, English philosopher, 1997 6 ACKNOWLEDGEMENTS

The present study was carried out in the Department of Pharmacology and Toxicology, University of Kuopio, during the years 2003 – 2008. I want to express my deepest gratitude to my principal supervisor Professor Pekka T. Männistö, who made this thesis possible. His encouragement, support and vast expertise have guided me throughout this project. Furthermore, even though he left to work in the University of Helsinki in 2004, he has always been able to find time for me. It has been a great privilege to work with Pekka. I wish also to thank my second supervisor, Anne Lecklin, Ph.D. for being my local adviser here in Kuopio. I express my gratitude to the official reviewers Docent Pekka Rauhala and Docent Seppo Kaakkola for their valuable comments and constructive criticism for improving the manuscript of this thesis. Furthermore, I am very grateful to Ewen MacDonald, Ph.D. for revising the language. I am honored to have Professor Raimo K. Tuominen as my opponent. I am indebted to my all co-authors for their significant contributions to this work. Especially, I wish to thank my collaborators in PTM’s team in Viikki, University of Helsinki; J. Arturo García-Horsman, Ph.D., Mikko Käenmäki, M.Sc., Bernardino Ossola, M.Sc. and Marjo Piltonen, M.Sc. I want to express my special thanks to Marjo Piltonen for her help and friendship during these studies and for her kind hospitality during my several visits to Helsinki. I also wish to thank Marko Huotari, Ph.D., for introducing me the world of the stereotaxis during my early days in the Department. I sincerely thank all of the present and former personnel of Department of Pharmacology and Toxicology. I wish to thank my colleagues, particularly Markus Forsberg, Ph.D., Aaro Jalkanen, M.Sc., Pasi Lampela, Ph.D., Šárka Lehtonen, Ph.D., Timo Myöhänen, Ph.D., Katja Puttonen, M.Sc., Minna Rahnasto, M.Sc., Kaisa Salminen, M.Sc., Timo Sarajärvi, M.Sc., Marjo Tampio, M.Sc., and Jarkko Venäläinen, Ph.D., for their support and friendship. I wish to express my special and sincere thanks to Markus Forsberg for being a mentor particularly during the time when I was completing this thesis, and for many discussions regarding scientific and not-so- scientific matters. I am grateful for Pirjo Hänninen and Jaana Leskinen for their excellent technical assistance and advice, and I wish to thank Leena Oksanen for all the help during these years. I owe special thanks to the personnel of National Laboratory Animal Center for their seamless collaboration. I also wish to thank all of those who may not find their names here, but who contributed in some way during these years. I warmly thank all my dear friends outside the university, especially Merja Rytkönen, Kristiina “Krisu” Saarelainen, Leena Tähtivaara and Anu von Delvig with their families, for sharing the joys and sorrows for many years. In addition, you all have supported me through this long process – I am so fortunate to have friends like you! I express my warmest gratitude to my family; my parents, Irma and Seppo, and my little brothers, Mikko, Antti and Olli for their endless encouragement, care, support and understanding – you have always had confidence in me. Special thanks to Antti for many joyful moments and for your curiosity about scientific issues – there really is a bit of art in everything, even in science! Per aspera ad astra. Finally, I am ready for the new and fascinating challenges which lay ahead of me in the brighter future. This work was supported in part by grants from the Finnish Cultural Foundation of Northern Savo, the Finnish Parkinson Foundation, University of Kuopio and the Foundation of Aleksanteri Mikkonen, which are greatly appreciated.

Kuopio, November 2008

Tiina Kääriäinen ABBREVIATIONS

AP Anterior-posterior AUC Area under the concentration-time curve CCW Counter-clockwise, contralateral rotations CMC Carboxymethylcellulose CNS Central nervous system DAT Dopamine transporter DDC Dopa decarboxylase 5,7-DHT 5,7-dihydroxytryptamine DOPAC 3,4-dihydroxyphenylacetic acid DV Dorsal-ventral GABA Gamma-aminobutyric acid G-protein Guanine nucleotide-binding protein GTP Guanine nucleotide triphosphate 5-HIAA 5-hydroxyindoleacetic acid 5-HT 5-hydroxytryptamine, serotonin HPLC High performance liquid chromatography HVA Homovanillic acid i.c.v. Intracerebroventricular i.p. Intraperitoneal L Lateral L-dopa L-3,4-dihydroxyphenylalanine MFB Medial forebrain bundle MPP+ 1-methyl-4-phenylpyridinium MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine OD Optical density 6-OHDA 6-hydroxydopamine 8-OH-DPAT 8-hydroxy-2-(di-n-propylamino)tetralin PCPA 4-chloro-DL- PD Parkinson’s disease ROS Reactive oxygen species s.c. Subcutaneous SERT Serotonin transporter SN SNl Substantia nigra pars lateralis SNpc Substantia nigra SNr Substantia nigra pars reticulata SSRI Selective serotonin reuptake inhibitor TAAR Trace amine-associated receptor TH Tyrosine hydroxylase TrH Tryptophan hydroxylase VTA Ventral tegmental area LIST OF ORIGINAL PUBLICATIONS

This doctoral dissertation is based on the following publications, referred to in the text by Roman numerals I – IV.

I Tiina M. Kääriäinen, J. Arturo García-Horsman, Marjo Piltonen, Pekka T. Männistö: L-dopa induced desensitization depends on 5-HT imbalance in hemiparkinsonian rats. NeuroReport, in press.

II Tiina M. Kääriäinen, J. Arturo García-Horsman, Marjo Piltonen, Marko Huotari, Pekka T. Männistö: Serotonergic activation after 2-week intrastriatal infusion of L- dopa and slow recovery of circling in rats with unilateral nigral lesions. Basic and Clinical Pharmacology and Toxicology 102: 300-307, 2008.

III Tiina M. Kääriäinen, Marko Lehtonen, Markus M. Forsberg, Jouko Savolainen, Mikko Käenmäki, Pekka T. Männistö: Comparison of the effects of deramciclane, and buspirone on extracellular dopamine and its metabolites in striatum and nucleus accumbens of freely moving rats. Basic and Clinical Pharmacology and Toxicology 102: 50-58, 2008.

IV Tiina M. Kääriäinen, Marjo Piltonen, Bernardino Ossola, Heli Kekki, Šárka Lehtonen, Terhi Nenonen, Anne Lecklin, Atso Raasmaja, Pekka T. Männistö: Lack of robust protective effect of quercetin in two types of 6-hydroxydopamine-induced parkinsonian models in rats and dopaminergic cell cultures. Brain Research 1203: 149-159, 2008.

CONTENTS

1 INTRODUCTION ...... 17 2 REVIEW OF THE LITERATURE ...... 19 2.1 Overview of central dopaminergic systems ...... 19 2.1.1 Dopaminergic neurons in the brain...... 19 2.1.2 Nigrostriatal dopaminergic system ...... 19 2.1.3 Mesolimbic and mesocortical dopaminergic systems ...... 20 2.1.4 Other dopaminergic pathways ...... 22 2.1.5 Dopamine transporters ...... 23 2.1.6 Dopamine receptors ...... 23 2.2 Overview of central serotonergic system ...... 24 2.2.1 Serotonergic neurons in the brain ...... 24 2.2.2 Rostral serotonergic system ...... 25 2.2.3 Caudal serotonergic system ...... 25 2.2.4 5-HT transporters ...... 26 2.2.5 5-HT receptors ...... 26 2.2.5.1 5-HT1 receptors ...... 26 2.2.5.2 5-HT2 receptors ...... 27 2.2.5.3 5-HT3 receptors ...... 29 2.3 Modulation of brain dopaminergic transmission by the serotonergic system .. 29 2.3.1 Anatomic interactions within serotonergic and dopaminergic neuronal pathways in the CNS ...... 29 2.3.1.1 The basis of interactions ...... 29 2.3.1.2 Direct serotonergic innervation to dopaminergic neurons ...... 30 2.3.1.3 Indirect serotonergic innervation to dopaminergic neurons via GABAergic innervation ...... 31 2.3.2 Serotonergic and dopaminergic interactions at the receptor level ...... 33 2.3.2.1 The mechanisms of receptor-interactions ...... 33 2.3.2.2 Modulation via 5-HT1,5-HT2 and 5-HT3 receptor subtypes ...... 35 2.3.2.3 Modulation via transporters ...... 40 2.4 Modulation of brain serotonergic transmission by the dopaminergic system .. 42 2.5 Dopaminergic and serotonergic changes in most common -induced animal models of Parkinson’s disease ...... 43 2.5.1 6-OHDA ...... 43 2.5.2 MPTP ...... 44 2.5.3 ...... 44 2.6 Serotonin/dopamine interactions in some disease states in the CNS...... 45 2.6.1 General ...... 45 2.6.2 Parkinson’s disease ...... 45 2.6.2.1 5-HT in Parkinson’s disease ...... 45 2.6.2.2 5-HT receptors in Parkinson’s disease...... 48 2.6.3 Schizophrenia ...... 50 2.6.3.1 Dopamine and D2 receptors in schizophrenia ...... 50 2.6.3.2 5-HT receptors in schizophrenia ...... 51 2.6.4 Depression ...... 52 2.6.4.1 The role of dopamine ...... 52 2.6.4.2 5-HT and D2 receptors in activity ...... 54 3 AIMS OF THE STUDY ...... 55 4 MATERIALS AND METHODS ...... 56 4.1 Animals ...... 56 4.2 Drugs ...... 56 4.3 6-OHDA-lesions ...... 57 4.3.1 6-OHDA-lesion of the right medial forebrain bundle (I, II, IV) ...... 57 4.3.2 6-OHDA-lesion of the striatum (IV) ...... 57 4.4 Measurement of rotational behavior (I, II, IV) ...... 58 4.5 Unilateral intrastriatal infusions of L-dopa (I, II) ...... 59 4.5.1 Acute single intrastriatal infusion of L-dopa (I) ...... 59 4.5.2 Continuous 2-week intrastriatal infusion of L-dopa (II) ...... 60 4.6 Brain microdialysis in conscious rats (III) ...... 61 4.7 Quercetin treatment schedules (IV) ...... 62 4.8 Analytical procedures (I – IV) ...... 63 4.8.1 Determination of striatal dopamine, 5-HT and their metabolites in rat tissue samples (I, II, IV) ...... 63 4.9 Nigral and striatal TH-positive cell assays (IV) ...... 64 4.10 Western immunoblotting (I, II) ...... 64

4.11 Dopamine D2 receptor binding and 5-HT uptake assay (II)...... 64 4.12 Data analysis and statistics ...... 65 4.12.1 Microdialysis data (III) ...... 65 4.12.2 Statistical analyses (I – IV) ...... 65 5 RESULTS ...... 67 5.1 The effect of acute and 2-week intrastriatal L-dopa infusion treatment on 6- OHDA-lesioned rats (I, II) ...... 67 5.1.1 The effect of intrastriatal L-dopa treatment on rotational behavior induced by acute intraperitoneally administered L-dopa ...... 67 5.1.2 The effect of intrastriatal L-dopa treatment on striatal levels of dopamine, 5-HT and their metabolites ...... 69 5.1.3 The effect of intrastriatal L-dopa treatment on striatal TrH and TH ...... 71 5.1.4 The effect of 2-week intrastriatal L-dopa treatment on striatal 5-HT uptake

and dopamine D2 receptor binding ...... 72 5.2 The effect of 5-HT-ligands on the extracellular levels of dopamine, DOPAC and HVA in rat nucleus accumbens shell and striatum (III) ...... 73 5.3 The effect of quercetin in unilateral 6-OHDA-lesions of two types of parkinsonian rat models (IV) ...... 75 5.3.1 Levels of dopamine, 5-HT and their metabolites in intact and lesioned striata ...... 75 5.3.2 TH-positive cell staining in intact and lesioned striata and substantia nigra ...... 76 5.3.3 Rotational behavior of unilaterally lesioned rats ...... 77 6 DISCUSSION ...... 80 6.1. General ...... 80 6.2 The effect of acute and 2-week intrastriatal L-dopa infusions on 6-OHDA- lesioned rats (I, II) ...... 81 6.3 The effect of 5-HT-ligands on the extracellular levels of dopamine, DOPAC and HVA in rat nucleus accumbens shell and striatum (III) ...... 87 6.4 Lack of protective effect of quercetin in two types of unilateral 6-OHDA- lesions (IV) ...... 90 7 CONCLUSIONS ...... 95 8 REFERENCES ...... 97 ORIGINAL PUBLICATIONS ...... 125

17

1 INTRODUCTION

Dopamine and serotonin (5-hydroxytryptamine, 5-HT) are two of the most intensively studied in the brain due to their participation in several crucial brain functions as well as their involvement in various neurological and mental disorders. Anatomical, electrophysiological, and biochemical data all confirm the important interplay of the serotonergic and dopaminergic pathways. In particular, the modulatory role of the serotonergic system on dopaminergic neurotransmission in the central nervous system (CNS) has been studied widely in animal and human studies. These interactions are supported by neuroanatomical data which have revealed important serotonergic projections from the dorsal raphe nucleus to dopaminergic cell bodies in the ventral tegmental area (VTA) and in the substantia nigra (SN) and to their respective target areas (e.g. nucleus accumbens, prefrontal cortex and striatum) (for review see Barnes and Sharp 1999). The serotonergic innervation mediates both excitatory and inhibitory effects on dopaminergic tone in the rat brain (Yoshimoto and McBride 1992; De Deurwaerdère et al. 1998; Gervais and Rouillard 2000). Various subtypes of 5-HT receptors (particularly 5-HT1B, 5-HT2A, 5-HT2C and 5-HT3) have been shown to modulate dopamine release. Regulation of dopamine neurotransmission may occur both directly by 5-HT receptors located on the dopaminergic neurons themselves, and indirectly via 5-HT heteroreceptors present on gamma-aminobutyric acid (GABA) - containing interneurons (Stanford and Lacey 1996). Less is known about the dopaminergic regulation of the serotonergic neurons, despite the morphological evidence of reciprocal projections between dopaminergic cell body areas (SN, VTA) and dorsal raphe nuclei (van der Kooy and Hattori 1980; Hervé et al. 1987) as well as the presence of D2-like receptors in dorsal raphe (Bouthenet et al. 1987). There is a physiologically relevant dopaminergic regulation of ascending serotonergic pathways in the CNS (Hery et al. 1980; Ferré et al. 1994; Callaghan et al. 2005). However, the importance of brain 5-HT/dopamine interactions still needs further clarification. In addition to its physiological importance, the interaction between dopaminergic and serotonergic neurotransmission is important from a therapeutic point of view. Dopaminergic processes involving serotonergic interactions in the CNS are also putative targets for therapeutic tools in disorders related to alterations in dopaminergic neurotransmission, such as Parkinson’s disease (PD) and schizophrenia (Dewey et al. 1995). Furthermore, a role of the relative shortage of dopamine in symptoms and the use of dopamine uptake blockers in the therapy of depression have been postulated (D'Aquila et al. 2000; Zangen et al. 2001). Specific animal models of brain disorders, such as PD, have provided valuable information of the interactions of neurotransmitter systems in the CNS. In these animal models, also changes in serotonergic system can often be seen. The new knowledge from animal studies has already led to some novel 18

therapeutic applications of existing drugs and also new approaches for the development of drug therapies. The following review of the literature focuses on the modulatory interactions between dopamine and 5-HT in the CNS, particularly on the effect of 5-HT on the three major brain dopaminergic neuron systems: nigrostriatal, mesolimbic and mesocortical tracts. Special emphasis is placed on certain 5-HT receptor subtypes, i.e. 5-HT1, 5-HT2 and 5-HT3, due to their expression in those brain areas containing dopaminergic cell bodies or nerve terminals with a high probability of involvement in the modulation of dopamine release. Finally, some clinically relevant applications related to interactions of brain 5-HT and dopamine in three common brain disorders, PD, schizophrenia and depression, will be discussed. The objectives of the experimental part of this thesis were to study the role of striatal 5-HT in the unilateral PD model in the rat, and to assess the ability of 5-HT2A/2C and 5-HT1A receptor ligands to modulate accumbal and striatal dopamine release and metabolism in normal rats. In addition, the advantages and disadvantages of two types of 6-hydroxydopamine (6-OHDA)-induced lesion models, associated with different levels of 5-HT loss, were evaluated in neuroprotection studies. 19

2 REVIEW OF THE LITERATURE

2.1 Overview of central dopaminergic systems

2.1.1 Dopaminergic neurons in the brain

Dopamine is one of the two major in the central nervous system, and was first found in brain in 1939 (see Grace and Bunney 1985). After the proposal by Carlsson and colleagues (1957; 1959) that dopamine could act as a neurotransmitter instead of being a simply precursor for and noradrenaline, it was discovered that the striatum contains 70 – 80% of the total brain dopamine, and the depletion of striatal dopamine is essential in the pathogenesis of PD (see Hornykiewicz 1973). Dopamine is also known to play significant roles in many crucial brain functions such as motor behavior, motivation, reward and cognition. Dopamine neurons in the central nervous system can be categorized on the basis of their site of origin and targets into different systems (Moore and Bloom 1978; Grace and Bunney 1985; Björklund and Dunnett 2007). The nigrostriatal, mesocortical and mesolimbic dopaminergic systems are probably the best characterized due to their involvement of important CNS functions as well as in brain disorders such as PD and schizophrenia. These major brain dopaminergic systems (together known as mesotelencephalic system) arise from three main dopaminergic cell groups, which were initially designated by Dahlström and Fuxe (1964) as areas A8, A9 and A10 in the ventral midbrain. The A8 refers to the retrorubral area, the A9 and A10 to the SN and VTA, respectively (Figure 1A). The groups A9 and A10 will be further discussed.

2.1.2 Nigrostriatal dopaminergic system

The basal ganglia consist of the caudate nucleus and the putamen (which are known together as dorsal striatum) together with external and internal divisions of globus pallidus. SN (area A9) located in the ventral midbrain, is divided into dorsal cell rich region, the pars compacta (SNpc), a more diffuse and cell poor region pars reticulata (SNr), and a small cell cluster called the pars lateralis (SNl) (Fallon and Loughlin 1985; Heimer et al. 1985). The dopaminergic cell bodies of the SN and medial VTA innervate the caudate-putamen, which forms the nigrostriatal pathway (Ungerstedt 1971; Lindvall and Björklund 1974) (Figure 1A). The ascending projections of dopaminergic cells in the SN and VTA travel through the heavily myelinated fiber bundles of the medial forebrain bundle (MFB) (Ungerstedt 1971; Fallon and Loughlin 1985; MacLean 1985) (Figure 2). Dopaminergic nigrostriatal fibers are thin, varicose and they travel over long distances throughout the striatum (Smith and Kieval 2000). This ascending pathway is 20

the main component participating in a regulation of motor control and degeneration of dopaminergic nigrostriatal pathway leading to motor deficits seen in PD (see Hornykiewicz 1973; Sian et al. 1999). In addition, this structure may be important for maintaining normal cognitive behavior and perception. It has been claimed that striatal dopaminergic hyperactivity possibly underlies the positive symptoms of schizophrenic patients (Laruelle et al. 1999).

2.1.3 Mesolimbic and mesocortical dopaminergic systems

The mesolimbic dopaminergic ascending pathway consists of long neurons originating from VTA of the ventral mesencephalon (area A10) and projecting to the limbic structures; the nucleus accumbens core and shell, olfactory tubercle (often referred to as ventral striatum), amygdala, septum and hippocampus (Dahlström and Fuxe 1964; Ungerstedt 1971; Heimer et al. 1985) (Figure 1A). The mesocortical dopaminergic pathway also originates from the VTA (A10) and terminates in the medial prefrontal cortex and cingulate suprarhinal and entorhinal cortices. In parallel with mesolimbic dopaminergic neurons, mesocortical axons also represent the long type neurons (Dahlström and Fuxe 1964; Fuxe et al. 1974; Fallon and Loughlin 1985; Weinberger 1987). The ascending projections of the midbrain dopaminergic neurons to the prefrontal cortex and nucleus accumbens play significant roles in motivation, reward and cognitive functions (Carr and Sesack 2000b). It has been hypothesized that dysfunction of one or more of the parts of the mesolimbic system may be involved in the pathogenesis of schizophrenia. In addition, hypoactivity of mesocortical dopaminergic neurons has been linked to the negative symptoms and impaired cognition occurring of the schizophrenia (Fallon and Loughlin 1985; Weinberger 1987). There is a considerable overlap between the VTA cells projecting to the target areas of mesolimbic and mesocortical systems. Because of this overlap, these two systems are sometimes collectively referred to as the mesocorticolimbic dopamine system (see Wise 2004). 21

Figure 1. (A) Major ascending dopaminergic pathways in the rat central nervous system (CNS); 1. nigrostriatal pathway (white), 2. mesolimbic pathway (black), 3. mesocortical pathway (grey). The dopaminergic cell body areas and terminal regions as well as the distribution of serotonin 5-HT1, 5-HT2 and 5-HT3 receptors in different brain areas are shown. A8: Retrorubral area; A9: Substantia nigra (SN), A10: Ventral tegmental area (VTA). (B) Main ascending serotonergic pathways in the CNS; serotonergic cell body areas (B1 – B9) in the brainstem as well as terminal regions of the 5-HT neurons and the distribution of dopamine D1 – D5 receptors are shown (for references, see text). 22

Figure 2. Scheme for the ascending dopaminergic (thick lines) and serotonergic pathways (thin lines) of the medial forebrain bundle (MFB) in the rat central nervous system, illustrating the close proximity of these pathways travelling in the MFB. The cell body areas and the main terminal fields are seen. Modified from Fallon and Loughlin (1985), Heimer et al. (1985), MacLean et al. (1985) and Törk (1990).

2.1.4 Other dopaminergic pathways

The tuberoinfundibular and tuberohypophyseal dopaminergic system consists of the intermediate length dopaminergic neurons originating from the arcuate and periventricular nuclei within the hypothalamus (A12). These neurons project to the median eminence and pituitary (Björklund et al. 1970). This system regulates the neurosecretion as well as the production and release of hypothalamic and pituitary hormones (Moore and Bloom 1978; Tuomisto and Männistö 1985). Dopamine also exists in the retinal cells. A major part of the dopaminergic cell bodies reside in the inner part of the inner nuclear layer of the retina. There, the main types of dopaminergic cells are the interplexiform and amacrine-like cells, projecting to the inner and outer plexiform cell layers of the retina with ultrashort projections (Moore and Bloom 1978; Fallon and Loughlin 1985). These dopaminergic cells participate in the visual sensory input through dendritic interactions with other retinal neurons (Fallon and Loughlin 1985). In the CNS, there are also other neuronal pathways where dopamine is present. Those include the periglomerular dopamine cells originating in the olfactory bulb with local ultrashort connections, the incertohypothalamic system within hypothalamus and 23

lateral septal nuclei, the medullar periventricular dopamine neurons, and the diencephalospinal dopamine projection neurons (Grace and Bunney 1985).

2.1.5 Dopamine transporters

The effects of dopamine are mainly terminated by high affinity dopamine uptake sites, which are also important in maintaining transmitter homeostasis and dopaminergic tone in the CNS. The re-uptake of dopamine into the terminal and removal from the synaptic cleft is mediated by the dopamine transporter (DAT) located in neuronal membranes on the dopamine nerve terminals. DAT is able to transport dopamine into and out of the terminal depending on the concentration gradient (Horn 1990; Suaud- Chagny et al. 1995; Zahniser et al. 1999).

2.1.6 Dopamine receptors

Dopamine is known to be involved in several essential brain functions, such as locomotor behavior, cognition, motivation and neuroendocrine secretion, with its actions mediated via dopamine receptors (Jaber et al. 1996; Schwartz et al. 1998). These receptors are known to belong to the large guanine nucleotide triphosphate (GTP) binding protein (G-protein) -coupled receptor family, and the receptors can be further classified into two categories; postsynaptic receptors and presynaptic receptors (autoreceptors). Postsynaptic receptors are located on the postsynaptic target areas of dopaminergic neurons while autoreceptors are located either on the dopaminergic cell body or on presynaptic terminals (Grace and Bunney 1985; Jaber et al. 1996; Lachowicz and Sibley 1997).

Postsynaptic dopamine receptors can be classified as D1-like or D2-like receptors according to their distinct biochemical characteristics (Schwartz et al. 1998). D1-like receptors include the D1 and D5 receptors mediating the activation of adenylate cyclases via coupling to a Gs protein. D2-like receptors consist of the D2, D3 and D4 receptors, inhibiting adenylate cyclases via coupling to Gi/Go proteins (Jaber et al. 1996; Schwartz et al. 1998). The dopamine receptor distribution in the CNS is described in Figure 1B. Dopamine receptors are found in the projection areas of midbrain dopaminergic neurons (Figure 1B). In striatum, dopamine acts through both D1- and D2-like receptors.

D1-like dopamine receptors are mainly localized in GABA-containing striatal neurons that project to globus pallidus and SN and contain neuropeptides such as dynorphin and substance P. D2-like dopamine receptors are primarily situated in GABAergic striatal neurons projecting to globus pallidus and co-localized with enkephalin (Grace and Bunney 1985; Wamsley et al. 1989; Gerfen et al. 1995; Aubert et al. 2000). In addition to the nigrostriatal system, D1 receptors are found in frontal cortex, nucleus accumbens, olfactory tubercle, amygdala, thalamus, VTA area and choroid plexus (Seeman 1980; 24

Wamsley et al. 1989; Jaber et al. 1996; Sealfon and Olanow 2000). D1 receptor sites are not found on dopaminergic neurons, but are located on the postsynaptic neurons (Seeman 1980).

The distribution of D2 receptors is very similar to that of D1 receptors, the major differences being a lower level in the cerebral cortex and the presence of presynaptic receptors on striatal dopaminergic neuron terminals (Hurley and Jenner 2006). In addition to striatum and SN, dopamine D2 receptors are also found on corticostriatal terminals (Grace and Bunney 1985), limbic areas such as nucleus accumbens, hypothalamus and olfactory tubercle (Seeman 1980; Wamsley et al. 1989; Jaber et al. 1996; Sealfon and Olanow 2000) as well as in the VTA area (Schwartz et al. 1998). They are also present in dorsal raphe nuclei (Bouthenet et al. 1987).

Dopaminergic autoreceptors are classified as D2 -like receptors and they are located in several regions of the dopaminergic cell, including the axon terminals (terminal receptors), soma and dendrites (somadendritic receptors). Somadendritic receptors are important in regulating cell firing rate, whereas terminal receptors regulate dopamine synthesis and release (Seeman 1980; Grace and Bunney 1985). The dopamine autoreceptors are more sensitive to dopamine than the postsynaptic dopamine receptors (Skirboll et al. 1979). The activation of autoreceptors by endogenous dopamine or dopamine inhibits the release of dopamine as well as dopamine synthesis by tyrosine hydroxylase (TH) enzyme via feedback regulation (Seeman 1980). However, these receptors are known to undergo rapid desensitization (tachyphylaxis) with repeated administration of dopamine agonists (Grace and Bunney 1985).

2.2 Overview of central serotonergic system

2.2.1 Serotonergic neurons in the brain

The location of large neuron cells of the brainstem with uncertain projections was first reported in the early 1910’s by Ramón y Cajal (see Jacobs and Azmitia 1992). These neurons were later identified as serotonergic raphe nuclei cells by Dahlström and Fuxe (1964). For many years investigators were aware of two substances: 1) a chemical named enteramine, which was found by Erspamer and had an ability to increase intestinal motility, and 2) a blood-borne compound that produced vasoconstriction. In the late 1940’s, the structure of the single compound, serotonin (5-HT), producing both of the abovementioned effects, was clarified by Rapport and co-workers. The finding that 5-HT exists widely in the mammalian CNS led to conclusion that 5-HT was acting as a neurotransmitter (for reviews see Jacobs and Azmitia 1992; Whitaker-Azmitia 1999). While 5-HT is abundantly present both in the CNS and periphery, the brain 5-HT represents less than 2% of total body 5-HT content since more than 95% of 5-HT is found in platelets and the gastro-intestinal tract (Erspamer 1957; Sanders-Bush and 25

Mayer 2001). In the CNS, 5-HT is recognized as one of the most important neurotransmitters. The central serotonergic system is known to regulate numerous physiological functions, such as appetite, circadian rhythm, locomotor activity, body temperature, memory, sexual behavior, vigilance and nociception. It also plays a role in different disease states, such as migraine, depression, schizophrenia, and aggressivity. The serotonin neuron cell body-groups were designated as groups B1 – B9 by Dahlström and Fuxe (1964). The 5-HT cells are located in the brain stem near the midline and are divided into two distinct subdivisions; the rostral and caudal divisions. The rostral raphe nuclei contain the dorsal raphe groups (B6, B7) and the median raphe groups (B5, B8), which project to partly overlapping areas in the forebrain. The caudal system (B1 – B3) project axons mainly to the spinal cord and to the periphery (Dahlström and Fuxe 1964; Ungerstedt 1971; Törk 1990) (Figure 1B). Additionally, 5- HT cells are found in area postrema, caudal locus coeruleus and within the interpeduncular nucleus (Dahlström and Fuxe 1964).

2.2.2 Rostral serotonergic system

The rostral part of the serotonergic system comprises of the caudal linear nucleus, the dorsal raphe nucleus and median raphe nucleus. The dorsal raphe nucleus (B6, B7) is the most prominent of these nuclei and it is located in the ventral part of the periaqueductal gray matter of the midbrain with the extending parts of the pons (Figure 1B). Caudal linear nucleus (B8) is a part of the ventromedial mesencephalic tegmentum, and its neurons have similar efferents to those of dorsal raphe nucleus. The cells of median raphe nucleus (B5, B8) are arranged into two adjacent regions, the midline and the more loosely arranged cells outside the midline. The B9 group of 5-HT neuron cluster is located next to the medial lemniscus (Dahlström and Fuxe 1964; Hillarp et al. 1966; Jacobs and Azmitia 1992). The cell bodies of the rostral division of serotonergic system enter through the MFB and innervate multiple regions of the CNS with specific innervation patterns. Those include particularly the cerebral and cerebellar cortices, limbic structures and basal ganglia (Dahlström and Fuxe 1964; Ungerstedt 1971; Steinbusch 1981; Jacobs and Azmitia 1992) (Figures 1B and 2).

2.2.3 Caudal serotonergic system

The caudal serotonergic system consists of raphe pallidus nucleus (B1), raphe obscurus nucleus (B2), raphe magnus nucleus (B3) and the small 5-HT cell cluster situated within the raphe obscurus nucleus (B4) (Figure 1B). Most of the 5-HT neuron cell bodies of the caudal system are located principally within the medulla oblongata 26

and pons with projections extending to the gray matter of the spinal cord (Dahlström and Fuxe 1964; Hillarp et al. 1966; Jacobs and Azmitia 1992).

2.2.4 5-HT transporters

Brain 5-HT homeostasis is primarily regulated by the Na+ and Cl- dependent serotonin transporters (SERT), which are located in the presynaptic membrane on the serotonergic nerve terminals. These high affinity uptake sites remove 5-HT from the extracellular space into the presynaptic serotonergic nerve terminal, and are able to transport 5-HT in either direction, depending on the concentration gradient (Blakely et al. 1994; Homberg et al. 2007).

2.2.5 5-HT receptors

In the CNS, there are at least 14 genetically, pharmacologically and functionally distinct 5-HT receptor subtypes belonging to seven families; 5-HT1 - 5-HT7 (see Barnes and Sharp 1999; Hoyer et al. 2002). The majority of the 5-HT receptor subtypes are G- protein coupled metabotropic receptors except for the 5-HT3 receptors, which are -gated ion channels (Derkach et al. 1989). As reviewed by Barnes and Sharp (1999), 5-HT receptors are divided into distinct subtypes according to their coupling to the second messengers in the G-protein system and their amino acid sequence homology.

The subsequent discussion concerning the 5-HT1-3 receptors is based on their postulated modulatory effect on brain dopaminergic transmission. The distribution of 5-

HT1, 5-HT2 and 5-HT3 receptors in the CNS is described in Figure 1A.

2.2.5.1 5-HT1 receptors

All five members of the 5-HT1 subfamily (5-HT1A, B, D, E, F) are negatively coupled to adenylate cyclase via Gi -like proteins of G-protein family. The 5-HT1A receptor subtype also activates a receptor-operated K+ channel and inhibits a voltage-gated Ca2+ channel (Hoyer et al. 1994; Barnes and Sharp 1999).

5-HT1A receptors

After the identification of the 5-HT1A binding site, the 5-HT1A receptor was the first fully sequenced 5-HT receptor (Albert et al. 1990). 5-HT1A receptors are expressed both as auto- and heteroreceptors, mainly somadendritically on neuronal cell bodies and dendrites (Riad et al. 2000), and they exert inhibitory effects on neuronal firing

(Bockaert et al. 2006). The distribution of 5-HT1A receptors is similar in many 27

mammalian species, including humans (Hoyer et al. 1986; Pazos et al. 1987). There is an abundance of these receptors in hippocampus, lateral septum, amygdala, neocortex, hypothalamus and raphe nuclei (Marcinkiewicz et al. 1984; Pazos and Palacios 1985;

Pazos et al. 1987; Radja et al. 1992). In contrast, the levels of 5-HT1A binding sites in the basal ganglia and cerebellum are almost undetectable (Pazos and Palacios 1985; Pazos et al. 1987; Barnes and Sharp 1999). However, more recent studies have suggested that these receptors also exist in primate striatum (Frechilla et al. 2001).

5-HT1B receptors

5-HT1B receptors are located on the nerve terminals and are involved in presynaptic control of neurotransmitter release (Sari et al. 1999; Riad et al. 2000). In rats, high or intermediate levels of 5-HT1B receptors are expressed in the VTA, SN and basal ganglia areas (Pazos and Palacios 1985; Sari et al. 1999) as well as in the cortex, hippocampus, olfactory tubercle, entopeduncular nucleus, the superficial gray layer of the superior colliculus and the cerebellum (Bruinvels et al. 1994; Sari et al. 1999; Sari 2004).

Previously, the human 5-HT1D receptor was considered to be a species analog of 5-

HT1B receptors found in rodents (Waeber et al. 1990). Subsequent studies revealed the heterogeneity of 5-HT1D receptors: 5-HT1DĮ and 5-HT1Dȕ receptors were identified in human brain (Oksenberg et al. 1992). According to current knowledge, both 5-HT1B and

5-HT1D receptors are expressed in many species, including humans (Hartig et al. 1996;

Doménech et al. 1997; Middlemiss et al. 1999). The human 5-HT1Dȕ receptor and the rodent 5-HT1B receptor are coded by an identical gene and thus likely have the same biological function (Waeber et al. 1990; Hartig et al. 1996; Raiteri 2006). As recently reviewed by Fink and Göthert (2007), 5-HT1B and 5-HT1D receptors exhibit similar pharmacological properties and evidently mediate the same effects in rodents and humans, but partly differ from each other in their brain distribution and function.

2.2.5.2 5-HT2 receptors

There are three subtypes of 5-HT2 receptors (5-HT2A, B, C) linked to the enzyme phospholipase C via Gq -like G proteins with the generation of two second messengers, diacylglycerol (activates protein kinase C) and inositol trisphosphate (releases intracellular stores of Ca2+) (Hoyer et al. 2002).

5-HT2A receptors

5-HT2A receptors exist mainly on cell dendrites and soma as well as to a lesser extent on axon terminals (Jakab and Goldman-Rakic 1998; Cornea-Hébert et al. 1999). They facilitate or stimulate neuron function as well as neuronal depolarization 28

(Bockaert et al. 2006). 5-HT2A receptors in a rat brain are mainly located in the frontal cortex, basal ganglia, and limbic areas such as olfactory tubercle, hippocampus, nucleus accumbens and amygdala, as well as in cerebellum, brainstem and spinal cord (Pazos et al. 1985; Cornea-Hébert et al. 1999). In humans, 5-HT2A receptors densely exist in all neocortical regions, whereas lower densities are found in basal ganglia, hippocampus and thalamus (Hall et al. 2000).

5-HT2B receptors

There is a restricted distribution of 5-HT2B receptors in the CNS. A limited number of 5-HT2B receptors has been found in the cells in the medial amygdala, lateral septum and hypothalamus as well as in cell fibers (but not in cell bodies) in the frontal cortex and spinal cord in rat (Duxon et al. 1997; Sanders-Bush and Mayer 2001). In human brain, the distribution of 5-HT2B receptors is also very restricted, only a few of these receptors are found in cortex (Bonhaus et al. 1995).

5-HT2C receptors

A high density of binding sites in choroid plexus was found in early autoradiographic studies performed with 5-HT1 but not 5-HT2 receptor ligands. Thus, it was presumed that these receptors would belong to the class of 5-HT1 receptors, and were subsequently named as 5-HT1C (Pazos et al. 1984; Pazos and Palacios 1985).

Later, the former 5-HT1C receptor was reclassified as 5-HT2C due to several similarities with the 5-HT2 receptor subclass (Hoyer et al. 1994). 5-HT2C receptors are localized both pre- and postsynaptically (López-Giménez et al. 2001), and they contribute substantially to the serotonergic regulation of many behavioral and physiological processes (Giorgetti and Tecott 2004). 5-HT2C receptor mRNA is expressed in several regions in the brain, both in catecholaminergic cells and in serotonergic neurons

(Hoffman and Mezey 1989). In addition to choroid plexus, the 5-HT2C receptors are widely distributed in several brain areas. Those include the areas containing nigrostriatal and mesocortical cell bodies, the SN and VTA, respectively (Molineaux et al. 1989;

Eberle-Wang et al. 1997). 5-HT2C receptors also exist in the terminal regions, i.e. in the striatum and frontal cortex (Eberle-Wang et al. 1997; Clemett et al. 2000), as well as in nucleus accumbens, hippocampus, amygdala and piriform cortex (Hoffman and Mezey 1989; Eberle-Wang et al. 1997; Sanders-Bush and Mayer 2001). Furthermore, intermediate levels are present in dorsal raphe nucleus (Hoffman and Mezey 1989). The expression of 5-HT2C receptors as well as receptor mRNA in human brain resemble the corresponding situation in rodents (Pazos et al. 1987; Pasqualetti et al. 1999). 29

2.2.5.3 5-HT3 receptors

5-HT3 receptors mediate fast excitatory responses to 5-HT. 5-HT3 receptors belong to the ligand-gated ion channel receptor superfamily based on their electrophysiological features. They trigger a rapid depolarization via a transient inward current and subsequent opening of the cation channels (influx: Na+, Ca2+, efflux: K+) in neurons; this is a rapid response (Derkach et al. 1989; Hoyer et al. 2002). Both presynaptic and postsynaptic 5-HT3 receptors are known to exist; presynaptic 5-HT3 receptors are involved in neurotransmitter release, whereas postsynaptic receptors are preferentially expressed on interneurons (Nayak et al. 1999; Zhou and Hablitz 1999; Van Hooft and Wadman 2003).

5-HT3 receptors are found in the forebrain, brainstem and spinal cord, particularly in cortex, olfactory nucleus, hypothalamus, hippocampus, amygdala, striatum, nucleus accumbens and area postrema as well as facial and trochlear nerve nuclei (Tecott et al. 1993; Morales et al. 1998; see Fink and Göthert 2007). In general, neurons showing 5-

HT3 receptor immunoreactivity are located within cortical, mesolimbic and motor regions indicating the possible involvement of 5-HT3 receptor in cognition, emotional and locomotor behavior (Tecott et al. 1993; Morales et al. 1998).

2.3 Modulation of brain dopaminergic transmission by the serotonergic system

2.3.1 Anatomic interactions within serotonergic and dopaminergic neuronal pathways in the CNS

2.3.1.1 The basis of interactions

Behavioral and neurochemical studies have shown that the serotonergic system is able to modulate the activity of the dopaminergic system in the brain via a variety of mechanisms. These interactions seem to be very complex and occur at the level of cell bodies and the nerve terminals and via both pre- and postsynaptic mechanisms. The anatomical basis of the modulation of dopamine release by endogenous 5-HT are achieved by the connections between serotonergic and dopaminergic neural pathways in different brain areas. In this context, two types of the serotonergic innervation will be discussed; the connections to dopaminergic neurons and their terminal areas, and the connections to non-dopaminergic cells, such as inhibitory GABA-containing neurons which subsequently innervate dopaminergic neurons. 30

2.3.1.2 Direct serotonergic innervation to dopaminergic neurons

The serotonergic innervation of the midbrain dopaminergic neurons is one of the densest 5-HT innervations in the brain (Dray et al. 1976; Lavoie and Parent 1990; Vertes 1991; Vertes et al. 1999). The cell bodies and terminal regions of all three major brain dopaminergic pathways are innervated by 5-HT neurons originating from the raphe nuclei and this represents the foundation for the serotonergic modulation of the dopaminergic transmission (reviewed in Barnes and Sharp 1999; Fink and Göthert 2007) (Figures 1B and 2). It has been shown that the serotonergic innervation mediates both excitatory and inhibitory effects on dopaminergic tone in different brain areas (Fallon and Loughlin 1985; Yoshimoto and McBride 1992; De Deurwaerdère et al. 1998; Gervais and Rouillard 2000). Serotonergic neurons from medial and dorsal raphe nuclei project densely to the cell bodies of dopaminergic neurons in SN (Dray et al. 1976; Fallon and Loughlin 1985; Moukhles et al. 1997) and VTA (Hervé et al. 1987), which are the respective origins of the nigrostriatal and mesocorticolimbic dopaminergic pathways. There seems to be a different modulation in SN and VTA by projections from dorsal raphe; in the SN, the majority of dopaminergic neurons are inhibited by electrical stimulation of dorsal raphe (Kelland et al. 1990; Gervais and Rouillard 2000), whereas VTA dopaminergic neurons are mainly excited after electrical dorsal raphe stimulation (Gervais and Rouillard 2000). Dorsal raphe neurons also innervate the target areas of the above-mentioned dopaminergic projections, i.e. striatum, nucleus accumbens and prefrontal cortex (Heimer et al. 1985; Törk 1990). Additionally, striatum receives collaterals from the majority of the 5-HT raphe neurons innervating the SN (van der Kooy and Hattori 1980; Moukhles et al. 1997). In contrast, there are also dopaminergic afferent neurons from SN and VTA to the dorsal raphe nucleus (Moore and Bloom 1978).

Direct interactions of 5-HT on dopamine synthesis and storage

The serotonergic system is also enzymatically linked into the dopamine synthesis pathway. In the CNS, the 5-HT synthesizing enzyme, aromatic amino acid decarboxylase (AADC), is the same enzyme as dopa decarboxylase (DDC), which decarboxylates dihydroxyphenylalanine (i.e. dopa) to dopamine. This enzyme is widely distributed; it is found in the periphery as well as in the brain within and serotonin-containing neurons. Andrews and co-workers (1978) used striatal synaptosomes and noted that the presence of 5-HT seemed to be linked to dopamine synthesis. Synthesis of dopamine from L-tyrosine was significantly and dose- dependently inhibited due to inhibition of TH enzyme in striatal dopaminergic synaptosomes in the presence of 5-HT, whereas L-tyrosine uptake was not affected. 31

Furthermore, 5-HT inhibited in a concentration-dependent manner dopa decarboxylation with no effect on neuronal uptake of dopa (Andrews et al. 1978). It has been shown that L-3,4-dihydroxyphenylalanine (L-dopa) competitively decreases the uptake of L-tryptophan and this can reduce the synthesis of 5-HT. After peripheral L-dopa administration in rats, brain 5-HT turnover was accelerated, increasing the 5-hydroxyindoleacetic acid (5-HIAA) levels (Allikmets and Zharkovsky 1978). However, after simultaneous administration of L-tryptophan and L-dopa, the increase in dopamine levels was less marked, pointing to the ability of serotonergic neurons to convert L-dopa to dopamine (Allikmets and Zharkovsky 1978). Moreover, the exogenous L-dopa has been observed to reduce the hydroxylation of L-tryptophan in microdialysis study in rats (Hashiguti et al. 1993). The authors suggested that an increase in L-dopa levels could interfere with the transport of L-tryptophan via blood- brain barrier and/or the uptake by 5-HT neurons, and thereby decrease the L-tryptophan levels. Instead, 5-hydroxytryptophan administration decreased the hydroxylation of L- tyrosine by TH in rats (Hashiguti et al. 1993). The effects of 5-HT on dopaminergic neurotransmission have been examined in several studies by blocking 5-HT synthesis with a tryptophan hydroxylase (TrH) enzyme inhibitor, p-chlorophenylalanine (PCPA). While PCPA significantly depletes brain 5-HT and 5-HIAA, PCPA has also been shown to increase striatal dopamine levels and the turnover rate of dopamine (Fuenmayor and Bermudez 1985). A direct or indirect effect of PCPA on tyrosine hydroxylation has been postulated (Waldmeier 1980). The inhibition of 5-HT synthesis has been reported to decrease dopa accumulation in striatum which probably reflects decreased activity of TH (Persson and Johansson 1978). However, PCPA reduces the endogenous levels of tyrosine indicating that PCPA may also affect the transport of the amino-acid from plasma to brain since the plasma levels of tyrosine were unaffected (Tagliamonte et al. 1973). The changes in dopaminergic function after PCPA administration may also be secondary to a functional imbalance between the serotonergic and dopaminergic systems (Tagliamonte et al. 1973). It appears that serotonergic neurons are also involved in the storage of dopamine synthesized from exogenous L-dopa in the CNS. In rats, after administration of L-dopa with the DDC inhibitor carbidopa, dopamine could be detected in the cell bodies of raphe nuclei as well as in striatal and cerebral serotonergic cell fibres. This effect was not seen without exogenous L-dopa (Arai et al. 1995).

2.3.1.3 Indirect serotonergic innervation to dopaminergic neurons via GABAergic innervation

There is a well-known inhibition of dopaminergic function mediated by GABAergic neurons in the CNS. The descending inhibitory striatonigral GABAergic 32

pathway arises from the caudate-putamen innervating globus pallidus, SN and VTA (Oertel et al. 1982; Fallon and Loughlin 1985; Grace and Bunney 1985). Additional parallel GABAergic pathways project from the nucleus accumbens to the VTA (Fallon and Loughlin 1985). Ventrotegmental GABAergic neurons send collaterals that synapse locally on dopaminergic neurons in the VTA as well as projections terminating in the prefrontal cortex (Carr and Sesack 2000a) and nucleus accumbens (Van Bockstaele and Pickel 1995). GABAergic neurons participate in the regulation of dopaminergic activity via their local axon collaterals and by regulation of the activity of dopaminergic terminal areas through their projecting axons (Van Bockstaele and Pickel 1995; Carr and Sesack 2000a). In addition to direct innervation to dopaminergic areas, serotonergic system can control dopaminergic output by GABAergic projection neurons (Moukhles et al. 1997) or via 5-HT heteroreceptors expressing on GABAergic interneurons (Stanford and Lacey 1996). The activation of somadendritic or presynaptic 5-HT receptors causes inhibition of GABAergic neurons and further disinhibition of dopamine release (Pehek et al. 2001). 5-HT receptors may also increase the activity of GABAergic neurons resulting in inhibition of dopamine release (Bubar and Cunningham 2007). It seems that several 5-HT receptor subtypes (at least 5-HT1A, 5-HT1B, 5-HT2A and 5-HT2C) are involved in the regulation of dopaminergic activity in brain through the GABAergic system (Figures 3 and 4). These are further discussed in a section 2.3.2.

Figure 3. Schematic model of serotonergic modulation of other types of neurons. The presynaptic heteroreceptor is activated by 5-HT released from neuron I (white). This receptor is located on the nonserotonergic neuron II (grey), from which the respective neurotransmitter is released to act on neuron III (black). 5-HT may inhibit, facilitate, or stimulate neurotransmitter release from neuron II. Modified from Fink and Göthert (2007). 33

2.3.2 Serotonergic and dopaminergic interactions at the receptor level

2.3.2.1 The mechanisms of receptor-interactions

At the receptor level, there are at least two different types of mechanisms by which a serotonergic neuron can affect other neurons. 5-HT can produce direct effects via presynaptic heteroreceptors (Göthert 1990), and indirect effects via 5-HT receptors on interneurons (Stanford and Lacey 1996). The serotonergic neuron is able to release 5- HT from the axo-axonal synapse which can directly bind into the 5-HT receptor located on the axon of a non-serotonergic neuron. This presynaptic 5-HT receptor belongs to a category of heteroreceptors, which by definition are stimulated by neurotransmitters other than those released by the host neuron. Thus, 5-HT may inhibit, facilitate or stimulate the neurotransmitter release from this host neuron. The terminal of the second neuron forms synapses with the third neuron etc. (Figure 3) (see Göthert 1990; Fink and Göthert 2007). Presynaptic 5-HT heteroreceptors have a role in the local fine regulation of the neurotransmitter release from the axon terminal of host neuron. In response to action potentials invading the axonal varicosities, these heteroreceptors are able to either facilitate or inhibit the transmitter release. On the other hand, those 5-HT receptors that are located in the somadendritic region, can modify the function of the whole neuron with all axonal branches (see Göthert 1990; Fink and Göthert 2007).

Various subtypes of 5-HT receptors (particularly 5-HT1, 5-HT2, and 5-HT3) have been shown to be involved in the modulation of dopamine release (e.g. Chen et al.

1991b; Lucas and Spampinato 2000; Riad et al. 2000). Both 5-HT1A and 5-HT1B receptors seem to have important roles as auto- and heteroreceptors, controlling the release of 5-HT as well as of other neurotransmitters in brain (Riad et al. 2000). Furthermore, the results of several electrophysiological and neurochemical studies point to the significant involvement of brain serotonergic system via 5-HT2 receptors on dopaminergic function in several brain areas. In particular, 5-HT2 receptors have been shown to be involved in the inhibitory control of 5-HT on dopaminergic activity (Di

Giovanni et al. 1999). However, there are differences between 5-HT2 receptor subtypes in their actions on dopaminergic neurotransmission. 5-HT2A and 5-HT2C receptors seem to have opposite actions on central dopaminergic function; 5-HT2C receptors appear to exert effects in basal conditions whereas 5-HT2A receptors intervene only when dopaminergic neurons are activated (Lucas and Spampinato 2000; Porras et al. 2002).

There is also evidence that 5-HT3 receptors have a role in regulating dopaminergic transmission in the CNS (Jiang et al. 1990). In addition to 5-HT receptor mediated dopamine release, there is evidence that 5-

HT and some 5-HT3 receptor agonists are able to mediate dopamine outflow through DAT (Yi et al. 1991; Jacocks and Cox 1992; Zazpe et al. 1994). Furthermore, the serotonergic system can modulate dopaminergic transmission via 5-HT heteroreceptors 34

present on GABAergic interneurons (Stanford and Lacey 1996). The activation of somadendritic or presynaptic 5-HT receptors causes inhibition of GABAergic neurons and further disinhibition of dopamine release (Pehek et al. 2001; see Fink and Göthert 2007). 5-HT receptors may also increase the activity of GABAergic neurons, leading to further inhibition of dopamine release (Bubar and Cunningham 2007). It seems that several 5-HT receptor subtypes (at least 5-HT1A, 5-HT1B, 5-HT2A and 5-HT2C) are involved in the regulation of dopaminergic activity in brain via the GABAergic system (Figure 4). These are further discussed in section 2.3.2.2.

Figure 4. Schematic model that describes the role of GABAergic neurons (light grey) in the modulation of neurotransmitter release from dopaminergic neuron II. Neuron II subsequently releases its neurotransmitter upon a neuron III (black). 5-HT acts via somadendritic or presynaptic heteroreceptors on the GABAergic interneurons that innervate either the somadedritic or terminal region of the neuron II. 5- HT may inhibit, facilitate, or stimulate GABA release from the interneuron, thus facilitating or inhibiting neurotransmitter release from neuron II. The effect of 5-HT on neuron II is modulated by the inhibitory GABAergic interneuron. Modified from Fink and Göthert (2007). 35

2.3.2.2 Modulation via 5-HT1, 5-HT2 and 5-HT3 receptor subtypes

5-HT1A receptors

Somadendritic 5-HT1A receptors, both auto- and heteroreceptors, are ideally situated to mediate the effects of 5-HT on neuronal firing (Riad et al. 2000). They are densely located in raphe nuclei as well as in cortical and limbic areas (Pazos and Palacios 1985).

In the rat VTA, systemic but not local administration of the selective 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) evoked a significant excitatory effect on the basal activity of dopaminergic neurons (Prisco et al. 1994). Furthermore, selective lesions of 5-HT neurons by the neurotoxin 5,7- dihydroxytryptamine (5,7-DHT) abolished completely the excitatory effect of 8-OH-

DPAT on dopamine neurons. The authors concluded that the excitatory effect of 5-HT1A receptor stimulation was probably indirect since local administration had no effect, and VTA dopaminergic neurons are probably under a tonic inhibitory influence from 5-HT terminals originating in the raphe nuclei (Prisco et al. 1994). A 5-HT1A agonist- mediated increase in dopaminergic output in VTA dopaminergic neurons after systemic administration of these ligands was detected also in another study by Lejeune and Millan (1998).

5-HT1A receptor agonists have been shown to increase dopamine levels in the target areas of mesolimbic dopamine neurons; a selective agonist MKC-242 and a partial agonist buspirone has been shown to induce an increase in extracellular dopamine levels in hippocampus (Sakaue et al. 2000) and in nucleus accumbens (Gobert et al. 1999), respectively.

A 5-HT1A receptor-mediated stimulation of dopamine release in prefrontal cortex has been demonstrated in several studies (Rollema et al. 1997; 2000; Sakaue et al. 2000; Díaz-Mataix et al. 2005). Sakaue et al. (2000) have shown that administration of either systemic 5-HT1A agonist of partial agonist (MKC-242 and buspirone, respectively) could increase cortical extracellular dopamine levels. The activation of 5-HT1A receptors by systemic subtype selective 5-HT1A agonist BAYx3702 has been demonstrated to enhance the activity of VTA dopaminergic neurons and subsequently mesocortical dopamine release (Díaz-Mataix et al. 2005). In addition, the local administration of BAYx3702 into frontal cortex also increased the local extracellular dopamine concentration in normal rat and mouse, but not in 5-HT1A receptor knock-out mice (Díaz-Mataix et al. 2005). 5-HT1A receptor-mediated cortical dopamine release seems to be a typical property of several atypical antipsychotics (Rollema et al. 1997; 2000). In the SN, the systemic administration of 8-OH-DPAT has been found to stimulate the firing rate of dopaminergic neurons in the rat (Kelland et al. 1990). However, the 36

results concerning the effects of 5-HT1A receptor agonists on dopamine release in striatum are inconsistent. The in vitro results of Sarhan et al. (1999) described that 8- OH-DPAT did not have any effect on the [3H]-dopamine overflow from striatal synaptosomes. This is in parallel with in vivo results that the selective agonist MKC-242 as well as buspirone have failed to alter striatal dopamine release after their systemic administration (Gobert et al. 1999; Sakaue et al. 2000). However, Ng et al. (1999) reported that intrastriatal administration of the 5-HT1 agonist 5-carboxamidotryptamine dose-dependently increased extracellular dopamine levels, which was partially, but not fully, abolished by a selective 5-HT1A antagonist, 5-HT1A.

5-HT1B receptors

Since 5-HT1 receptors inhibit the neuronal firing (Bockaert et al. 2006), it is plausible that the activation of somadendritic or presynaptic 5-HT1A or 5-HT1B receptors on inhibitory GABAergic interneurons leads to stimulation of dopamine release. In particular, 5-HT1B receptors have been shown to mediate GABAergic inhibition in the

CNS. 5-HT1B agonism inhibited GABA release from rat VTA slices in vitro, whereas 5-

HT1A receptor stimulation or blockade had no effect (Yan and Yan 2001b). Furthermore, intracellular recording studies performed by Johnson and colleagues (1992) in SN and VTA midbrain slices demonstrated the 5-HT-induced activation of 5-

HT1B receptors and further inhibition of GABA release i.e. loss of GABAB receptor subtype tone. Local infusion of 5-HT1B receptor agonist CP93129 into the rat VTA significantly decreased VTA levels of GABA with a concomitant increase in dopamine release in both in VTA and nucleus accumbens, reflecting increased dopaminergic activity in the mesolimbic pathway (Yan and Yan 2001a). These effects were blocked by the 5-HT1B antagonist SB216641 (Yan and Yan 2001a). Accumbal dopamine release can also be facilitated by 5-HT1B agonism under stimulated conditions; cocaine-induced dopamine release was further elevated after intra-VTA administration of the 5-HT1B agonist, CP93129, with concomitant ventrotegmental GABA release (O'Dell and Parsons 2004). With respect to the other dopaminergic terminal areas, increased striatal extracellular dopamine levels have been detected after local infusion of the 5-HT1B receptor agonist, anpirtoline, into VTA (Ng et al. 1999). In frontal cortex, locally administered 5-HT1B agonist significantly increased cortical dopamine levels, which was effectively blocked by the 5-HT1B/D antagonist GR127935 (Iyer and Bradberry

1996). 5-HT1B receptors are also localized as presynaptic heteroreceptors on the dopaminergic axon terminals. They have been shown to participate in the inhibition of K+ -induced release of dopamine from rodent striatal synaptosomes in several in vitro experiments (Sarhan et al. 1999; Sarhan and Fillion 1999; Sarhan et al. 2000), suggesting that 5-HT1B heteroreceptors directly modulate dopamine release in striatum. 37

5-HT2A receptors

The systemic administration of 5-HT2A receptor antagonists (e.g. MDL100907, SR46349B) did not affect basal extracellular dopamine levels either in nucleus accumbens, striatum or frontal cortex (De Deurwaerdère and Spampinato 1999; Gobert et al. 2000; Porras et al. 2002). Several studies have shown, however, that 5-HT2A receptors can alter dopamine release under stimulated conditions. Local cortical 5-HT2A antagonism seems to have an inhibitory effect on stimulated dopamine release. Under activated conditions, cortical 5-HT2A receptors facilitate the release of frontocortical + dopamine. K -stimulated or 5-HT2A agonist DOI -induced dopamine increase was attenuated by local administration of a highly selective 5-HT2A antagonist MDL100907 into medial prefrontal cortex (Gobert and Millan 1999; Pehek et al. 2001; Pehek et al. 2006). MDL100907 attenuated the frontocortical release of dopamine also when 5-HT transporters were blocked by systemic administration of . The blockade of transporter function results in an increase in the synaptic concentration of 5-HT which in turn leads to the increased brain serotonergic activity (Zhang et al. 2000). Brain 5-HT may further stimulate dopamine release and thus increase the dopaminergic signal via a

5-HT2A receptor-related mechanism (Pehek et al. 2001). Pehek and colleagues (2006) have suggested that cortical 5-HT2A receptors do not generally modulate basal release in frontal cortex since local administration of 5-HT2A antagonists has no effect on cortical basal dopamine levels.

5-HT2A receptors have been shown to have facilitating effects on dopamine transmission under stimulated conditions also in striatum and nucleus accumbens. In nucleus accumbens, systemic administration of the 5-HT2A antagonist SR46349B significantly reduced the excitatory effect of dorsal raphe stimulation on dopamine release (De Deurwaerdère and Spampinato 1999). -induced dopamine release in nucleus accumbens and striatum was significantly reduced by SR46349B

(Porras et al. 2002) and enhanced by the 5-HT2A receptor agonist DOI without any effect on basal dopamine release in these areas (Ichikawa and Meltzer 1995).

5-HT2B and 5-HT2C receptors

On the basis of several electrophysiological and microdialysis studies, it seems that the central serotonergic system exerts tonic inhibitory control of both mesolimbic and nigrostriatal dopaminergic pathway activity, particularly via the 5-HT2C receptor subtype (Soubrié et al. 1984; De Deurwaerdère and Spampinato 1999; Di Giovanni et al. 1999; Porras et al. 2002; Berg et al. 2006). Peripheral administration of the 5-HT2C/2B antagonist SB206553 dose-dependently increased the basal firing rate of dopaminergic neurons arising from VTA and SN with a concomitant increase in basal dopamine release in both the nucleus accumbens and the striatum (Di Giovanni et al. 1999; Gobert 38

et al. 2000; Porras et al. 2002). The dopamine levels in nucleus accumbens and striatum and the firing rate of VTA dopaminergic neurons were likewise reduced by treatment with the preferential 5-HT2C receptor agonist, Ro60-0175, whereas this inhibitory action was blocked by the 5-HT2C antagonist, SB242084 (Di Matteo et al. 2000a; 2000b).

While the levels of 5-HT2B receptors in these brain areas are low in the rat (Duxon et al.

1997), and the selective 5-HT2B antagonist SB204741 failed to have any effect on striatal and accumbal dopamine levels (Gobert et al. 2000), the increase in dopamine levels in these areas may be mediated via 5-HT2C receptors. More recently, Berg et al.

(2006) have demonstrated a 5-HT2C -induced dopamine release in rat nucleus accumbens. The results of microdialysis study by Pozzi et al. (2002) indicated that endogenous

5-HT exerts only a low tone on 5-HT2C receptors in frontal cortex under basal conditions, whereas the stimulation of these receptors by Ro60-0175 inhibits stress- induced dopamine release. However, preferential 5-HT2C agonists (such as Ro60-0175 and SB206553) and selective 5-HT2C antagonist SB242084 were able to inhibit and enhance the cortical dopamine levels, respectively (Millan et al. 1998; Gobert et al. 2000; Pozzi et al. 2002). This indicates that the mesocortical dopaminergic pathway may well be under the inhibitory control of 5-HT2C receptors (Millan et al. 1998).

The sites of action of 5-HT2A/2B/2C receptors

5-HT2A receptors are located presynaptically on the dopaminergic nerve terminals and appear to facilitate dopamine release, particularly when dopaminergic activity is enhanced. The 5-HT2A receptors are present on dopaminergic and non-dopaminergic neurons in the VTA area (Nocjar et al. 2002) as well as in striatum (Pazos et al. 1985). In prefrontal cortex, they are located on the apical dendrites of the pyramidal cells as well as on the GABAergic interneurons (Willins et al. 1997; Santana et al. 2004). 5-

HT2A receptors located on the GABAergic interneurons are believed to be able to regulate the neuronal activity and subsequent dopamine release (Pehek et al. 2001).

In comparison to the effects of 5-HT2A receptors, 5-HT2C receptors seem to exert a tonic, suppressive effect on the activity of mesocortical as well as mesolimbic and nigrostriatal dopaminergic pathways, this being likely mediated by an indirect effect at the level of dopaminergic cell bodies (Gobert et al. 2000; Di Matteo et al. 2001; Alex et al. 2005). On the other hand, 5-HT2B receptors are not expressed in many areas of a rat brain (Duxon et al. 1997), and 5-HT2B receptors do not seem to have major effects on dopaminergic neurotransmission (Gobert et al. 2000).

5-HT2C receptors are found basically in all brain regions containing dopaminergic cell bodies and terminals of mesocorticolimbic system (Molineaux et al. 1989; Eberle- Wang et al. 1997; Clemett et al. 2000), and thus they are able to modulate dopaminergic neurotransmission both at the level of the cell bodies and the nerve terminals. The exact 39

sites where 5-HT2C -type receptors mediate the regulation of dopaminergic neurotransmission, are not known (see Fink and Göthert 2007). 5-HT2C receptor- mediated GABAergic inhibition of dopaminergic system has been examined in several studies. 5-HT2C receptors localized on the GABAergic (inter)neurons likely activate GABAergic neurotransmission (Di Matteo et al. 2001; Giorgetti and Tecott 2004). The mRNA as well as the 5-HT2C receptor protein have been detected in both dopaminergic and GABAergic neuron subpopulations in the rat VTA and SN (Eberle-Wang et al. 1997; Clemett et al. 2000; Bubar and Cunningham 2007). Brain 5-HT may directly stimulate GABAergic neurons by activation of 5-HT2C receptors (Stanford and Lacey 1996). Furthermore, they may evoke GABAergic inhibition of dopaminergic neurons by being constitutively active (i.e. the capacity to regulate cellular signaling systems in the absence of occupancy by a ligand) (Berg et al. 2005).

5-HT2C receptors located on GABAergic neurons seem to be the main site of action for the inhibition of dopaminergic neurotransmission (Bubar and Cunningham 2007) rather than the 5-HT2C receptors localized on the dopaminergic neurons (Giorgetti and Tecott 2004). The electrophysiological in vivo studies of Prisco et al. (1994) and Di Giovanni et al. (2001) have indicated that the microiontophoretic application of the nonselective 5-HT2C agonist m-chloro- into the VTA induced an activation of the non-dopaminergic (presumably GABAergic) neurons both in the VTA and SN, and decreased the firing rate of ventrotegmental dopaminergic neurons.

In contrast, the local intrastriatal administration of the 5-HT2B/2C antagonist SB206553 has been shown to inhibit dopamine release (Lucas and Spampinato 2000; Alex et al. 2005). This points to a direct tonic control of striatal dopamine release by 5-

HT2C receptors (Lucas and Spampinato 2000), which are likely located on the dopaminergic neurons themselves (Eberle-Wang et al. 1997; Clemett et al. 2000). Systemic administration of antagonist SB206553 resulted in increase of accumbal and striatal extracellular dopamine levels (Di Giovanni et al. 1999) implying that the 5-HT2C receptors may exert opposite effects on dopamine release depending their localization (Pozzi et al. 2002).

In general, the lack of fully selective 5-HT2C receptor ligands has been a problem in assessing the more specific functional role of this receptor subtype (Hoyer et al. 2002; see Fink and Göthert 2007). However, very recently a novel and highly selective 5-

HT2C agonist, was introduced (Smith et al. 2008; Thomsen et al. 2008). Currently, there are no studies which would have assessed the effects on lorcaserin on brain dopamine neurotransmission.

5-HT3 receptors

There is evidence that 5-HT3 receptors have a role in regulating dopaminergic transmission in the CNS. 5-HT3 receptors are likely expressed as presynaptic 5-HT3 40

heteroreceptors on the dopaminergic nerve terminals (Nayak et al. 1999). The stimulation of 5-HT3 receptors by 5-HT3 agonists has been demonstrated to enhance dopamine release both in vitro (Yi et al. 1991; Jacocks and Cox 1992; Zazpe et al. 1994) and in vivo (Jiang et al. 1990; Chen et al. 1991b). The effects of 5-HT ligands on dopaminergic neurotransmission seem to vary in different brain areas. Central 5-HT3 receptors have been demonstrated to control the mesocorticolimbic dopaminergic pathway; administration of 5-HT3 agonists increase dopamine levels in vivo both in nucleus accumbens and prefrontal cortex (Jiang et al. 1990; Chen et al. 1991b). However, facilitatory control of endogenous 5-HT via an action on 5-HT3 receptors in this dopaminergic pathway apparently takes place only when both central dopaminergic and serotonergic tones are concomitantly increased (De

Deurwaerdère et al. 1998; 2005). The ability of 5-HT3 receptors to modulate striatal dopamine release has been a matter of debate. There are results supporting a facilitative role for 5-HT3 receptors on striatal dopamine release in striatal slices in vitro (Blandina et al. 1989) as well as in microdialysis studies with rats (Kankaanpää et al. 1996; Porras et al. 2003). But many other studies have concluded that the nigrostriatal dopaminergic system is probably insensitive to 5-HT3 receptor modulation (Jacocks and Cox 1992;

Zazpe et al. 1994; Bonhomme et al. 1995; De Deurwaerdère et al. 1998). 5-HT3 receptors are also found in GABA-containing interneurons, where they may enhance the GABA-mediated inhibitory processes, such as dopamine release (Bloom and Morales

1998). Thus, the involvement of 5-HT3 receptors in the modulation of nigrostriatal dopaminergic tone remains to be clarified.

The mechanisms underlying the 5-HT3 mediated dopamine release are not fully understood. There are suggestions that 5-HT3 related dopamine transmission is not totally mediated through 5-HT3 receptors; carrier-mediated mechanisms may also be involved (Yi et al. 1991; Jacocks and Cox 1992; Zazpe et al. 1994). Some studies have indicated that the blockade of the dopamine transporter by selective DAT inhibitors but not by the administration of 5-HT3 antagonists could inhibit 5-HT or 5-HT3 agonist- induced dopamine release (Jacocks and Cox 1992; Santiago et al. 1995). Thus, DAT may have a crucial role in 5-HT3 receptor mediated dopamine release.

2.3.2.3 Modulation via transporters

Dopamine transporter

It has been demonstrated in several studies that 5-HT is able to modulate dopamine release. In vitro, perfusion of rat striatal slices with 5-HT increased the basal release of endogenous dopamine (Blandina et al. 1989) as well as [3H]-dopamine from striatal synaptosomes (Yi et al. 1991). Local administration of 5-HT in vivo also elevated 41

dopamine levels in nucleus accumbens (Parsons and Justice 1993) and striatum (Yadid et al. 1994; Bonhomme et al. 1995; De Deurwaerdère et al. 1996). There may also be a direct involvement of dopamine uptake sites in the 5-HT stimulated dopamine release (Yi et al. 1991; De Deurwaerdère et al. 1996). 5-HT seems to be able to penetrate into dopaminergic terminals and once there to increase extracellular dopamine content by displacing dopamine from its vesicular storage pools (Andrews et al. 1978). Furthermore, dopamine uptake blockers such as nomifensine have been shown to suppress the effect of 5-HT on dopamine release in vitro (Yi et al. 1991) a finding replicated in in vivo microdialysis studies (De Deurwaerdère et al. 1996). This points to the involvement of a carrier mediated process in the effect of 5-HT on striatal dopamine outflow in vivo. After being transported to the synaptic cleft, 5-HT may evoke increased efflux by dopamine transported in a reversed direction via DAT (Yi et al. 1991; De Deurwaerdère et al. 1996).

5-HT transporter

Selective serotonin reuptake inhibitors (SSRI) that block SERT have been described to affect dopaminergic transmission in a variety of ways. In addition to the increase of 5-HT levels, local administration of 5-HT uptake blockers, alaproclate and fluoxetine, increased extracellular dopamine levels in striatum (Yadid et al. 1994; Matsumoto et al. 1999). Yadid et al. (1994) concluded that the DAT did not seem to be involved in this effect, because the specific dopamine uptake blocker GBR-12909 was not able to suppress the increased dopamine efflux. Furthermore, cumulative amounts of extracellular striatal dopamine derived from exogenous L-dopa have been found in the 6-OHDA-lesioned rat in the presence of SSRI-inhibition by intrastriatal administration of fluoxetine, suggesting a role of SERT in dopamine uptake (Kannari et al. 2006). Thus, the exogenous L-dopa-derived dopamine in the striatum could be taken up and then act as a false neurotransmitter instead of 5-HT. It was postulated that the uptake of dopamine by SERT may have a significant role in the clearance of extracellular dopamine in severely dopamine denervated striatum (Kannari et al. 2006). While PCPA does not directly interact with SERT, brain 5-HT depletion by PCPA leads to changes in SERT gene expression in raphe nuclei (Yu et al. 1995). Significant elevations in SERT mRNA but not the transporter protein have been detected in dorsal raphe nucleus one week after PCPA administration (Rattray et al. 1996). In studies by Rattray et al. (1996), [3H]-citalopram binding which reflects the levels of SERT, were decreased by 10 – 20% in striatum and frontal cortex at two weeks (but not at one week) after PCPA administration. However, while cortical [3H]-5-HT uptake was concomitantly decreased by 20%, striatal [3H]-5-HT uptake was not affected. This was interpreted to indicate that striatum exhibits only a low sensitivity to abovementioned effects of 5-HT depletion by PCPA (Rattray et al. 1996). 42

After peripheral administration, the effects of SSRIs on the dopaminergic system seem to be region-specific. While increased extracellular dopamine levels have been detected in frontal cortex after subcutaneous (s.c.) administration of fluoxetine (Zhang et al. 2000), a dose dependent inhibition of the firing rate of dopamine-containing neurons has been observed in VTA (Di Mascio et al. 1998) but not in SN (Prisco and Esposito 1995) after administration of SSRIs. The fluoxetine-induced inhibition of the ventrotegmental dopamine neuron firing rate appeared to be transient, since after three- week chronic treatment, the additional acute fluoxetine exposure no longer evoked any ventrotegmental dopaminergic neuron inhibition. Certain subtypes of 5-HT receptors are likely involved, whereas pretreatment with 5-HT2C/2B receptor antagonist blocked the acute inhibitory effect of fluoxetine (Prisco and Esposito 1995). Prisco and Esposito (1995) proposed that the inhibitory effect of SSRIs on the ventrotegmental dopaminergic neuron firing rate is not directly mediated via SERT, but that 5-HT2C receptors are concomitantly involved. Thus, one could hypothetize that SSRIs increase the 5-HT concentrations, which subsequently lead to inhibition of dopaminergic neuron activity via activation of 5-HT2C receptors. During chronic SSRI treatment which evoked prolonged elevated synaptic 5-HT concentrations, 5-HT2C receptors probably became downregulated, resulting in a diminished effect on dopaminergic neuron activity.

2.4 Modulation of brain serotonergic transmission by the dopaminergic system

Dorsal raphe serotonergic neurons modulate dopaminergic neurotransmission in the CNS. However, less is known about possible dopaminergic regulation of the serotonergic neurons despite morphological evidence of reciprocal projections between dopaminergic cell body areas (SN, VTA) and dorsal raphe nuclei (Moore and Bloom

1978; van der Kooy and Hattori 1980; Hervé et al. 1987) and the location of D2-like receptors in dorsal raphe (Bouthenet et al. 1987). There is some evidence that dopamine/5-HT interactions may be physiologically relevant for the control of ascending serotonergic pathways (Hery et al. 1980; Ferré et al. 1994). Administration of amphetamine into dorsal raphe increases local extracellular dopamine and 5-HT levels (Ferré et al. 1994), and local administration of the dopamine agonist elevates the raphe 5-HT concentration (Ferré and Artigas 1993). Systemic administration of apomorphine increases the extracellular 5-HT in dorsal raphe with a concomitant decrease in striatal dopamine. This decrease was prevented by treatment with the 5-HT1A antagonist, evidence of increased stimulation of somadendritic raphe 5-HT1A autoreceptors. It has been postulated that there is an inhibitory dopaminergic control of ascending serotonergic pathways, which is mediated through the elevated extracellular 5-HT concentrations at the somadendritic level in 43

dorsal raphe nucleus (Ferré and Artigas 1993; Ferré et al. 1994). The DAT-related mechanisms may also be involved in the steady state concentration of striatal 5-HT and 5-HIAA. In normal rats, subchronic peripheral treatment with a selective dopamine uptake blocker decreased both striatal dopamine and 5-HT levels with a concomitant increase in 5-HIAA concentrations (Sivam 1995). Furthermore, the results of Callaghan et al. (2005) indicated that the increased striatal 5-HT clearance rate was due to significant 5-HT uptake through DAT, since this effect could be blocked by local administration of a DAT inhibitor. Intranigrally administered L-dopa has been shown to increase nigral 5-HT levels, suggesting that dopamine receptors may regulate nigral 5-HT release either directly on nigral 5-HT terminals or indirectly via other nigral inhibitory mechanisms, such as GABA (Thorré et al. 1998). Furthermore, Thorré and colleagues (1998) have proposed that dopamine released from dendrites may take part in the transfer of information to serotonergic systems innervating basal ganglia.

2.5 Dopaminergic and serotonergic changes in most common neurotoxin- induced animal models of Parkinson’s disease

2.5.1 6-OHDA

A selective catecholaminergic neurotoxin 6-OHDA is one of the most common used to experimentally model nigral dopaminergic degeneration in vivo. In rodents, 6-OHDA is injected unilaterally into striatum, SN or the ascending MFB destroying nigral dopaminergic neurons and dopaminergic terminals in the striatum, leading to significant depletion of ipsilateral striatal dopamine (Kumar et al. 1995; Kirik et al. 1998; Blum et al. 2001). 6-OHDA is taken via DAT into dopaminergic terminals where it can induce oxidative stress, the generation of free radicals and inhibition of mitochondrial function leading to cell death (Zigmond et al. 1992; Kumar et al. 1995; Choi et al. 1999). 6-OHDA-infusion into the MFB produces almost complete depletion of striatal dopamine and its metabolites in the lesioned side, whereas striatal 6-OHDA- lesion decreases dopamine levels to a lesser extent, by about 50% (Yuan et al. 2005). The effects of 6-OHDA-lesions on the serotonergic system in adult rats are controversial; 5-HT levels have either remained unchanged (Erinoff and Snodgrass 1986; Tanaka et al. 1999) or declined from 30 to 50% (Abrous et al. 1990; Karstaedt et al. 1994). Furthermore, regional differences in the patterns of 5-HT innervation have been reported after unilateral 6-OHDA-lesion in rats (Takeuchi et al. 1991; Zhou et al. 1991) (see section 6.2). 44

2.5.2 MPTP

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) produces in humans an acute parkinsonian syndrome that is virtually indistinguishable from idiopathic PD (Langston et al. 1983; Ballard et al. 1985). Indeed, this toxin is widely used to model PD in non-human primates and mice (Albanese et al. 1993; Taylor et al. 1997; Przedborski and Vila 2003). MPTP crosses the blood-brain barrier and is converted mainly in glial cells by monoamine oxidase B into its effective form, 1-methyl-4- phenylpyridinium ion (MPP+) (Blum et al. 2001). MPP+ is a substrate for DAT and therefore easily accumulates preferentially in dopaminergic cells by selective uptake. In cells, MPP+ inhibits the activity of mitochondria leading to cell death through a drop in cellular adenosine triphosphate (ATP) levels and formation of reactive oxygen species (ROS) (Blum et al. 2001). MPTP treatment leads to 80 – 90% bilateral striatal dopamine depletion and a decline in striatal dopamine metabolites in mice (Mayer et al. 1986; Fukuda et al. 1988). Furthermore, striatal TH activity decreases by >90% (Matsuda et al. 1986; Mayer et al. 1986). There also indications that MPTP affects the brain serotonergic system. In mice, striatal 5-HT levels were reduced (by 25%) after one-week MPTP treatment (Hara et al. 1987; Fukuda et al. 1988). Moreover, the density of 5-HT immunoreactive-like nerve fibers was markedly reduced in the striatum of MPTP-treated mice (Fukuda et al. 1988). MPTP has also been observed to elevate striatal TrH for at least one month after the last injection, along with an increase in 5-HIAA in mice (Fukuda et al. 1988). It seems that the MPTP-induced changes in serotonergic system are transient since the declined 5-HT levels had recovered to normal within three months (Fukuda et al. 1988). Interestingly, Rozas et al. (1998) have observed a marked bilateral 5-HT hyperinnervation in striatum more than three months after MPTP lesion resembling the 5-HT neuron sprouting reported in 6-OHDA-lesioned rats (e.g. Zhou et al. 1991). This may well indicate a compensatory mechanism through serotonergic system to counteract striatal 5-HT and even dopamine loss.

2.5.3 Rotenone

The common pesticide, rotenone, appears to reproduce many characteristics of PD in rats, including selective nigrostriatal dopaminergic neuron degeneration and motor deficits (Sherer et al. 2003). Like MPTP, it inhibits mitochondrial function and induces the formation of ROS leading to dopaminergic neurodegeneration. In contrast to MPTP, rotenone also facilitates the formation ubiquitin and Į-synuclein-positive cytoplasmic inclusions (Betarbet et al. 2000). These are also present in Lewy bodies, cytoplasmic fibrillar inclusions found in brains of PD patients (Baba et al. 1998). 45

In rats, striatal dopamine has been decreased by 50%, its metabolites by >90% (Alam and Schmidt 2002) and striatal TH by >50% (Alam and Schmidt 2002; Höglinger et al. 2003) after chronic (•4 weeks) peripheral rotenone treatment. It is not clear if rotenone induces additional serotonergic damage. After intraperitoneal administration in rats, there were no changes in striatal 5-HT (Alam and Schmidt 2002), but a decrease (by 34%) in striatal 5-HT fibers was observed (Höglinger et al. 2003). Even after intranigral infusion of rotenone, only high doses decreased (by 40%) ipsilateral striatal 5-HT levels but failed to affect on 5-HT levels in striatum or SN when infused into dorsal raphe (Saravanan et al. 2005). However, it has been demonstrated recently that rotenone is able to induce dose-dependent and selective serotonergic cell death in midbrain neuronal cell cultures (Ren and Feng 2007).

2.6 Serotonin/dopamine interactions in some disease states in the CNS

2.6.1 General

The interactions between the dopaminergic and serotonergic systems in the CNS seem to play a significant role in the pathogenesis and drug treatment of many CNS disease states. Thus understanding the mechanisms underlying the interactions between brain 5-HT and dopamine is important for several reasons. The etiology and progression of several CNS diseases may be related to alterations in the biochemical mechanisms that modulate neurotransmission, and many psychiatric and neurologic diseases seem to involve multiple deficits in neurotransmitter systems. Furthermore, many neurotransmitter-specific drugs, such as the atypical antipsychotics, may induce undesirable adverse-effects related to their interaction with multiple neurotransmitters. The therapeutic effects of these drugs are possibly related to their distinctive ability to alter several neurotransmitter pathways that are functionally linked to the target system (Dewey et al. 1995). Here, some clinically relevant applications of 5-HT/dopamine - interactions in three common brain disorders, PD, schizophrenia and depression, are discussed.

2.6.2 Parkinson’s disease

2.6.2.1 5-HT in Parkinson’s disease

PD is a progressive neurologic disorder characterized by a selective degeneration of nigrostriatal dopaminergic pathway originating from the SN causing severe striatal dopamine depletion. This evokes the typical motor deficits seen in PD patients: the resting tremor, rigidity, akinesia and bradykinesia (see Sian et al. 1999). The standard symptomatic drug treatment of PD is the replacement therapy with the dopamine 46

precursor, L-dopa, combined with a peripheral DDC inhibitor (Olanow 2004). It is generally concluded that parkinsonian symptoms do not appear until 70 – 80% of the nigral dopamine neurons and an equivalent percentage of striatal dopamine have been lost (Jellinger 1989). Nigrostriatal dopamine neuron loss leads to compensatory upregulation of striatal dopamine receptors (see Sian et al. 1999). Since a proportion of presynaptic D2 receptors are lost due to degeneration of the dopaminergic nerve terminals, the increase in striatal D2 receptors probably result mainly from upregulation of postsynaptic striatal

D2 receptors. Following antiparkinsonian drug treatment, the elevated striatal D2 receptors are downregulated to normal levels and often decreased levels (Table 1) while

D1 receptors usually remain unaltered or show modest increase (see Hurley and Jenner 2006). There is a substantial amount of clinical evidence that the brain serotonergic system is also affected in PD (Jellinger 1991; Fox and Brotchie 2000b; Haapaniemi et al. 2001; Kerenyi et al. 2003; Kish et al. 2008). The mean values of dorsal raphe cell loss in PD are reported to range from 20 to 40% (Jellinger 1991). Reduced striatal 5-HT and 5- HIAA levels (Scatton et al. 1983; Birkmayer and Birkmayer 1987; Kish et al. 2008), a decrease in TrH and a reduction in density of SERT have been observed in the striata of PD patients (Kerenyi et al. 2003; Kish et al. 2008) (Table 1). Reduced levels of all of these key markers for the serotonergic system support the view that striatal serotonergic abnormalities occur in PD (Kish et al. 2008). While serotonergic projections from the dorsal raphe nuclei innervate all components of the basal ganglia circuitry (Lavoie and Parent 1990), it is plausible that 5-HT plays a role in the regulation of movements by the basal ganglia (Jacobs and Fornal 1997; Scholtissen et al. 2006b). Furthermore, abnormalities in 5-HT transmission may participate in the neuronal mechanisms underlying both basal ganglia related disorders, such as PD, and the complications encountered in the treatment of these disorders, e.g. L-dopa-induced dyskinesia seen in PD (Nicholson and Brotchie 2002). Furthermore, 5-HT is known to influence cognitive symptoms (Schmitt et al. 2000). However, there are currently no drugs improving PD symptoms which act by directly affecting the brain serotonergic system in clinical use. 47

Table 1. Changes in dopamine and serotonin markers as well as D2, 5-HT1A and 5-HT2A,2C receptors in discrete brain regions in Parkinson’s disease. Striatum Substantia Frontal Raphe nuclei nigra cortex Dopamine a,b,c -80 – 98% > -90% -60 – 75% DOPAC a -90% HVA a,c -55 – 70% -40% TH d,e -20 – 50% > -90% DAT c,d,f,g -25 – 90% -50% 5-HT a,b,c -60 – 70% -55% -45% 5-HIAA a,c -30 – 40% -25% TrH c -30 – 60% SERT c,g,h -30 – 50% -10% i,j D2 - Untreated +15 – 30% - Treated d,k -25 – 40% -50% l,m 5-HT1A +35 – 45% -30% l 5-HT2A +25 – 35% n 5-HT2C +110% Values are percentage change vs. healthy normal subjects and calculated from data of concentrations of amines and their metabolites, enzyme protein levels, transporter sites and receptor binding capacities. Untreated refers to drug-naïve PD patients; Treated refers to PD patients chronically treated with antiparkinsonian drugs. Data from a Scatton et al. (1983); b Birkmayer and Birkmayer (1987); c Kish et al. (2008); d Murray et al. (1995); e Ryoo et al. (1998); f Nurmi et al. (2000); g Kerenyi et al. (2003); h Guttman et al. (2007); i Rinne et al. (1995); j Ichise et al. (1999); k Brooks et al. (1992); l Chen et al. (1998); m Doder et al. (2003); n Fox and Brotchie (2000).

Reduced 5-HT levels in the CNS are often associated with a risk for depression. Indeed, depression is one of the most common comorbid disorders seen in PD patients (Scholtissen et al. 2006b), occurring in approximately 45% of all PD patients (for review, see Lemke 2008). However, little is known about the pathophysiology of PD related depression. In some in vivo studies, 5-HT has shown an ability to inhibit striatal dopamine release, and thus a decrease in brain 5-HT levels has been considered as a secondary event in PD (Mayeux 1990; Hornykiewicz 1993; Jacobs and Fornal 1993). Accordingly, the serotonergic hypothesis of depression in PD regards the reduced serotonergic tone as an adaptation to the low striatal dopamine levels conferring a risk for depression (Mayeux et al. 1984; Mayeux 1990; Leentjens et al. 2006). Furthermore, 5-HT is known to influence cognitive symptoms (Schmitt et al. 2000). Interestingly, 48

both 5-HIAA and homovanillic acid (HVA) have reported to be decreased more in the brain of depressed PD patients than in the non-depressed PD patients (Mayeux et al. 1984). The VTA dopaminergic neurons are evidently also affected in PD (Jellinger 1991). The alternative hypothesis, the dopaminergic hypothesis for depression in PD, thus states that dopamine deficiency in mesolimbic and mesocortical dopaminergic system can lead to malfunction of self-reward system, constituting a potential risk for depression (see Lieberman 2006). Drug treatment of depression in PD may be difficult due to presumed multiple neurotransmitter deficits (Hanagasi and Emre 2005). Dopamine agonists, such as pramipexole and ropinirole, are useful in improving depressive symptoms in PD when given as adjunctive treatment or alone (Ostow 2002; Rektorová et al. 2003). In case of serotonergic agents, SSRIs are widely used to treat PD-related depression. Such treatment has been effective and has only rarely led to deterioration of motor symptoms of PD (Ceravolo et al. 2000; Tesei et al. 2000; Dell'Agnello et al. 2001; Weintraub et al. 2006).

2.6.2.2 5-HT receptors in Parkinson’s disease

5-HT1A receptors

In PD, the presynaptic 5-HT1A receptor binding in the raphe nuclei is decreased as demonstrated by Doder et al. (2003) (Table 1), and the authors suggested that this may be due to loss of raphal 5-HT cell bodies. On the contrary, postsynaptic 5-HT1A receptor levels in frontal cortex seem to be increased, probably representing receptor upregulation in response to 5-HT deficit (Chen et al. 1998). 5-HT1A receptor stimulation with 8-OH-DPAT or buspirone has been found to alleviate the fluctuation in striatal dopamine levels occurring after L-dopa treatment and to reduce L-dopa-induced dyskinesias (Tomiyama et al. 2005; Eskow et al. 2007), as well as enhancing motor activity (Eskow et al. 2007; Mignon and Wolf 2007) in a hemilesioned 6-OHDA-rat model of PD. Bezard et al. (2006) have demonstrated that 5-HT1A agonists prevent neuronal damage and thus delay the worsening of motor symptoms in experimental conditions. These findings are in line with the observations that the full 5-HT1A agonist (EMD128130), which also displays antagonist properties at dopamine D2 receptors, improves L-dopa -induced motor complications in rat and primate models of PD (Bibbiani et al. 2001), as well as preventing tardive dyskinesia in rodents (Rosengarten et al. 2006). On the basis of preclinical and phase II studies, sarizotan was hypothetized to be able to ease the dyskinesia in PD patients, and it was tested in double-blind trials as an adjunct therapy to L-dopa (Olanow et al. 2004; Bara-Jimenez et al. 2005; Goetz et al. 2007). The recent results indicate that sarizotan was able to decrease the Unified 49

Parkinson’s Disease Rating Scale (UPDRS) scores, as well as increasing on-time without dyskinesias during L-dopa-therapy (Goetz et al. 2007). However, due to lack of sufficient efficacy to attenuate dyskinesias in PD compared to placebo in human phase III studies, the further development of the compound as an antidyskinetic agent was discontinued (Merck KGaA, 2006).

5-HT2A/2C receptors

There are only a few reports about brain 5-HT2 receptor changes in PD. In the 6-OHDA rat model of PD, nigrostriatal cell loss induces an upregulation of 5-HT2A receptor mRNA, but a downregulation of 5-HT2C receptor mRNA in the striatum (Zhang et al.

2007). Chen et al. (1998) have found increased levels of 5-HT2A receptors in frontal cortex of PD patients, probably due to upregulation to compensate a decrease in 5-HT levels (Table 1). (ACP-103), a potent 5-HT2A and antagonist (Vanover et al. 2006), has been shown to attenuate L-dopa-induced dyskinesias in the monkey MPTP-model of PD, as well as to suppress parkinsonian-like tremor in rats (Vanover et al. 2008). Pimavanserin also possess antipsychotic effects in experimental rodent models (Gardell et al. 2007), and is currently in phase III development for treatment of PD related psychosis (ACADIA Pharmaceuticals Inc., 2008a).

In the 6-OHDA-rodent model of PD, 5-HT2C receptors play a significant role in modulating basal ganglia output. Blockade of these receptors results in increased locomotor behavior and enhanced the actions of dopamine replacement therapy (Fox et al. 1998; Fox and Brotchie 2000a). The 5-HT2C receptor binding in the SN is also found to be increased in post-mortem brains of PD patients suffering from L-dopa-induced dyskinesias. Thus, it has been claimed that there is a compensatory up-regulation of receptor levels in the response to a decreased stimulation of endogenous dopamine, and subsequent abnormalities of 5-HT2C mediated neurotransmission in basal ganglia (Fox and Brotchie 2000b). To date, there have been no further clinical studies examining the effects of 5-HT2C ligands on PD patients.

5-HT3 receptors

The 5-HT3 receptor antagonist, , seems to be useful in treating tardive dyskinesia as well as the psychotic symptoms in schizophrenic patients (Sirota et al. 2000; see below). Ondansetron has also markedly improved the psychotic symptoms, such as visual hallucinations and paranoid delusions, in an advanced stage of PD (Zoldan et al. 1995). Furthermore, this drug apparently does not have any antagonistic properties at dopamine receptors since it did not worsen the basic symptoms in PD, and it furthermore did not counteract the efficacy of L-dopa. Indeed, Zoldan et al. (1995) 50

suggested that the beneficial effect of ondansetron in PD related psychosis may be exclusively mediated via inhibition of 5-HT3 receptors. Thus, it could produce antipsychotic effects without inducing sedation or extrapyramidal symptoms (Sirota et al. 2000).

2.6.3 Schizophrenia

2.6.3.1 Dopamine and D2 receptors in schizophrenia

The classical dopamine hypothesis of schizophrenia proposes that hyperactivity of brain dopamine transmission induces the positive symptoms, such as delusions, hallucinations, and a marked disturbance of thought processess (Laruelle et al. 1999). The negative symptoms of schizophrenia include affective flattening, anhedonia, apathy and poverty of speech (Alex and Pehek 2007). Current evidence suggests that in schizophrenia there is excessive dopaminergic activity in mesostriatal and mesolimbic regions (Moghaddam and Bunney 1990; Meltzer 1999; Alex and Pehek 2007). Dysregulation of dopaminergic transmission seems to be pronounced during episodes of illness exacerbation in comparison to the periods of remission of the disease (Laruelle et al. 1999). The older (so-called typical) antipsychotics, such as and , affect the neurochemistry of the nigrostriatal and mesolimbic dopaminergic systems, whereas drugs seem to be more selective in the mesocorticolimbic dopaminergic system (Moghaddam and Bunney 1990; Nomikos et al. 1994). Hypofunction of the mesocortical dopaminergic pathway has been postulated to be related to the negative symptoms and cognitive deficits in schizophrenia (Weinberger 1987). The beneficial effects of atypical antipsychotics in schizophrenia may be associated with their ability to affect dopaminergic neurons and to elevate baseline dopaminergic tone in frontal cortex. Indeed, most of the atypical antipsychotics preferentially increase mesocortical dopamine levels, but show less effects on striatal and accumbal dopamine levels (Moghaddam and Bunney 1990; Chen et al. 1991a; Chen et al. 1992; Alex and Pehek 2007).

Increased D2 receptor stimulation can be detected both in neuroleptic-naive patients as well as in previously treated chronic patients experiencing an episode of illness exacerbation (Abi-Dargham et al. 2000). Hyperactivity of the dopaminergic system as a result of increased D2 receptor -mediated transmission has been proposed to underlie the positive symptoms because these symptoms respond better to D2 receptor blockade than do the negative or cognitive symptoms (Joyce 1993; Laruelle et al. 1999; Abi-Dargham et al. 2000). The ability of antipsychotic drugs to alleviate the positive symptoms of schizophrenia is well correlated with their ability to bind to D2 receptors. drugs bind with good affinity to dopamine D2 receptors, blocking 51

dopaminergic actions in striatum. However, the main problem encountered in antipsychotic treatment with these older antipsychotics is the occurrence of extrapyramidal motor side-effects, including and tardive dyskinesia, due to dopamine receptor blockade in striatum (Meltzer 1999; Alex and Pehek 2007). Furthermore, these drugs are not effective in treating either the negative or the cognitive symptoms (Marsden 2006). In contrast, newer atypical antipsychotic agents, such as , , , and , have less antagonistic effects on D2 receptors. While these atypical antipsychotics bind to several receptor types, their ability to alter dopamine release has been claimed to be mainly attributable to their antagonist or inverse agonist effects on 5-HT receptors (Pehek et al. 2001), including 5-HT1A, 5-

HT2A, 5-HT2C and 5-HT3 receptors (Meltzer 1999; Meltzer et al. 2003; Marsden 2006).

2.6.3.2 5-HT receptors in schizophrenia

It seems that 5-HT has a role in schizophrenia, 5-HT1A and 5-HT2 receptor levels have been shown to be altered in several areas of post-mortem brains of schizophrenic patients (Hashimoto et al. 1993; Joyce 1993). Furthermore, an increased 5-HT uptake has been detected in striatum, whereas a marked decline in 5-HT uptake occurs in cortex (Joyce 1993; Kahn and Davidson 1993). Decreased densities of 5-HT2A receptors are found in cortical areas of schizophrenic patients (Hashimoto et al. 1993). Specifically these receptors have been implicated in the genesis and treatment of psychosis and mood disturbances as well as in the treatment of positive and negative symptoms and the extrapyramidal side effects induced by antipsychotics (Schotte et al.

1996; Jakab and Goldman-Rakic 1998). Furthermore, there is a theory that 5-HT2C receptors are involved in the etiology of psychosis (Herrick-Davis et al. 2000) and in mediating the effects of atypical antipsychotics on mesocortical dopamine systems (Di Matteo et al. 2002). It has been hypothesized that a relatively high receptor affinity particularly at 5-

HT2A receptors compared to the D2 receptor forms the basis of the main differences between atypical and typical antipsychotic drugs (Schotte et al. 1996). The ability of atypical antipsychotics to alter dopaminergic function and to alleviate schizophrenic symptoms may be due to their actions on one or more 5-HT receptor subtypes (Nomikos et al. 1994; Alex and Pehek 2007). These drugs have high affinities for 5-

HT2A receptors, and many of them are 5-HT2C inverse agonists and/or 5-HT1A agonists (Meltzer 1999; Weiner et al. 2001; Pehek et al. 2006). With respect to the atypical antipsychotics in clinical use, clozapine, olanzapine and sertindole have equivalent affinities to 5-HT2A and 5-HT2C receptors, whereas risperidone, quetiapine, and are more selective for the 5-HT2A receptor subtype (Meltzer 1999; Stark et al. 2007). 52

There is evidence that antidepressant drugs possessing 5-HT2A/2C receptor antagonist activity, such as Į1- and Į2- adrenoceptor antagonist, , may have beneficial effects as an adjunctive type therapy in schizophrenia to potentiate the effect of the antipsychotic drugs (Wiker et al. 2005). , which was first introduced as an antidepressant, has a very similar receptor binding profile (5-HT2/D2) to that of atypical antipsychotics in humans (Kapur et al. 1999) and possesses antipsychotic-like activity in vivo (Wadenberg et al. 2000). A selective 5-HT2A inverse agonist pimavanserin (ACP-103), which is currently in phase III development for treatment of PD related psychosis (ACADIA Pharmaceuticals Inc., 2008a), has been shown to exert an antipsychotic effect in experimental models (Li et al. 2005; Gardell et al. 2007). Pimavanserin is now under development as a co-therapy for schizophrenia (ACADIA Pharmaceuticals Inc., 2008b). Some of the new antipsychotics, such as aripiprazole, exert significant agonist activity at 5-HT1A receptors (Bardin et al. 2006; Mamo et al. 2007; Stark et al. 2007). 5-

HT1A receptor-mediated cortical dopamine release is a typical property of several atypical antipsychotics (Rollema et al. 1997; 2000). In particular, 5-HT1A agonism, combined with D2 antagonism, seems to be important in the actions of many atypical antipsychotics (Rollema et al. 1997; Meltzer 1999). Meltzer et al. (2003) also stated that the combined effect 5-HT1A agonism and 5-HT2A/D2 -antagonism may produce an atypical antipsychotic capability.

Schizophrenia may represent also a potential indication for 5-HT3 ligands, since 5-

HT3 receptor activation leads to dopamine release, and 5-HT3 receptor antagonists have shown antipsychotic activity (Hagan et al. 1993; Sirota et al. 2000; Hoyer et al. 2002).

The 5-HT3 receptor antagonist, ondansetron, which is used to prevent nausea and vomiting, has been reported to alleviate neuroleptic-induced tardive dyskinesia and psychotic symptoms in schizophrenic patients (Sirota et al. 2000). In another study, the negative symptoms and parkinsonism-like adverse effects were attenuated in chronic, treatment-resistant patients when ondansetron was co-administered with haloperidol (Zhang et al. 2006). Ondansetron may also have a role in improving the memory deficits related to schizophrenia (Levkovitz et al. 2005).

2.6.4 Depression

2.6.4.1 The role of dopamine

Pharmacologic treatments of depression mainly affect either the reuptake or degradation of 5-HT or noradrenaline. Although neurobiological theories of depression have mostly focused on disturbances in serotonergic and noradrenergic systems, increasing evidence suggests that dopamine also plays a role in symptoms as well as in the benefits of antidepressant therapy (Ninan 1999; D'Aquila et al. 2000; Zangen et al. 53

2001). There are two possible roles of dopamine; in the pathophysiology of depression and in the mechanism of action of (D'Aquila et al. 2000). Antidepressants are often used to treat also anxiety since they are effective in treating co-existing depressive and psychic symptoms. Furthermore, they have shown efficacy in anxiety even in the absence of depressive symptoms (Dazzi et al. 2001; Allgulander and Sheehan 2002). Thus, these disorders may share some common neurobiological pathways (Dazzi et al. 2001; Alex and Pehek 2007). Many of the symptoms of depression resemble the negative symptoms of schizophrenia (e.g. apathy, psychomotor retardation), and thus may be related to dysfunction of the mesocortical dopamine system in the prefrontal cortex. There is substantial evidence indicating that also dopamine plays an important role as a mediator of an antidepressant response. Atypical antipsychotics are increasingly used to treat depression, particularly as an adjunct therapy (Brugue and Vieta 2007; Montgomery 2008). While the hippocampus and prefrontal cortex are undoubtedly involved in the pathophysiology of depression (Nestler et al. 2002), it has been suggested that the mesolimbic dopamine system with nucleus accumbens-VTA dopaminergic circuit also contributes to these effects (D'Aquila et al. 2000; Nestler and Carlezon 2006). Dopaminergic neurons originating in the VTA and projecting into the nucleus accumbens as well as the prefrontal cortex may be involved in the control of reward- related behaviour and motivation. These functions are impaired in depression (D'Aquila et al. 2000; Beaufour et al. 2001; Zangen et al. 2001; Nestler and Carlezon 2006) further evidence for a role of dopaminergic disturbances in the pathophysiology of depressive symptoms. Many of the currently used antidepressants selectively inhibit the reuptake of 5-HT and/or noradrenaline. A role for dopamine uptake in the mechanisms of antidepressive drugs has been proposed, since several antidepressants e.g. , nomifensine and have also the ability to block the action of the DAT (Zahniser et al. 1999; Baldessarini 2001; Gershon et al. 2007). A combined noradrenaline and 5-HT uptake inhibitor has also been shown to increase dopamine levels in the frontal cortex (Kihara and Ikeda 1995). Indeed, due to the putative involvement of dopamine in depression, so-called triple reuptake inhibitors (such as PRC200-SS, DOV102677) are being developed as a new class of antidepressants (Popik et al. 2006; Liang et al. 2008). Those drugs elevate synaptic levels of 5-HT, noradrenaline and dopamine particularly in the frontal cortex, and they are hypothetized to produce a more rapid onset of action and a better efficacy than the currently used antidepressants selectively affecting only 5-HT or noradrenaline neurotransmission (Skolnick et al. 2003; Popik et al. 2006; Shaw et al. 2007; Liang et al. 2008). 54

2.6.4.2 5-HT and D2 receptors in antidepressant activity

In the mesolimbic system, 5-HT1A receptors are abundant, suggesting that they may play a role in emotional responses (Hoyer et al. 1994). Both 5-HT1A and 5-HT1B receptors are known to be involved in the anxiety and depression (reviewed in Fink and

Göthert 2007). For example, 5-HT1A partial agonist buspirone is in clinical use for the treatment of anxiety, but the 5-HT1A agonists have also demonstrated antidepressant activity in animal models of depression (Wieland and Lucki 1990). Furthermore, many antidepressants are used to treat anxiety in human patients (see Rasmussen 2006). 5-

HT2C receptors, which have been shown to modulate dopaminergic neurotransmission (see section 2.3.2.2), have also been implicated in the mechanisms of both depression (Baxter et al. 1995) and anxiety (Kennett et al. 1997; Bagdy et al. 2001; Bourin and Hascoët 2001). In fact, several widely used antidepressants possess antagonist properties at 5-HT2 receptors; e.g. the prototypical SSRI fluoxetine is a 5-HT2C receptor subtype antagonist (Ni and Miledi 1997) and the tricyclic antidepressants and also block 5-HT2 receptors (Sánchez and Hyttel 1999). There is evidence that amitriptyline can evoke accumbal dopamine release, possibly via 5-HT2C receptors (Di Matteo et al. 2000b). Chronic antidepressant treatment has been shown to increase accumbal dopamine

D2 receptor mRNA and D2-like binding in rats (Ainsworth et al. 1998), and the dopamine agonist pramipexole has been demonstrated to possess antidepressant actions in humans (Ostow 2002). Indeed, two antiparkinsonian agents, pramipexole and ropinirole, have affinity for D2-like receptors and lack significant affinity for central 5- HT receptors. Both of these drugs have displayed promising antidepressive activity in preliminary studies in clinical trials (Goldberg et al. 1999; Ostow 2002; Cassano et al.

2005). Therefore, the sensitivity of D2-like receptors may contribute to antidepressant properties. In conclusion, it seems that serotonergic and dopaminergic interactions play a significant role in the pathophysiology of depression and anxiety. However, the role of dopamine evidently is under appreciated. It is essential to understand the function of dopamine in the pathophysiology of these disorders since drug therapies modulating dopaminergic neurotransmission may provide adjunctive or even alternative options for the treatment of depression. 55

3 AIMS OF THE STUDY

The objective of the present study was to investigate the interaction of the 5-HT and dopamine systems in rat brains with a unilateral dopamine deficiency and after administration of 5-HT ligands. One special focus was to investigate the influence of two types of 6-OHDA-lesions on striatal 5-HT levels and to study whether the putative neuroprotective effect of quercetin would depend on the severity of dopaminergic lesions with the concomitant existence of different levels of serotonergic deficiency.

The specific aims of this study were:

1. To assess the short-term effect of acute intrastriatal L-dopa infusion on rotational responses and striatal dopaminergic and serotonergic function of 6-OHDA-lesioned rats with concomitant tryptophan hydroxylase inhibition (I).

2. To assess the long-term effect of continuous 2-week intrastriatal L-dopa infusion on rotational responses and striatal dopaminergic and serotonergic function of 6- OHDA-lesioned rats (II).

3. To assess the dose margin between 5-HT receptor- and dopamine D2 receptor-

mediated dopamine efflux and metabolism of the acutely administered 5-HT2A/2C antagonist, deramciclane, in the striatum or nucleus accumbens. The effects were

compared to those obtained with two other 5-HT ligands a) ritanserin, a 5-HT2A/2C

antagonist and b) buspirone, a 5-HT1A partial agonist (III).

4. To evaluate if there are any differences between two types of 6-OHDA-lesion models associated with different levels of 5-HT deficiency, in neuroprotection studies with quercetin (IV). 56

4 MATERIALS AND METHODS

4.1 Animals

Male Wistar rats were supplied by the National Laboratory Animal Center, University of Kuopio, Finland (I – IV) or the Viikki Animal Center, Helsinki (IV). The rats were housed in stainless steel cages and kept on a 12-h light/dark cycle (lights on at 7:00 a.m.) at an ambient temperature of 22 ± 1oC. Pelleted food (Lactamin R36, Lactamin AB, Södertälje, Sweden) and tap water were available ad libitum. At the time of the microdialysis experiments (III), the rats weighed 190 – 370 g, and at the time of the lesioning 190 – 230 g. The total number of animals used was 520. Before the surgery, the rats were housed in stainless steel cages in groups of 3 – 5 rats/cage. After the surgery, the animals were kept in individual stainless steel cages for one week for recovery and subsequently in groups of 3 – 4 rats. During the 2-week infusion period with osmotic minipumps (II) and after the implantation of guide cannula (III), the rats were also housed individually. All the experiments were carried out between 7:00 a.m. and 7:00 p.m. All procedures with animals were made according to European Community Guidelines for the use of experimental animals and reviewed by the Animal Ethics Committee of the University of Kuopio (I – IV) or Helsinki (IV) in conformance with current legislation and approved by the local Provincial Government.

4.2 Drugs

6-OHDA HCl, apomorphine HCl, L-dopa methyl ester HCl, 4-chloro-DL- phenylalanine methyl ester (PCPA), quercetin dihydrate and carboxymethylcellulose (CMC) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Chloral hydrate was purchased from Merck KgaA (Darmstadt, Germany) and buprenorphine from Reckitt & Colman (Hull, England) (II, III) or Schering-Plough (Kenilworth, NJ, USA) (I, IV). D- amphetamine sulphate was from Helsinki University Pharmacy (Helsinki, Finland) (IV). Carbidopa, lidocaine and were obtained from Orion Pharma (Espoo, Finland). In paper (III), buspirone hydrochloride, deramciclane fumarate (1R,2S,4R)-(- )-dimethylamino-exthoxy)-2-phenyl-1,7,7-trimethylbicyclo [2.2.1]heptane 2-(E)- butenedioate (1:1), ritanserin (Sigma-Aldrich) and D-amphetamine (Sigma-Aldrich) were provided by Orion Pharma. Unless otherwise mentioned, the doses of drugs refer to free bases. 57

4.3 6-OHDA-lesions

4.3.1 6-OHDA-lesion of the right medial forebrain bundle (I, II, IV)

The lesion procedure (MFB lesion) was performed as described previously by Huotari et al. (2000). 6-OHDA was dissolved in isotonic NaCl solution containing 0.2 mg/ml of ascorbic acid (2.5 µg of 6-OHDA in 1 µl). Prior to the administration of 6- OHDA, the rats were anesthetized with chloral hydrate (350 mg/kg, i.p.). The volume of the 6-OHDA-infusion was 4 µl and it was dispensed over an 8-min period. The coordinates for the final infusion site in the right MFB near to SN measured from bregma and from the skull surface were (AP -4.4, L -1.2, DV -8.3) (Paxinos and Watson 1986). Before the surgery, lidocaine was applied to the scalp and to the surface of the skull as local anesthesia to relieve postoperative pain. Also a single dose of buprenorphine (0.02 mg/kg, s.c.) was given after the surgery.

4.3.2 6-OHDA-lesion of the striatum (IV)

The stereotaxic operation (striatal lesion) was the same as described above, except for the infusion sites and the concentration of 6-OHDA-solution. For the quadruple infusion into the striatum, four needles (29 G) were lowered directly to the infusion sites using a custom-made infusion instrument where the needles were fixed to a single unit block (Figure 5A and C). Each needle was connected to a microsyringe with separate tubing and run by a motor-driven slow-motion syringe pump. The final coordinates for intrastriatal 6-OHDA-infusion measured from bregma (AP and L) and from the dura surface (DV) were (AP +1.3, L -2.6, DV -5.0); (AP +0.4, L -3.2, DV -5.0); (AP -0.4, L - 4.3, DV -5.0) and (AP -1.3, L -4.5, DV -5.0) (Paxinos and Watson 1986). A total of 7.0 µg of 6-OHDA in 3.5 µl dissolved as described above was infused into each location over a 7-min period at a flow rate 0.5 µl/min. The needles were retained in position for 4 min after the infusion of 6-OHDA. The pain medication was the same as above (4.3.1). 58

Figure 5. Schematic figures of custom made instruments (A) for the quadruple 6- hydroxydopamine (6-OHDA)-infusion into the striatum (each needle is connected to separate tubing) and (B) for local intrastriatal L-dopa infusions simultaneously into four sites (the needles are connected to one tube). 1. infusion needles; 2. infusion tubes; 3. an osmotic minipump. (C) Figure illustrating the approximate location (grey oval) on the surface of a rat skull for the placement of instrument aiming intrastriatal L-dopa- or 6-OHDA-infusions.

4.4 Measurement of rotational behavior (I, II, IV)

Rotational behavior of the 6-OHDA-lesioned rats was tested to assess 1) the successfulness of the 6-OHDA-lesion, 2) the effect of subsequent treatments (I, II, IV), 3) the effect of intraperitoneal (i.p.) L-dopa to induce rotation before and after acute or continuous 2-week intrastriatal L-dopa replacement therapy (I, II) and 4) the effect of quercetin treatment (IV). To assess the successfulness of the lesion, the rotational responses of rats were tested with apomorphine (0.1 mg/kg, s.c.) six (I) or 14 days (II) after administration of 6-OHDA. After drug injection, the rats were placed into individual hemispherical plastic bowls (diameter 35 cm) and fitted into harnesses connected to an automatic eight-channel rotometer (Coulbourn Instruments, Inc., Allentown, PA, USA or Roto- Rat, Med-Associates, Inc., St. Albans, VT, USA). The rotometer registered both ipsilateral and contralateral full rotations. In the acute intrastriatal L-dopa treatment study (I), the apomorphine test was used as a preliminary test, and owing to the short postlesion time, the rats showing more than 25 full contralateral circlings in a 60-min test session were accepted for further tests. L- 59

dopa/carbidopa -induced rotational behavior was then performed as primary rotation test at one and two weeks postlesion. L-dopa methyl ester HCl (10 mg/kg) was dissolved in 0.9% NaCl and carbidopa (30 mg/kg) was suspended in 0.9% NaCl containing few drops of Tween 80 and administered i.p. After the second rotation test with L-dopa, the rats which rotated at least 30 full contralateral rotations in a 120-min test session were randomly divided into two groups. Then, PCPA methyl ester HCl (250 mg/kg, i.p.) or 0.9% NaCl as vehicle control was administered to the rats (n = 10 in both groups). In the 2-week continuous L-dopa treatment study (II), those rats showing more than 100 full contralateral circlings in a 60-min test session were accepted for further tests. One month after the 6-OHDA-lesion, L-dopa/carbidopa-induced rotational behavior of rats was assessed as described above at one-week interval. For continuous intrastriatal L-dopa treatment, osmotic minipumps were implanted. The L-dopa/carbidopa-induced rotation tests were then continued at one-week intervals until day 84 from pump implantation. On each test day during and after intrastriatal L-dopa treatment, the rats were allowed to adapt to the rotometer bowls for 1 h before L-dopa/carbidopa administration. In each group, the results were compared against the mean of two pre- operative L-dopa/carbidopa -induced rotation tests, i.e., those performed one week and one day before pump implantation, which was further referred to as day 0. To assess the effect of quercetin on rotational behavior (IV), apomorphine-induced (0.1 mg/kg, s.c.) contralateral or D-amphetamine-induced (2.5 mg/kg, i.p.) ipsilateral circling behavior of rats was tested 7 and 14 days after 6-OHDA-infusion.

4.5 Unilateral intrastriatal infusions of L-dopa (I, II)

4.5.1 Acute single intrastriatal infusion of L-dopa (I)

Three days after peripheral administration of PCPA or vehicle, the rats were anaesthetized with chloral hydrate (350 mg/kg, i.p.) and placed into a stereotaxic apparatus (Stoelting or Kopf, USA). For the intrastriatal L-dopa infusion, a stainless steel guide cannula (made from a 21 G needle) was lowered 2.0 mm above the infusion site. An infusion needle (made from a 29 G needle) was lowered directly to the final infusion coordinates measured from bregma (AP: 0.2, L: -2.8, DV: -7.5) (Paxinos and Watson 1986), connected to a microsyringe and run by a motor-driven slow-motion syringe pump (CMA/102, CMA Microdialysis, Solna, Sweden). L-dopa was dissolved in isotonic NaCl solution (10 µg of L-dopa in 2 µl) and infused into the striatum over a 4-min period. The needle was retained in position for 4 min after the infusion. The coordinates for the infusion place were the same as used in study (III), and were verified as described in section 6.4. At 16 h after the intrastriatal L-dopa-infusion, L-dopa/carbidopa-induced (10/30 mg/kg, i.p.) rotational behavior was tested. Subsequently, one half of the rats (n = 5) 60

were sacrificed and the other half was further tested for the rotation one week later after the L-dopa-infusion and subsequently sacrificed. In each group, the results were compared against the result of L-dopa/carbidopa-induced rotation test performed three days before the intrastriatal L-dopa infusion (referred as Day 0). The full time schedule of the acute intrastriatal L-dopa treatment study is shown in Figure 6A. In the rat brain microdialysis, it has been shown that a drug administered via a perfusion fluid can diffuse for at least 1 – 2 mm from the probe into the brain tissue (de Lange et al. 1995). Thus, it may be estimated that by a single L-dopa infusion a similar kind of distribution can be reached.

4.5.2 Continuous 2-week intrastriatal infusion of L-dopa (II)

L-dopa was dissolved in isotonic NaCl solution containing 1 mg/ml of ascorbic acid. Osmotic minipumps (Alzet model 2002, Alza, Palo Alto, CA, USA) were filled with L-dopa solutions and incubated at +37°C overnight in 0.9% NaCl before implantation. The pumps were implanted subcutaneously into the scapular area of the rat under chloral hydrate anesthesia (350 mg/kg, i.p.). Each pump was attached to a catheter with a custom made instrument with four infusion needles (Figure 5B and C) and implanted surgically using a stereotaxic device to enable local drug infusions simultaneously into four sites of the lesioned striatum at a rate of 0.5 µl/h for 14 days. The coordinates for intrastriatal infusion were measured from bregma (AP 1.2, L -2.4, DV -6.0); (AP 0.5, L -3.5, DV -7.0); (AP 0.0, L -2.2, DV -6.5) and (AP -0.8, L -4.0, DV -6.5) (Paxinos and Watson 1986). The doses of L-dopa used for continuous intrastriatal infusion were 0, 1, 3 or 10 µg/h. After the 2-week infusion period, the pumps and the infusion needles were removed under chloral hydrate anesthesia. The verification for the accurate position of infusion needles in the striatum was made by macroscopic examination from slices of dissected brain. The full time schedule of continuous 2-week intrastriatal L-dopa treatment study is shown in Figure 6B. Hargraves and Freed (1987) evaluated the spread of the intrastriatally infused [3H]- dopamine-solution from the osmotic minipumps by measuring the radioactivity at several time points after the local one-site infusion (0.5 µl/h). They found that tritium counts were detectable beyond 6 mm from the infusion site and that the distribution of the tracer was very similar after infusions from one to seven days. It seems thus evident that in our study, the intrastriatally infused L-dopa for two weeks into four sites with the total volume of 0.5 µl/h may well have diffused into a wide area troughout the striatum. 61

A 6-OHDA- Intrastriatal lesion PCPA or vehicle L-dopa-infusion Ļ Ļ Ļ Days -17 -11 -10 -3 0 ~16 h 1

Ĺ Ĺ Ĺ Ĺ Rotation test with Rotation tests with intraperitoneal subcutaneous L-dopa / carbidopa apomorphine

Intrastriatal L-dopa-infusion B 6-OHDA- Pump Pump lesion implantation removal Ļ Ļ Ļ Days -35 -28 -21 -14 -7 0 7 14 21 28 35 84

Ĺ Ĺ Ĺ Ĺ Ĺ Ĺ Ĺ ĹĹ Rotation test with Rotation tests with intraperitoneal L-dopa / carbidopa subcutaneous apomorphine

Figure 6. The time courses of (A) the acute and (B) the continuous 2-week intrastriatal L-dopa treatment experiments.

4.6 Brain microdialysis in conscious rats (III)

Rats were anaesthetized with chloral hydrate (350 mg/kg, i.p.). Each rat was placed in a Kopf stereotaxic apparatus and a guide cannula was implanted (Figure 7) through a burr hole into striatum (MAB 6.9.IC, AgnTho’s AB, Lidingö, Sweden) with the tip extending 3.5 mm below the dura at final coordinates (tip of the probe) measured from bregma (AP: 0.2, L: 2.8, DV: -7.5) and into nucleus accumbens (MAB 4.10.IC, AgnTho’s AB) with the tip extending 6.0 mm below the dura at coordinates measured from bregma (AP: 1.7, L: -0.9, DV: -8.0) (Paxinos and Watson 1986). The pain medication was the same as described in section 4.3.1. The coordinates for the accurate probe placement was verified in preliminary experiments from slices of dissected brain after infusion of methylene blue into the respective coordinates in nucleus accumbens shell and striatum. Five to ten days after the surgery, dialysis probes (MAB 6, 4 mm exposed membrane, 0.57 mm outer diameter, 15 000 Daltons cut off, AgnTho’s AB) were placed into striatum and into nucleus accumbens shell (MAB 4, 2 mm exposed membrane, 0.2 mm outer diameter, 6 000 Daltons cut off, AgnTho’s AB) of a conscious rat through guide cannulae. The probes were perfused with artificial cerebrospinal fluid with a flow 62

rate of 2 µl/min. During the microdialysis experiment, each rat was housed in an individual plastic bowl (diameter 400 mm). After a 120 min wash-out period, the dialysate was collected as 20-min fractions and divided into two aliquots. The first aliquot was stored at +4oC and assayed for dopamine within 24 h. The other aliquot was stored at -70oC until analyzed for 3,4-dihydroxyphenylacetic acid (DOPAC) and HVA. The drugs were suspended in 0.5% CMC dissolved in 0.9% NaCl. Treatments were

(doses refer to the salts) 5-HT2A/2C antagonist deramciclane fumarate 3 mg/kg (7.2

µmol/kg), 10 mg/kg (24 µmol/kg) and 30 mg/kg (72 µmol/kg), 5-HT2A/2C antagonist ritanserin 1 mg/kg (2.1 µmol/kg), a partial 5-HT1A agonist buspirone hydrochloride 5 mg/kg (12 µmol/kg). An indirect dopamine agonist D-amphetamine sulphate 2 mg/kg (5.4 µmol/kg), was used as a positive control. After 60-min baseline collection, the vehicle or drugs were given i.p. in a volume of 5 ml/kg and the samples were collected for 240 min. The levels of dopamine, DOPAC and HVA in rat microdialysis samples were measured by two high performance liquid chromatography (HPLC) methods with electrochemical detection (III).

Figure 7. The schematic figure of the microdialysis guide cannula and probe placement in (A) nucleus accumbens shell and (B) striatum. Both of the probes were implanted into the right and left hemisphere in striatum and nucleus accumbens of the same animal, respectively. The probe implantation sites as measured relative to bregma are shown. The sizes of the probes are in a true relation to brain size. The probe placement was verified as described in section 4.6. Modified from Paxinos and Watson (1986).

4.7 Quercetin treatment schedules (IV)

In studies with quercetin, rats were given either vehicle (0.25% CMC) or various doses of quercetin i.p. in a volume of 0.5 ml per 100 g of body weight once a day for 63

one week before (pretreatment study) or one week after (post-treatment study) the 6- OHDA-infusion either into MFB or four sites into the striatum (Figure 8). In the combination treatment study, the study protocol was the same as above except that rats were given either vehicle or quercetin twice a day for one week before and for one week after the 6-OHDA-infusion.

Quercetin once a day A before lesion or after lesion

-7 0 +7 +14 days

6-OHDA lesion Apomorphine- or D-amphetamine- a) s. nigra induced rotation measurements b) striatum B

-7 0 +7 +14 days

Quercetin 100 mg/kg twice a day

Figure 8. Time schedule of the animal experiments in quercetin study (IV; Figure 3). Quercetin was administered once a day (A) as a pretreatment one week before 6- hydroxydopamine (6-OHDA) or as a post-treatment one week after the 6-OHDA or (B) twice a day as a combination treatment one week before and for one week after the 6-OHDA.

4.8 Analytical procedures (I – IV)

4.8.1 Determination of striatal dopamine, 5-HT and their metabolites in rat tissue samples (I, II, IV)

The rats were sacrificed by decapitation after the last rotation experiment. The brains were removed and both striata were dissected on ice immediately after decapitation. The dissected tissues were frozen in liquid nitrogen and stored at -70oC until sample preparation and assay for dopamine, DOPAC, HVA, 5-HT and 5-HIAA by 64

HPLC using electrochemical detection as earlier described by Huotari et al. (2002) and Airavaara et al. (2006) (Table 2).

4.9 Nigral and striatal TH-positive cell assays (IV)

The numbers of striatal and nigral TH-positive cells were estimated by using immunohistochemical staining and assay methods as earlier described (Bradford 1976; Sauer et al. 1995; Lindholm et al. 2007; Mijatovic et al. 2007) (Table 2).

Table 2. The methods used to analyze biogenic amines, overall protein levels and TH enzyme from the striatal samples in studies (I), (II) and (IV).

Method References HPLC with electrochemical detection (I, II, IV) Huotari et al. (2002) (II), Airavaara et al. (2006) (I, IV) Protein assay (I, II) Bradford (1976) Stereological analysis of TH-positive cells (IV) Sauer et al. (1995) Striatal densitometry measurements (IV) Lindholm et al. (2007) TH-immunohistochemistry (IV) Mijatovic et al. (2007)

4.10 Western immunoblotting (I, II)

Western immunoblotting was used to determine the enzyme protein levels of TH and TrH in striatal samples of 6-OHDA-lesioned rats after acute (I) or 2-week (II) intrastriatal L-dopa treatment. Western blots for TH and TrH were run in parallel experiments in triplicate including intact and lesion controls. The same protein amounts were used in all wells. Total striatum protein was electrophoresed on SDS-PAGE gels, transferred to nitrocellulose membranes and incubated with primary monoclonal TH and TrH antibodies, washed and exposed to secondary antibody. TH and TrH were visualized by chemiluminescence using a Super Signal West Femto kit (Pierce, Rockford, IL, USA).

4.11 Dopamine D2 receptor binding and 5-HT uptake assay (II)

3 Specific binding of [ H]- reflecting the amount of dopamine D2 receptors and 5-HT uptake was determined in 6-OHDA-lesioned, L-dopa infusion-treated rats using striatal synaptosomal preparation. The [3H]-spiperone binding assay was performed in 96-well plates, each well containing assay buffer, 25 µg of protein, 0.3 nM [3H]-spiperone, and when required to determine non-specific binding. Assay plates were incubated for 90 min at 25°C. Samples were filtered through 0.1% 65

polyethyleneimine pre-treated Wallac FilterMat C (Milton Keynes, UK), and washed with cold assay buffer. Bound radioligand was measured with a Wallac Micro Beta counter. To assess 5-HT uptake and non-specific uptake, 5-hydroxy[G-3H] and cold 5-HT were used, respectively. The assay was performed in 96-well plates, each well containing assay buffer, 25 µg of protein, 30 nM radioactive 5-HT, and 2.1 µM 5- HT when required. The further incubation, sample preparation and the measurement of bound radioligand were made as described above.

4.12 Data analysis and statistics

4.12.1 Microdialysis data (III)

The mean of three baseline samples (-60 to -20 min) was calculated for each rat as a baseline value (= 100%) and the alterations of extracellular dopamine, DOPAC and HVA concentrations in dialysis samples after drug administration were expressed as a percentage of this value. The area under the concentration-time curve (AUC) values (% of baseline × min) of dopamine, DOPAC and HVA were calculated with the trapezoid rule from time zero to 240 min (AUC0-240 min) using GraphPad Prism 3.02 software.

4.12.2 Statistical analyses (I – IV)

One-way ANOVA was used to test the statistical significance of differences between the effects of various treatments on rotational behavior (II, IV), differences between optical density (OD) measurements for striatal TrH and TH (I, II), in specific

D2 receptor binding as well as 5-HT uptake (II) between the treatment groups. Further comparisons between groups were performed with Tukey’s post hoc test. Part of the rotational data was analyzed using paired t-test (I). The significance of differences between the changes in the concentrations of striatal amines (dopamine, 5-HT) and their metabolites (DOPAC, HVA, 5-HIAA) after intrastriatal L-dopa treatment or quercetin between intact and lesioned striatum was analyzed by unpaired t-test. The same test was used with Bonferroni’s adjustment for multiple comparisons to analyze significance of differences between treatment groups (I, II, IV). The effect of drug treatment vs. baseline on extracellular levels of dopamine, DOPAC and HVA was tested with ANOVA for repeated measures (III). Further comparisons between different fractions and baseline (i.e. 20 min – 240 min vs. baseline) were carried out using non-orthogonal contrasts with Bonferroni adjustment for multiple comparisons. One-way ANOVA was used to analyze the statistical significance of differences between the AUC0-240 min values of dopamine, DOPAC and HVA in nucleus accumbens or striatum after different treatments (III). Further 66

comparisons between treatment groups were carried out using Tukey’s HSD or Games- Howell post hoc test. Unpaired t-test was applied to test the significance of difference between the AUC0-240 min values in nucleus accumbens and striatum (III). The criterion for statistical significance was p<0.05. 67

5 RESULTS

5.1 The effect of acute and 2-week intrastriatal L-dopa infusion treatment on 6-OHDA-lesioned rats (I, II)

5.1.1 The effect of intrastriatal L-dopa treatment on rotational behavior induced by acute intraperitoneally administered L-dopa

Acute infusion of L-dopa (10 µg) into 6-OHDA-lesioned striatum 16 h prior to the rotation experiment significantly decreased the total amount of contralateral rotations (day 1; p<0.05) as well as the peak rotation induced by acute L-dopa/carbidopa (10/30 mg/kg, i.p.) in control animals as (Figure 9). The duration of action of peripherally administered L-dopa appeared to be slightly reduced on day 1 (but not longer on day 7) in animals whose 5-HT synthesis was blocked by PCPA 4 days before the test. However, the intrastriatal L-dopa-treatment did not have any significant effect on the total amount of contralateral rotations in those rats. Furthermore, the intensity of circling was essentially the same as on day 1 in both groups, when tested again later on day 7 on one half of the rats. Continuous 2-week intrastriatal L-dopa infusion treatment decreased the contralateral rotations induced by acute L-dopa/carbidopa (10/30 mg/kg, i.p.) with apparent dose-dependency. While the vehicle or the lowest dose of intrastriatal L-dopa (1 µg/h) did not have any effect on the L-dopa/carbidopa -induced contralateral rotational behavior, the dose of 3 µg/h tended to decrease the intensity of circling (Figure 10). However, this reduction was not statistically significant until three weeks after the 2-week L-dopa treatment period (on day 35), when the rotational behavior was decreased by 69 ± 8% (p<0.05) compared to the circling before the L-dopa treatment (day 0 in Figure 10). At the dose of 10 µg/h of L-dopa, the contralateral rotational behavior significantly decreased during the L-dopa infusion treatment period. However, the number of rotations was at its lowest one week after the treatment period (day 21), when the circling was reduced by 91 ± 4% (p<0.001). The significant decrease (by 62 ± 14%, p<0.05) in the rotational behavior in this group was still seen 10 weeks after the cessation of the L-dopa infusion treatment (day 84). 68

Day 1 post-infusion (a) n 50 Control; =10 (b) Control; n=10 400 45 40 Day 0 35 300 30 25 200 20 15 Day 1 100 * 10 5 Full CCW rotations/10 min rotations/10 CCW Full 0 0 Full CCW rotations in 120 min 120 in rotations CCW Full 0 10 20 30 40 50 60 70 80 90 100 110 120 Day 0 Day 1 Time (min)

(c) PCPA; n =10 (d) PCPA; n=10 50 400 45 40 Day 0 300 35 30 25 200 20 15 100 10 5 Day 1 Full CCW rotations/10 min

0 Full CCWrotations in 120 min 0 0 10 20 30 40 50 60 70 80 90 100 110 120 Day 0 Day 1 Time (min)

Day 7 post-infusion (e) Control, n=5 50 (f) Control; n =5 400 45 40 Day 0 35 300 30 25 200 20 15 10 100 5

Full CCWrotations/10 min Day 7 0 0 Full CCW rotations in 120 min 120 in rotations CCW Full 0 10 20 30 40 50 60 70 80 90 100 110 120 Day 0 Day 7 Time (min)

(g) PCPA; n=5 (h) PCPA; n=5 50 400 45 40 Day 0 35 300 30 25 200 20 15 100 10 5 Day 7 Full CCW rotations/10 min rotations/10 CCW Full 0

Full CCW rotations in 120 min 120 in rotations CCW Full 0 0 10 20 30 40 50 60 70 80 90 100 110 120 Day 0 Day 7 Time (min)

Figure 9. The effect of acute intrastriatal L-dopa treatment on L-dopa/carbidopa-induced (10/30 mg/kg, i.p.) contralateral (CCW) rotations of unilaterally 6-hydroxydopamine-lesioned rats after vehicle (NaCl 5 ml/kg, i.p.; Control) or 4-chloro-DL-phenylalanine (250 mg/kg, i.p.; PCPA) - treatment (I; Figure 1). Three days after single i.p. injection of vehicle or PCPA, each rat received a single intrastriatal L-dopa-infusion (10 µg) into the lesioned side and it was tested for rotation at 16 h (Day 1; a – d; n = 10) and at seven days (Day 7; e – h; n = 5) after the intrastriatal L-dopa infusion. Half of the rats were sacrificed after the first rotation test. The values are expressed as mean ± S.E.M.; *p<0.05 vs. Day 0 which refers to the result of rotation test performed three days before the intrastriatal L-dopa treatment and 17 days from the 6- OHDA-infusion. 69

A B 1 µg/h 1800 0 µg/h 1800 1600 1600 infusion 1400 1400 infusion 1200 1200 1000 1000 800 800 in 240 min in 240 minin 240 600 600 400 400 200 200 Full contralateral circlings contralateral Full Full contralateral circlings contralateral Full 0 0 0 7 14 21 28 35 84 0 7 14 21 28 35 84 Time (days) after pump implantation Time (days) after pump implantation

D C 3 µg/h 10 µg/h 1800 1800 1600 1600 1400 infusion 1400 infusion 1200 1200 1000 1000 800 800 * in 240 minin 240 in 240 min in 240 600 600 ** ** 400 * 400 *** *** *** 200 200 Full contralateral circlings contralateral Full Full contralateral circlings 0 0 0 7 14 21 28 35 84 0 7 14 21 28 35 84 Time (days) after pump implantation Time (days) after pump implantation

Figure 10. The effect of continuous 2-week intrastriatal L-dopa treatment on L-dopa/carbidopa- induced (10/30 mg/kg, i.p.) rotational behavior (II; Figure 2). 6-hydroxydopamine-lesioned rats received vehicle solution (Control; A) or L-dopa (1, 3 or 10 µg/h; B, C and D, respectively) into the lesioned striatum as a continuous infusion via osmotic minipumps on days 1 – 14. The pumps were implanted on day 0, 35 days from the 6-OHDA-infusion. Control group n = 7; 1 µg/h n = 9; 3 µg/h n = 13; 10 µg/h n = 12. Values are expressed as mean ± S.E.M. of full contralateral circlings in 240-min rotation test, *p<0.05, **p<0.01 and ***p<0.001 vs. day 0.

5.1.2 The effect of intrastriatal L-dopa treatment on striatal levels of dopamine, 5-HT and their metabolites

In the acute L-dopa study, dopamine (p<0.001; Table 3), DOPAC and HVA (p<0.001 and p<0.01, respectively) levels were significantly decreased in lesioned striata vs. intact striata in both vehicle and PCPA treatment groups. The 6-OHDA-lesion -induced loss of dopamine and its metabolites was also seen in the 2-week L-dopa study in both intrastriatal vehicle- and L-dopa -treated groups; the levels of dopamine and DOPAC were decreased (p<0.001) in lesioned striata, and the striatal levels of HVA 70

were still practically undetectable in the lesioned side 70 days after the cessation of the infusion treatment. 5-HT levels were significantly decreased (p<0.01, Table 3) both 1 and 7 days after acute L-dopa infusion in the lesioned striata of vehicle-treated group. A concomitant decrease (p<0.05) in 5-HIAA levels was seen on day 7 (data not shown). In PCPA- treated group, 5-HT levels were significantly decreased in both sides due to synthesis inhibition (p<0.01) on day 1 vs. the respective striata of the vehicle group. The levels had partially recovered on day 7, and then there were no statistically significant differences between intact and lesioned sides. On day 7, only the 5-HT levels of intact side were significantly different (p<0.05) vs. the respective value of vehicle control group. In the 2-week L-dopa study, the 5-HT levels were significantly decreased in the lesioned striata vs. intact striata in all 2-week treatment groups (0 µg/h p<0.001, 1 and 3 µg/h p<0.01 and 10 µg/h p<0.05; Figure 11), whereas striatal 5-HIAA levels were significantly decreased only in lesioned side in the control group (p<0.01; Figure 11). However, no significant differences between the control and 2-week L-dopa treatment groups in 5-HIAA levels in lesioned striata were observed (data not shown).

Table 3. Striatal dopamine and 5-HT levels in normal rat or in intact and lesioned side after vehicle- or 4-chloro-DL-phenylalanine-treatment and intrastriatal L-dopa-infusion in 6- hydroxydopamine-lesioned rats (I; Table 1).

Dopamine 5-HT Intact Lesion Intact Lesion Vehicle Day 1 11.47 ± 0.52 0.11 ± 0.06*** 0.38 ± 0.07 0.11 ± 0.02** Vehicle Day 7 11.67 ± 0.56 0.08 ± 0.04*** 0.54 ± 0.06 0.18 ± 0.03*** PCPA Day 1 12.98 ± 0.86 0.42 ± 0.34*** 0.01 ± 0.01## 0.01 ± 0.01## PCPA Day 7 10.74 ± 2.42 0.08 ± 0.01** 0.29 ± 0.06# 0.29 ± 0.12 Control rat 11.53 ± 0.92 0.40 ± 0.04

Values are ng/mg of tissue weight; mean ± S.E.M.; n = 5. Vehicle: 0.9% NaCl; PCPA: 4- chloro-DL-phenylalanine. Day 1 and Day 7: one and seven days after intrastriatal L-dopa treatment, respectively; four and eleven days from the PCPA administration, respectively. Statistics: **p<0.01, ***p<0.001 vs. corresponding intact striatum; #p<0.05, ##p<0.01 vs. corresponding striata of vehicle group. 71

5-HT A B 5-HIAA 3.0 3.5 Left side 2.5 3.0 Right side †† 2.5 † 2.0 †† 2.0 1.5 ††† 1.5 †† 1.0 1.0 nmol/g wet tissue wet nmol/g 0.5 tissue wet nmol/g 0.5 0.0 0.0 01310 01310 Dose of L-dopa (µg/h) Dose of L-dopa (µg/h) infused for 2 weeks infused for 2 weeks

Figure 11. Striatal (nmol/g wet tissue) 5-HT (A) and 5-HIAA (B) levels of unilaterally lesioned rats (left = intact side; right = lesion side) treated with 2-week intrastriatal infusion of vehicle (0 µg/h) or L-dopa (1, 3 and 10 µg/h) 70 days after the cessation of the infusion treatment and 119 days from the 6-OHDA-infusion (II; Figure 3). 0 µg/h n = 4; 1 µg/h n = 7; 3 µg/h n = 7; 10 µg/h n = 10 rats per group. Values are expressed as mean ± S.E.M., †p<0.05, ††p<0.01, †††p<0.001 vs. the corresponding intact striatum.

5.1.3 The effect of intrastriatal L-dopa treatment on striatal TrH and TH

Western blot analysis was performed to evaluate whether the acute or 2-week intrastriatal L-dopa infusion had altered the striatal levels of TrH or TH. On day 1 (day 18 post-lesion), 16 h after acute intrastriatal L-dopa infusion, striatal TrH protein levels were not changed by the 6-OHDA-lesion or PCPA, or their combination. Essentially the same was observed on day 7 (day 25 post-lesion). However, there were no signs of increased TrH protein levels (data not shown). The amounts of detected TrH protein were very low, hardly above the background level, and therefore the quantitation remained uncertain. In the 2-week L-dopa treatment study, the levels of TrH were significantly increased in a dose dependent manner in the lesioned striata of all L-dopa treatment groups (1 µg/h p<0.01; 3 and 10 µg/h p<0.001) vs. the control group 70 days after the cessation of the infusion treatment (Figure 12). A clear dose-dependency was seen, TrH levels in the lesioned side of the 3 µg/h group were significantly increased (p<0.01) compared to 1 µg/h group. Furthermore, TrH was significantly higher (p<0.001) in the 10 µg/h group than that in the groups receiving 1 or 3 µg/h L-dopa. TrH levels were also significantly increased (p<0.001) in lesioned striata compared to the corresponding intact striata within the L-dopa treatment groups. In the acute L-dopa study, striatal TH protein levels were very low in the lesioned side in all groups as compared to the corresponding intact striata (data not shown). Also 72

in the 2-week L-dopa treatment study, the levels of TH were significantly reduced (p<0.001) in the lesioned striata of all groups (Figure 12).

A Tryptophan hydroxylase B Tyrosine hydroxylase 10000 ††† 10000 ††† §§§ Left side ‡ ‡ *** Right side ††† *** 1000 ** 1000

100 100 ††† Band densityBand Band density ††† ††† †††

10 10 Intact 0 1 3 10 Intact 0 1 3 10 Dose of L-dopa (µg/h) Dose of L-dopa (µg/h)

Figure 12. A quantitative densitometric analysis of Western blot bands illustrating tryptophan hydroxylase (A) and tyrosine hydroxylase (B) protein levels in striata of intact and unilaterally lesioned (left = intact side; right = lesion side) rats administered 2-week intrastriatal vehicle (control; 0 µg/h of L-dopa) or L-dopa (1, 3 and 10 µg/h) infusions 70 days after the cessation of the infusion treatment and 119 days from the 6-OHDA-infusion (II; Figure 4). Values are expressed as mean ± S.E.M., n = 6 in each group. Statistics: **p<0.01 and ***p<0.001 vs. 0 µg/h right side, ‡‡p<0.01 vs. 1 µg/h, §§§p<0.001 vs. 1 and 3 µg/h, †††p<0.001 vs. the corresponding intact striatum.

5.1.4 The effect of 2-week intrastriatal L-dopa treatment on striatal 5-HT

uptake and dopamine D2 receptor binding

We investigated the amount of 5-HT nerve endings in the lesioned striatum and the change over 2-week intrastriatal L-dopa infusion by measuring radioactive 5-HT uptake into crude synaptosomal preparations. 6-OHDA-lesion caused a significant decrease in 5-hydroxy[G-3H]tryptamine uptake compared to the corresponding intact striata (p<0.05, Figure 13) 70 days after the 2-week intrastriatal vehicle infusion. However, L- dopa infusion treatment produced a dose dependent recovery in 5-HT uptake (3 and 10 µg/h p<0.01 and p<0.001 vs. 0 µg/h, respectively). In order to determine whether the intrastriatal 2-week L-dopa treatment had 3 changed the number of striatal D2 receptors, the specific binding of [ H]-spiperone in the striatal crude cell free extract was measured 70 days after the cessation of 2-week intrastriatal infusion. The binding was significantly increased in lesioned striatum in the groups receiving 0 and 3 µg/h L-dopa (p<0.01 and p<0.001 vs. intact striatum, respectively; Figure 13). At the highest dose of L-dopa (10 µg/h), the binding was partially reversed and was not significantly different from the corresponding intact side. 73

5-HT uptake D2 receptors A B †† ††† 250 300 Left side Right side 250 200 *** ** 200 150 † H]tryptamine

3 150 100 100 50 (fmol/mg protein) 50 H]-spiperone binding 3 [

uptake (fmol/mg uptake protein) 0 5-hydroxy[G- 0 Intact 0 3 10 Intact 0 3 10

Dose of L-dopa (µg/h) Dose of L-dopa (µg/h)

Figure 13. The uptake (fmol/mg protein) of 5-hydroxy[G-3H]tryptamine (A) and specific binding (fmol/mg protein) of [3H]-spiperone (B) to crude cell free extract of intact (left side) and unilaterally 6-hydroxydopamine-lesioned (right side) rat striata 70 days after the cessation of 2-week intrastriatal infusion of vehicle (0 µg/h of L-dopa) or L-dopa (3 and 10 µg/h) and 119 days from the 6-OHDA-infusion (II; Figure 5). The intact group represents the 5-HT uptake or spiperone binding in healthy left and right striata. Values are expressed as mean ± S.E.M. (intact group n = 3; 0 µg/h n = 4; 3 µg/h n = 6; 10 µg/h n = 6, triplicate samples); **p<0.01 and ***p<0.001 vs. 0 µg/h right side; †p<0.05, ††p<0.01 and †††p<0.001 vs. the corresponding intact striatum.

5.2 The effect of 5-HT-ligands on the extracellular levels of dopamine, DOPAC and HVA in rat nucleus accumbens shell and striatum (III)

Deramciclane (3 and 10 mg/kg), or two reference compounds, the 5-HT2A/2C antagonist ritanserin (1 mg/kg) and the 5-HT1A partial agonist buspirone (5 mg/kg), did not significantly change the accumbal extracellular dopamine levels (III, Figure 1), whereas 30 mg/kg of deramciclane significantly increased the levels at 40 – 100 min and at 160 – 240 min (p<0.05 vs. baseline). Accordingly, the AUC0-240 min of dopamine was significantly higher (p<0.05) after deramciclane 30 mg/kg than after lower doses of deramciclane, but there was no difference to buspirone (Figure 14). (III, Figure 1). The

AUC0-240 min values of both DOPAC and HVA were significantly higher after deramciclane 30 mg/kg as compared to lower doses of deramciclane (p<0.01). After administration of buspirone, a significant (p<0.05 vs. baseline) increase in the accumbal levels of DOPAC at 40 min and HVA at 60 – 100 min was seen (III, Figure 2). In parallel, the AUC0-240 min of HVA was significantly higher after administration of buspirone than after any other treatment (p<0.05) excluding deramciclane 30 mg/kg. 74

Nucleus accumbens Striatum A Dopamine §§ 150000 130000 110000 * 40000 * * 30000 § 20000 § 10000 0 -10000 AUC (% of baseline x min) x baseline of (% AUC -20000 Control Dera3 Dera10 Dera30 Rita1 Busp5 Amph2

DOPAC B * 20000 †† ** ** † ‡ 10000

0

-10000 §

AUC (% of baseline x min) * § -20000 Control Dera3 Dera10 Dera30 Rita1 Busp5 Amph2

HVA C ††, § 20000

‡ † 10000

0

-10000 § ** ** §§ AUC (% of baseline x min) x baseline of (% AUC -20000 Control Dera3 Dera10 Dera30 Rita1 Busp5 Amph2

Figure 14. The AUC0-240 min (mean ± S.E.M.) of extracellular dopamine, DOPAC and HVA after intraperitoneal administration of deramciclane fumarate (3 mg/kg and 10 mg/kg, n = 9; 30 mg/kg, n = 8), D-amphetamine sulphate (2 mg/kg, n = 9), ritanserin (1 mg/kg, n = 9), buspirone hydrochloride (5 mg/kg, n = 8) and vehicle (5 ml/kg, n = 4 – 5) (III; Figure 3). Statistics: *p<0.05, **p<0.01 between the treatments shown by a bracket; †p<0.05, ††p<0.01 vs. all other treatments excluding buspirone hydrochloride (5 mg/kg) in one brain area (in nucleus accumbens shell or striatum); ‡p<0.05 vs. all other treatments excluding deramciclane fumarate (30 mg/kg) in one brain area; §p<0.05 and §§p<0.01 nucleus accumbens vs. striatum. 75

In striatum, none of the deramciclane doses studied, and neither buspirone nor ritanserin, had any significant effect on extracellular dopamine levels compared to the basal levels (III, Figure 1). After deramciclane 30 mg/kg treatment, the AUC0-240 min values of both DOPAC and HVA were significantly higher (p<0.05) than after any other treatment except buspirone, reflecting an overall increase in the striatal levels of

DOPAC and HVA. In parallel, the AUC0-240 min of DOPAC was higher after buspirone than after any other treatment (p<0.05) excluding deramciclane 30 mg/kg. Deramciclane 3 and 10 mg/kg did not show significant differences between the

AUC0-240 min of dopamine in nucleus accumbens and in striatum. After deramciclane 30 mg/kg or buspirone, the AUC0-240 min value of dopamine, was significantly higher

(p<0.05) in nucleus accumbens than in striatum. The same was observed in the AUC0-

240 min of HVA after administration of deramciclane 30 mg/kg. The positive control, D-amphetamine (2 mg/kg), significantly increased the extracellular dopamine levels in nucleus accumbens and in striatum (III, Figure 2). In parallel, the AUC0-240 min of dopamine was significantly higher (p<0.01) after administration of D-amphetamine compared to all other treatments (Figure 14) and was significantly higher (p<0.01) in striatum than in nucleus accumbens. On the contrary, the AUC0-240 min values of DOPAC (p<0.01) and HVA (p<0.01) were significantly lower after administration of D-amphetamine than obtained with the other treatments.

5.3 The effect of quercetin in unilateral 6-OHDA-lesions of two types of parkinsonian rat models (IV)

5.3.1 Levels of dopamine, 5-HT and their metabolites in intact and lesioned striata

Dopamine levels were significantly decreased by • 98% (p<0.001) in lesioned striata compared to intact striata in all groups of MFB-lesioned rats treated with quercetin or vehicle (Table 4). Subsequently, the levels of DOPAC and HVA were significantly decreased (p<0.001) by 71 – 99% and 79 – 99%, in lesioned striata in all groups. In striatal lesioned rats, dopamine levels were also significantly decreased in the lesioned side in the control groups (57 – 88%; p<0.05 vs. intact side), however, the decrease was smaller than that observed with MFB lesions. Striatal levels of DOPAC and HVA were also significantly decreased (by 39 – 82% and 59 – 79%, p<0.05, respectively). Notably, 5-HT (Table 4) and 5-HIAA levels in lesion side were significantly (p<0.05) decreased only by MFB lesions, but not by striatal lesions. Neither quercetin pretreatment (100 and 200 mg/kg, i.p.) nor combination treatment (100 mg/kg, i.p. twice daily one week before and after 6-OHDA-infusion) had any significant effect on the levels of dopamine, 5-HT or their metabolites. 76

Table 4. Concentrations of striatal dopamine and 5-HT in intact and lesion side of rats with unilateral medial forebrain bundle (MFB) or striatal lesion Quercetin (mg/kg) MFB lesion Striatal lesion

Dopamine Intact Lesion Intact Lesion 0 A) 8.64 ± 0.24 0.19 ± 0.17*** 11.5 ± 1.47 4.97 ± 1.33* B) 9.78 ± 0.84 0.03 ± 0.01*** 11.2 ± 0.44 1.30 ± 0.50** 100 A) 9.05 ± 0.38 0.03 ± 0.01*** 13.1 ± 0.70 1.58 ± 0.49*** B) 10.7 ± 0.62 0.02 ± 0.002*** 10.5 ± 0.36 1.01 ± 0.37*** 200 A) ND ND 10.9 ± 1.01 2.56 ± 0.30*** 5-HT 0 A) 0.18 ± 0.01 0.07 ± 0.03* 0.19 ± 0.03 0.18 ± 0.03 B) 0.15 ± 0.01 0.07 ± 0.02* 0.17 ± 0.01 0.17 ± 0.02 100 A) 0.18 ± 0.02 0.04 ± 0.02*** 0.21 ± 0.01 0.23 ± 0.03 B) 0.19 ± 0.01 0.05 ± 0.03** 0.13 ± 0.01 0.10 ± 0.01 200 A) ND ND 0.16 ± 0.03 0.16 ± 0.03

Values are ng/mg of tissue weight; mean ± S.E.M.; n = 5. A) one week quercetin i.p. treatment before 6-hydroxydopamine (6-OHDA); B) one-week treatment before 6-OHDA + one-week treatment after 6-OHDA. Statistics: *p<0.05, **p<0.01, ***p<0.001 versus respective intact side; ND, not determined.

5.3.2 TH-positive cell staining in intact and lesioned striata and substantia nigra

TH, the rate limiting enzyme in dopamine synthesis, was used as a marker for dopaminergic neurons in striatum and substantia nigra. In MFB-lesioned animals, both striatal and nigral TH were undetectable in the lesioned side, and the levels were not restored by quercetin. After striatal lesions, there was visible TH-staining even in the lesioned striatum (Figure 15). It appears that 100 mg/kg of quercetin had some restorative effect (n.s.) on striatal TH levels (Figure 15). In stereological analysis of TH- positive cells, there were no statistically significant differences in nigral TH-positive cell numbers between treatment groups of the striatal or MFB-lesioned rats. Furthermore, no statistically significant differences were observed in the OD values reflecting the density of striatal TH-fibers between quercetin- or vehicle-treated animals with the striatal lesion. 77

Figure 15. Representative photomicrographs (three sections for each treatment) of striatal (Anterior-posterior: +0.2 – +0.48) tyrosine hydroxylase -immunoreactive fibers in rats pretreated with vehicle (A) or quercetin 100 mg/kg (B) or 200 mg/kg (C) for 7 days before 6- hydroxydopamine (6-OHDA) -infusion into four sites of striatum (7 µg per each site) (IV; Supplementary Figure 1). The samples were taken 21 days after 6-OHDA infusion.

5.3.3 Rotational behavior of unilaterally lesioned rats

All MFB-lesioned rats pretreated with quercetin before intrastriatal 6-OHDA- infusion showed rotational behavior one and two weeks post-lesion after administration of apomorphine (0.1 mg/kg, s.c.) or D-amphetamine (2.5 mg/kg, i.p.) (Figure 16). In some rats, quercetin pretreatment at higher doses decreased (n.s.) contralateral rotations induced by apomorphine on days 7 and 14 after 6-OHDA-infusion. However, the effect of quercetin pretreatment was not significantly different at any dosage compared to the respective control group (0 mg/kg). In parallel, quercetin pretreatment did not have any significant effect on striatal 6-OHDA-lesions (IV; Figure 6). However, 100 mg/kg of quercetin had some attenuating effect (n.s.) on D-amphetamine-induced rotational 78

behavior which was not seen at a dose 200 mg/kg. After quercetin post-treatment, which was given for one week after the 6-OHDA-infusion into MFB, there was no significant effect on apomorphine-induced rotational behavior. In the combination treatment study, where quercetin (100 mg/kg) was administered twice a day one week before and one week after the 6-OHDA infusion into striatum or MFB, there were no significant differences between quercetin- or vehicle-treated groups in apomorphine- or D- amphetamine-induced circling (Figure 17).

A Apomorphine, week 1 B Apomorphine, week 2 800 800 700 700 600 600 500 500 400 400 300 300 200 200 Full contralateral Full contralateral circlings in 60 min circlings in 60min 100 100 0 0 0 50 100 200 0 50 100 200 Dose of quercetin (mg/kg) Dose of quercetin (mg/kg)

D-Amphetamine, week 1 D-Amphetamine, week 2

Dose of quercetin (mg/kg) Dose of quercetin (mg/kg) C 0 50 100 200 D 0 50 100 200 0 0

-500 -500

-1000 -1000 Full ipsilateral Full ipsilateral Full circlings in 120 min 120 in circlings circlings in 120 min

-1500 -1500

Figure 16. The effect of quercetin pretreatment (for one week before 6-hydroxydopamine (6- OHDA) -infusion) on apomorphine (0.1 mg/kg, s.c.) -induced contralateral rotational behavior (A and B) and D-amphetamine (2.5 mg/kg, i.p.) -induced ipsilateral rotational behavior (C and D) in 6-OHDA-rats with MFB lesion (IV; Figure 4). Tests were performed two and three weeks after the 6-OHDA-infusion. Apomorphine groups: 0 mg/kg n = 14; 50 mg/kg n = 10; 100 and 200 mg/kg n = 15. D-amphetamine groups: 0 mg/kg n = 16; 100 mg/kg n = 13; 200 mg/kg n = 5. Individual values of each rat as well as mean ± S.E.M. of each group are shown. 79

Dose of quercetin 0 mg/kg 100 mg/kg

Apomorphine Apomorphine A (MFB lesion) B 300 300 (STR lesion)

200 200 in 60 min in 60 100 min 60 in 100 Full contralateral circlings 0 Full contralateral circlings 0 Week 1 Week 2 Week 1 Week 2

D-Amphetamine D-Amphetamine (MFB lesion) (STR lesion)

C Week 1 Week 2 D Week 1 Week 2 0 0

-100 -100

-200 -200

-300 -300 in 120 min in 120 in 120 min -400 -400 Full contralateral circlings contralateral Full -500 circlings contralateral Full -500

Figure 17. The effect of quercetin combination treatment (twice a day for one week before and one week after 6-hydroxydopamine (6-OHDA) -infusion) on apomorphine (0.1 mg/kg, s.c.) - induced contralateral rotational behavior (A and B) and D-amphetamine (2.5 mg/kg, i.p.) - induced ipsilateral rotational behavior (C and D) in 6-OHDA-rats with striatal (STR) or MFB lesions (IV; Figure 5). Tests were performed two and three weeks after the 6-OHDA-infusion. Values are expressed as means ± S.E.M.; n = 4 – 6 per group. 80

6 DISCUSSION

6.1. General

It is known that there are interactions between dopaminergic and serotonergic systems in the CNS. This functional interplay represents a source of putative targets for drug therapies in disorders related to alterations in dopaminergic or serotonergic neurotransmission, such as PD, schizophrenia and depression. However, the mechanisms to explain the interactions between 5-HT and dopamine in these brain dysfunctions are not well-defined. In this study, we explored dopamine/5-HT interactions in rat brains with a unilateral dopamine depletion (rat PD model) and after systemic administration of 5-HT ligands. In the light of other animal studies, it is evident that the serotonergic innervation may compensate for the functional loss of dopaminergic neurons in PD (Arai et al. 1995; Maeda et al. 2005). Unilateral dopaminergic denervation in the MFB-lesioned rats can trigger the striatal hyperinnervation of 5-HT-immunoreactive fibers in adult rats (Maeda et al. 2003a). Moreover, it seems that 5-HT neurons are able to accumulate exogenous L-dopa and further decarboxylate it to dopamine (Arai et al. 1995; see Kostrzewa et al. 2005; Maeda et al. 2005). The efficacy of L-dopa becomes reduced during the progression of PD and the motor complications associated with L-dopa treatment, such as increased off-time and dyskinesias, occur more often. The integrity of the 5-HT system may thus be an important factor in the efficacy of L-dopa, helping to avoid the adverse effects of L- dopa (Carta et al. 2007). Indeed, the 5-HT2A antagonist pimavanserin is currently in clinical studies as co-therapy for L-dopa (ACADIA Pharmaceuticals Inc., 2008a). To date, however, there are no drugs directly acting on the serotonergic system in use in the treatment of PD. In contrast, the importance of the interaction of the serotonergic and dopaminergic systems is recognized in the treatment of schizophrenia, since atypical antipsychotics are known to affect both systems. In particular, 5-HT1A agonism and combined 5-

HT2A/D2 antagonism are thought to be involved in the actions of antipsychotic drugs (Meltzer et al. 2003). While 5-HT disturbances in the CNS are connected to depression and anxiety, there may also be a significant dopamine component involved in the pathophysiology of these diseases. The 5-HT1A agonists and 5-HT2A/2C antagonists, such as buspirone and ritanserin, respectively, are known to be able modify brain dopamine levels (Devaud et al. 1992; Gobert et al. 1999). Interestingly, it has been proposed that there is involvement of 5-HT1 and 5-HT2 receptor-mediated mechanisms in the treatment of depression (see section 2.6.4.2). All of these receptors have also been shown to modulate dopamine neurotransmission in vivo. Due to the unique 5-HT2/D2 receptor binding profile of anxiolytic deramciclane, we explored the dose margin 81

between 5-HT receptor- and dopamine D2 receptor-mediated dopaminergic effects of deramciclane in striatum or nucleus accumbens. Since the findings of the alterations of striatal 5-HT content in 6-OHDA-lesioned rats are controversial (e.g. Karstaedt et al. 1994; Tanaka et al. 1999), we wanted to study the influence of two types of 6-OHDA-lesions (MFB- and striatal lesion) on striatal 5-HT levels. There has been a debate if antioxidative compounds would have beneficial effects on 6-OHDA-induced neurodegeneration in vivo. The bioflavonoid, quercetin, which has shown antioxidative and some neuroprotective effects in vitro (Fiorani et al. 2001; Dajas et al. 2003), was tested in these two types of 6-OHDA-lesioned rat models with different degree of the dopaminergic and serotonergic neuron damage.

6.2 The effect of acute and 2-week intrastriatal L-dopa infusions on 6- OHDA-lesioned rats (I, II)

PD is characterized by a selective degeneration of the nigrostriatal dopaminergic neurons and a severe reduction in the striatal dopamine levels, but it has become apparent that also other neurotransmitter systems in addition to dopamine, such as the serotonergic system, may be involved in the pathophysiology of PD (Brotchie 2005). Indeed, the functional relationship between 5-HT and dopamine has been intensively studied in experimental PD. Previously, there have been several proposals about how the serotonergic system could compensate for the dopamine depletion or participate in the action of L-dopa (see Introduction). Here, we studied the short- and long-term effects of acute and 2-week intrastriatal L- dopa treatment, respectively, on unilaterally 6-OHDA-lesioned rats. As far as we are aware, this is the first reported study where a continuous 2-week intrastriatal L-dopa therapy via osmotic minipumps has been given to unilateral parkinsonian rats. Several indices reflecting the involvement of the serotonergic system in dopaminergic function of these rats were observed after intrastriatal L-dopa treatments, both in rotation tests and biochemical assays. The acute intrastriatal L-dopa at a dose of 10 µg decreased the contralateral rotations induced by acute systemic L-dopa/carbidopa after intrastriatal L- dopa administration, but this effect was prevented by 5-HT synthesis inhibition (I). Our results indicate that the striatal serotonergic imbalance between the intact and lesioned sides is associated with the decrease in L-dopa-induced contralateral rotation. The unilateral 6-OHDA-lesion induced a significant suppression of ipsilateral striatal 5-HT levels which was clearly observed in the 6-OHDA-lesioned control animals without PCPA treatment. However, this imbalance between intact and lesioned striata was abolished by inhibition of TrH enzyme as observed on days 1 and 7 when there were no significant differences in 5-HT levels between intact and lesioned sides of 6-OHDA- lesioned animals after PCPA-treatment. 82

Intrastriatally administered L-dopa seems to induce desensitization of striatal dopamine D2 receptors which evokes a significant decrease in contralateral rotation. The restoration of D2 receptor binding has previously been observed in 6-OHDA-lesioned rats after oral L-dopa treatment by Reches et al. (1984). Furthermore, a diminished contralateral rotation has been found after the short-term oral L-dopa in 6-OHDA-rats apparently due to loss of dopamine receptor sensitivity (Brannan et al. 1998). In our 2- week intrastriatal L-dopa treatment study, the highest dose of L-dopa (10 µg/h) induced 3 partial reversal of [ H]-spiperone binding to striatal D2 receptors of 6-OHDA-lesioned rat (II). The serotonergic system seems to contribute to the rotational behavior of unilaterally 6-OHDA-lesioned rats. The decreased rotational behavior observed after acute intrastriatal L-dopa treatment in control rats, but not in PCPA treated rats, is evidence that the desensitization of D2 receptors to L-dopa does not develop when TrH enzyme is inhibited (I). It seems that 5-HT participates in the regulation of the sensitivity of striatal postsynaptic D2 receptors in 6-OHDA-lesioned rats. Thus, intact 5- HT synthesis may be an important factor in modulating rotational behavior. On the basis of the present study, we are unable to determine the exact nature of the influence of 5- HT system on sensitization. However, it has been shown previously that the 5-HT system can influence brain dopamine receptors. In fact, evidently dopamine D1 receptor supersensitivity does not occur in neonatally 6-OHDA-lesioned rats when 5-HT fibers are bilaterally destroyed by intracerebroventricular (i.c.v.) injection of serotonergic neurotoxin 5,7-DHT (Kostrzewa 1995). Matsumoto et al. (1989) and Idova et al. (2004) have observed behavioral indices of increased dopamine D2 receptor activation after PCPA treatment in mice which they postulated were due to a decrease in brain serotonergic activity. Furthermore, D-amphetamine-induced rotation has been shown to increase significantly in PCPA treated, unilaterally 6-OHDA-lesioned mice (Milson and Pycock 1976). This finding supports an important role of 5-HT neurons in the dopamine receptor sensitization level and the rotational behavior after 6-OHDA-lesions. The lack of L-dopa-induced desensitization in rotation tests after acute intrastriatal L-dopa treatment is associated with the lack of imbalance in 5-HT concentration between intact and lesioned striata (I). When the effect of PCPA is fading and TrH enzyme is recovering, this activation of the 5-HT synthesis may be particularly intense on the lesioned side since the 5-HT levels in the 6-OHDA-lesioned striatum were equal to those in the intact striatum. TrH is found only in cells that synthesize 5-HT and its distribution is similar to that of 5-HT. 6-OHDA-lesions of the MFB deplete 5-HT and presumably also TrH in the serotonergic cells. Furthermore, L-dopa infusion evidently induced local formation of L-dopa-quinone and dopamine which have been shown to inactivate TrH in vitro (Kuhn and Arthur 1998; 1999). However, this acute dual serotonergic damage was apparently only moderate since in our acute study, 5-HT levels had recovered already on day 7 post-infusion, despite the PCPA treatment (I). TrH was also greatly increased in the 2-week L-dopa study (II). This increased 83

activation is in parallel with the results seen after the 2-week intrastriatal L-dopa treatment where striatal TrH levels on the lesioned side were significantly increased by day 70 post-infusion (II). In the 2-week L-dopa study, however, increased levels of TrH in the lesioned striatum were not directly reflected in the striatal 5-HT levels indicating that some other, possible compensatory, mechanisms like ipsilateral 5-HT neuron sprouting may occur after the intrastriatal L-dopa treatment (see below). Several long-term effects of 2-week intrastriatal L-dopa in hemilesioned rats were also observed both in rotational and biochemical studies. The 2-week intrastriatal L- dopa treatment at doses 3 and 10 µg/h in 6-OHDA-lesioned rats significantly and dose- dependently decreased the contralateral rotational behavior induced by peripherally administered L-dopa (II). A dose of 3 µg/h of L-dopa decreased the number of circlings, being statistically significant on day 35 (3 weeks post-infusion), and 10 µg/h of L-dopa significantly decreased rotation at all time points during and after infusion. This finding was unexpected, since the rotational behavior induced by peripheral L- dopa/carbidopa did not recover to the baseline during the 10 post-infusion weeks. Although this effect appeared to recover slowly, it was still detectable at 10 weeks post- infusion. In addition, a significant and dose-dependent increase in the striatal TrH levels was observed 10 weeks after the 2-week L-dopa treatment. Furthermore, the decreased amount of serotonergic uptake in the nerve endings in the lesioned striatum was dose- dependently reversed in L-dopa-treated rats paralleling the highly increased ipsilateral striatal TrH protein levels. There is evidence that a unilateral 6-OHDA-lesion induces hyperinnervation of 5-HT-immunoreactive fibers in adult rats. 5-HT fibers have been found to densely sprout throughout the ipsilateral striatum and in the SN one to two months after a unilateral 6-OHDA-infusion into the MFB or SN (Zhou et al. 1991; Guerra et al. 1997; Maeda et al. 2003a,b). Zhou et al. (1991) and Maeda et al. (2003a,b) concluded that sprouting of serotonergic neurons coexists with dopamine denervation (>95%) and almost total striatal dopamine loss. This serotonergic hyperinnervation may well serve as a substitute for the degenerated dopaminergic terminals, increasing for instance regarding uptake of exogenous L-dopa, conversion from L-dopa to dopamine as well as release and even storage of dopamine (Maeda et al. 2003b; Kostrzewa et al. 2005). In theory, these kinds of serotonergic compensatory mechanisms may partly substitute the loss of dopaminergic function. In our study, significantly increased striatal TrH levels and reversal of 5-HT uptake may reflect proliferation of 5-HT fibers (II). Notably, both of these parameters were dose-dependently enhanced by the 2-week intrastriatal L-dopa, suggesting a role of the continuous administration of L-dopa in changes of 5-HT neurons in the striatum after dopamine denervation. Interestingly, increased levels of brain derived neurotrophic factor (BDNF) mRNA were found after repeated L-dopa treatment (twice daily for five days) in the 6-OHDA-lesioned subthalamic nucleus, a part of the basal ganglia system, (Zhang et al. 2006). It is possible that L-dopa has some, currently unknown, modulative effects on 84

synaptogenesis and synaptic plasticity (Zhang et al. 2006), which may also partly explain the dose-dependent changes in the 5-HT system (TrH, 5-HT uptake) observed in our study after the 2-week L-dopa infusion (II). The significant increase in the striatal TrH after an L-dopa treatment did obviously not improve 5-HT synthesis, since the reduced 5-HT concentration was not restored to the levels of the intact side. Although there are no previous data of the functionality of the 5-HT fibers sprouting after 6-OHDA-lesions, it does seem that not all of these neurons are functionally active. That would provide an explanation for the sustained low 5-HT levels in our study (II). While 5-HIAA levels were significantly lower in lesioned striatum than in intact striatum of control rats, the levels had partially recovered after 2-week L-dopa treatment: there were no longer any significant differences between intact and lesioned side when this was measured 70 days post- treatment. One could suggest that these changes in 5-HIAA levels may reflect slightly enhanced striatal 5-HT turnover. Clinically, an enhanced 5-HT turnover rate (increased 5-HIAA/5-HT ratio) was observed in the striatum possibly resulting from a compensatory increase in the firing rate of the serotonergic neurons (Scatton et al. 1983; Kish et al. 2008). In contrast, after acute intrastriatal L-dopa treatment (I), marginally detectable striatal TrH levels were not affected by 6-OHDA-lesion and intrastriatal L-dopa, which is not in agreement with Western findings of the 2-week L-dopa study (II). However, it is difficult to estimate the situation immediately after the cessation of continuous 2-week infusion since our samples were collected much later, 70 days post-treatment. It is possible that the compensatory serotonergic activation postulated to occur in the 2-week study is not fully achieved by a short post-treatment time (1 or 7 days) after acute infusion. Furthermore, if increased TrH levels depend on both striatal dopamine depletion and L- dopa, it may be that prolonged L-dopa treatment time is needed to achieve the desired effect (I). Trace amines, such as and tryptamine, are closely associated by structure, metabolism and tissue distribution with the dopamine, noradrenaline and serotonin systems in the mammalian brain (Burchett and Hicks 2006). Their concentrations in the brain are only 1/1000 of those of the catecholamines (Berry 2004). Of the trace amine- associated receptor (TAAR) family, TAAR1 is distributed throughout the CNS in regions containing catecholaminergic neuron cell bodies and their projections, such as SN, VTA, dorsal raphe and striatum (Borowsky et al. 2001). However, very little is known about the regulation and function of these receptors (Sotnikova et al. 2008). There are some hypotheses that TAAR1 has a role in the modulation of functional processes of the brain dopaminergic systems (Geracitano et al. 2004), and it may have tonic inhibitory action on motor control (Sotnikova et al. 2008). Trace amines may cause indirect activation of dopamine D2 autoreceptors, further inhibiting dopaminergic 85

activity in VTA (Geracitano et al. 2004). Conversely, presynaptic D2 autoreceptor activation seems to inhibit the activity of TAAR1 (Xie and Miller 2007). Under normal conditions, tryptophan is preferentially converted to 5- hydroxytryptophan via TrH. However, the blockade of 5-HT synthesis by PCPA can elevate brain tryptamine levels; 65% increase has been detected 48 h after PCPA administration to rats (Saavedra and Axelrod 1973). Theoretically, this may lead to increased activity of trace amine-associated system in the brain, such as TAAR1 activation. However, significant disturbances of dopamine formation from L-dopa by either PCPA or TAAR1 seem unlikely to have occurred in our study, since the L- dopa/carbidopa-induced rotation of PCPA-treated rats was not significantly changed as compared to rotations before PCPA-treatment, indicating that there was adequate striatal dopamine formation after peripheral L-dopa administration. There are no reports describing significant alterations of 5-HT neurons to convert L-dopa to dopamine after PCPA. While PCPA depletes brain 5-HT, the firing rate of raphal 5-HT neurons does not seem to be modified (Chaput et al. 1990).

Little is known about the modulatory effects of trace amines on postsynaptic D2 receptors. Although it can be hypothesized that D2 autoreceptors are activated through

TAAR1 (Xie and Miller 2007), it should be noted that the D2 autoreceptor regulation in 6-OHDA-lesioned striatum is obviously significantly impaired due to lesions in dopaminergic nerve terminals (Borah and Mohanakumar 2007). The serotonergic and dopaminergic neurotransmitter systems seem to be anatomically and functionally linked (see sections 2.3.1 and 2.3.2). As discussed above, serotonergic innervation may compensate for the functional loss of dopaminergic neurons, e.g. by facilitating the conversion of L-dopa to dopamine and its further release (Maeda et al. 2005). The mainstay of symptomatic drug treatment of PD is still the dopamine precursor L-dopa co-administered with a peripheral DDC inhibitor. As the disease progresses, the efficacy of L-dopa becomes reduced and the motor complications associated with L-dopa treatment increase. There is evidence that 5-HT terminals in dopamine depleted striatum may convert exogenous L-dopa to dopamine with further facilitation of storage and release. Dopaminergic immunoreactivity has been detected in the cell bodies of raphe nuclei and in striatal and nigral serotonergic cell fibres after administration of L-dopa and carbidopa both in intact and 6-OHDA- lesioned rats (Arai et al. 1995; Maeda et al. 2005; Yamada et al. 2007). The function of the 5-HT system may thus be an important factor in the efficacy of L-dopa as well as in avoiding the adverse effects of L-dopa in PD patients (Carta et al. 2007). It seems obvious that a substantial amount of the L-dopa can be converted to dopamine in lesioned striatum, perhaps even with greater efficacy than in the intact striatum. After peripheral L-dopa administration, 5-times higher extraneuronal dopamine levels have been detected in the lesioned striata of 6-OHDA-rats than in striata of intact rats (Zetterström et al. 1986; Abercrombie et al. 1990). However, it has been hypothesized 86

that dopaminergic neurons can use also L-dopa itself as a neurotransmitter (Misu et al. 1996). Indeed, L-dopa has been reported to have various effects both in vitro and in vivo (see Misu et al. 2003). However, these mechanisms are unclear and need to be further explored. The progression of PD causes degeneration of the remaining dopaminergic terminals and loss of buffering capacity for exogenous L-dopa leading to fluctuation in striatal dopamine levels between dosing. Excessive dopamine levels cause dyskinesia and decreased levels off-states (see Nyholm 2007). Other types of cells, such as serotonergic neurons, take up L-dopa and convert it to dopamine, but the neurotransmitter cannot be properly stored. Instead, it leaks into the presynaptic area, stimulates dopamine receptors and is then lost (Melamed et al. 1980). It has been demonstrated that dopamine released from the serotonergic terminals acts as a false neurotransmitter and this inappropriate dopamine release can cause dyskinesia in 6- OHDA-rats (Carta et al. 2006; Carta et al. 2007). Thus, the 5-HT system has been implicated in the L-dopa-induced dyskinesias (Borah and Mohanakumar 2007; Carta et al. 2008). Although dopamine conversion and release through 5-HT neurons are evidently dysregulated, there seem to be alternative ways to improve the L-dopa response in vivo. Co-treatment with 5-HT1A receptor agonists and L-dopa has been found to alleviate the fluctuation in striatal dopamine levels, reduce dyskinesias and enhance the motor activity in the 6-OHDA-rats (Tomiyama et al. 2005; Eskow et al. 2007; Mignon and Wolf 2007) and in primate models of PD (Bibbiani et al. 2001).

Furthermore, the 5-HT2A receptor antagonist attenuated L-dopa-induced dyskinesias in the monkey MPTP-model of PD and suppressed parkinsonian-like tremor in rats (Vanover et al. 2008). Therefore, it seems that an increased serotonergic tone in the CNS is beneficial in improving L-dopa therapy. As a co-treatment, 5-HT ligands may lessen the need for L-dopa, leading to an improved motor control. The results of our present study further point to an involvement of the serotonergic system in the unilateral dopamine depletion and intrastriatal L-dopa treatment. Thus, the serotonergic system evidently is a putative target for developing new treatment strategies to PD. In advanced PD, the continuous dopaminergic stimulation treatment, mimicking normal dopaminergic tone in striatum, is a potential treatment strategy in improving motor control by decreasing the fluctuation of dopamine levels (see Nutt 2007; Nyholm 2007). Methodologically, our 2-week continous intrastriatal infusion treatment study design resembles the chronic dopaminergic stimulation. Since TH, dopamine and its metabolites remain significantly lowered on the lesion side in all groups of 6-OHDA-lesioned rats, the changes observed in rotational behavior were obviously not due to any dopaminergic recovery of the nigrostriatal system after acute or 2-week intrastriatal L-dopa treatment (I, II). Several in vitro studies have demonstrated that L-dopa and also extracellular dopamine are toxic to dopaminergic neuronal cell cultures (Mytilineou et al. 1993; Pardo et al. 1995; Jiang et al. 2008). Even 87

though enzymatic and non-enzymatic metabolism of L-dopa can produce hydrogen peroxide and free oxygen radicals, there is controversy about whether L-dopa itself can generate oxidative stress and cell damage in vivo (Perry et al. 1984; Ogawa et al. 1994; Camp et al. 2000). However, one could hypothesize that in unilateral 6-OHDA-lesioned rats, the remaining dopaminergic neurons in lesioned striatum may have impaired cellular defense mechanisms against oxidative stress (Ferrario et al. 2003). The 2-week L-dopa exposure on the 6-OHDA-lesioned striatum in the 2-week study may have even caused more extensive local lesions resulting in lower efficiency of acute systemic L- dopa/carbidopa in the rotation tests. This is probably the most likely explanation for the significantly decreased rotation observed after the 2-week intrastriatal L-dopa treatment (II). Thus, our neurochemical findings point to marked alterations in the serotonergic system in the striatum rather than to any significant recovery of the nigrostriatal dopaminergic system in response to long-term intrastriatal L-dopa treatment. A compensatory mechanism induced by continuously administered L-dopa, such as the long-term alterations in the serotonergic system found in the 2-week L-dopa study, to oppose the dopamine deficit may have occurred. The present results of both the acute and 2-week intrastriatal L-dopa treatment study are evidence for involvement of 5-HT neurons in dopaminergic imbalance that is responsible for the rotational behavior of unilaterally 6- OHDA-lesioned rats and that 5-HT neurons play a role in the actions of exogenous intrastriatal L-dopa in the dopamine depleted striatum (I, II).

6.3 The effect of 5-HT-ligands on the extracellular levels of dopamine, DOPAC and HVA in rat nucleus accumbens shell and striatum (III)

The putative anxiolytic drug, deramciclane, has a high affinity for 5-HT2A and 5-

HT2C receptors (Ki 8.7 – 27 nmol/l); it is an antagonist at both receptor subtypes and possesses inverse agonist properties at the 5-HT2C receptors. The receptor binding profile of deramciclane (Gacsályi et al. 1997) is rather similar to that of ritanserin, another 5-HT2A/2C antagonist (Leysen et al. 1985). Deramciclane also has moderate affinity for dopamine D2 receptors (Ki 113 nmol/l) and in this respect it differs from ritanserin, but it has no notable affinity for dopamine D1 receptors (Gacsályi et al.

1997). The partial 5-HT1A agonist, buspirone, has also a significant antagonist affinity at dopamine D2 receptors (Ki 9.2 and 13 nmol/l at 5-HT1A and D2 receptors, respectively)

(Algeri et al. 1988; Piercey et al. 1994). D2 receptor antagonists are capable of enhancing dopamine outflow in nucleus accumbens and striatum (Moghaddam and Bunney 1990; Baldessarini 2001). It has also been shown that drugs acting as antagonists/inverse agonists at 5-HT2 receptors have an ability to alter dopamine release in striatum and in nucleus accumbens (see section 2.3.2.2). Due to the unique D2/5-HT2 receptor binding profile of deramciclane, our aim was to assess the dose margin between 5-HT receptor- and dopamine D2 receptor-mediated dopaminergic effects of 88

deramciclane in striatum or nucleus accumbens (III). The effects were compared to those obtained by either buspirone or ritanserin. In the present study, small doses of deramciclane (3 or 10 mg/kg) had practically no effect on dopamine levels in either brain area. In contrast, a high single dose (30 mg/kg) of deramciclane significantly increased extracellular dopamine levels in nucleus accumbens, but there was no effect in striatum. Both striatal and accumbal extracellular levels of dopamine metabolites were also significantly increased. In general, systemic administration of 5-HT2C antagonists is known to increase dopamine release in nucleus accumbens, striatum and frontal cortex (Di Giovanni et al. 1999; Gobert et al. 2000; Pozzi et al. 2002). Since deramciclane has antagonistic properties at these receptors, it may increase accumbal dopamine levels via 5-HT2C antagonism at a high dose of 30 mg/kg. On the other hand, deramciclane is known to have affinity for dopamine D2 receptors, and notably, the dopamine metabolite profile (increased DOPAC and HVA levels) of deramciclane resembles that evoked by neuroleptics (Zetterström et al. 1984; Imperato and Di Chiara 1985). When dopamine receptors are blocked, more dopamine is synthesized and released to compensate for the receptor blockade, and also more dopamine metabolites are formed (Moghaddam and Bunney 1990; Gray and Connick 1998). This is opposite to the effects of indirect dopamine agonists and DAT inhibitors, which increase extracellular dopamine levels, and either decrease or have no effect on the levels of dopamine metabolites (Nomikos et al. 1990; Kuczenski et al. 1991; Keller et al. 1992; Tanda et al. 2005). The positive control, the indirect dopamine agonist, D- amphetamine sulphate, significantly increased dopamine levels in both brain regions studied confirming that our experimental set up was able to detect extracellular elevations in dopamine levels.

In some studies, mixed 5-HT2A/2C receptors antagonists, such as ritanserin (1.5 – 5 mg/kg), have been shown to increase extracellular dopamine levels after systemic administration in prefrontal cortex (Nomikos et al. 1994; Pehek and Bi 1997) and in nucleus accumbens but not in striatum (Devaud et al. 1992). Ritanserin may also have some selectivity for the mesolimbic dopaminergic projections over the nigrostriatal dopaminergic projection since it increases VTA dopaminergic neuron firing rate at a low dose (1 mg/kg) (Andersson et al. 1995) but in another study it had no effect on SN dopaminergic neurons in a dose range of 0.1 – 6.4 mg/kg (Shi et al. 1995). In our study, ritanserin 1 mg/kg tended to inhibit dopamine efflux in both brain areas studied, confirming some earlier reports where the effects of low doses of ritanserin have been modest on extracellular dopamine levels in either striatum or nucleus accumbens (Devaud et al. 1992; Andersson et al. 1995; Di Matteo et al. 1998). Furthermore, there was a trend to decreased levels of dopamine metabolites in both brain areas. Interestingly, a similar effect was seen with the lower doses of deramciclane (3 and 10 mg/kg), which slightly decreased the levels of dopamine and its metabolites in both brain areas. Systemic 5-HT2C antagonists increases dopamine outflow in nucleus 89

accumbens, striatum and frontal cortex (Di Giovanni et al. 1999; Gobert et al. 2000;

Pozzi et al. 2002), whereas 5-HT2A receptor antagonists apparently inhibit dopamine release under stimulated conditions in these brain areas (De Deurwaerdère and Spampinato 1999; Porras et al. 2002; Pehek et al. 2006). Combined blockade of 5-

HT2A/2C receptors, e.g. by low doses of ritanserin, probably masks the stimulatory effect of 5-HT2C receptor blockade on dopamine outflow (Di Matteo et al. 1998). Our results are in agreement with this observation. After buspirone administration, there was a trend to increased accumbal, but not striatal, dopamine levels. This is in agreement with earlier studies, where buspirone has been shown to significantly increase dopamine efflux in frontal cortex, slightly in nucleus accumbens and not at all in striatum at doses 2.5 – 10 mg/kg (Gobert et al.

1999; Sakaue et al. 2000). Systemic administration of the 5-HT1A agonist, 8-OH-DPAT, has been previously shown to increase accumbal dopamine levels via an excitatory effect on the ventrotegmental dopaminergic neurons (Prisco et al. 1994). In our study, dopamine metabolism was also stimulated by buspirone in both brain areas as reported earlier (at 10 mg/kg) by Algeri et al. (1988). Notably, there were similarities between the effects of a high dose of deramciclane (30 mg/kg) and buspirone in both brain areas studied. Both deramciclane and buspirone seem to be more effective in increasing accumbal than striatal dopamine levels. Both of these drugs have D2 receptor antagonist properties and thus the increased dopamine levels may well be a consequence of a dopamine D2 receptor blockade, particularly in nucleus accumbens. The Ki values of buspirone for 5-HT1A and D2 receptors are close to each other indicating that the antagonistic effects at D2 receptors occur at very similar dose range than is needed to stimulate 5-HT1A receptors (Piercey et al. 1994).

Buspirone is currently in clinical use for the treatment of anxiety. 5-HT1A agonists have shown antidepressant activity in animal models of depression (Wieland and Lucki 1990) and antidepressants are used to treat some cases of anxiety (see Rasmussen

2006). 5-HT2C receptors (Baxter et al. 1995; Bagdy et al. 2001; Bourin and Hascoët

2001) and dopamine D2 receptors (Ainsworth et al. 1998; Ostow 2002) may be involved in the mechanisms of depression and anxiety. Interestingly, the combined 5-HT2/D2 receptor action of atypical antipsychotics, such as quetiapine and olanzapine, can have beneficial effects also in the treatment of depression (Montgomery 2008). Generally, these drugs have high affinities for 5-HT2A receptors, and many of them are 5-HT2C inverse agonists and/or 5-HT1A agonists (Meltzer 1999; Weiner et al. 2001). Deramciclane has shown anxiolytic activity in several animal tests (Gacsályi et al. 1988; Gacsályi et al. 1996; Gacsályi et al. 1997). Indeed, doses of 3 and 10 mg/kg of deramciclane have been shown to produce plasma levels in rats that are close to or higher than the anxiolytic plasma levels in humans (Gacsályi et al. 1997; Kanerva et al. 1999; Nemes et al. 2000). Even doses 1/5th or 1/10th of those have been effective in several anxiolytic animal models (Gacsályi et al. 1988; Gacsályi et al. 1997). The lack 90

of effect of deramciclane at doses 5 – 20 mg/kg on L-dopa/carbidopa-induced circling behavior in 6-OHDA-rats has also been claimed to indicate that the drug possesses no significant antidopaminergic effects at this dose level (Gacsályi et al. 1997). In our study, the approximate dose margin between anxiolytic and antidopaminergic dose of deramciclane in rats was estimated as the ratio between the highest anxiolytic dose in previous animal tests (7 mg/kg; Gacsályi et al. 1997) and the antidopaminergic dose 30 mg/kg. In humans, there is no evidence of dopamine antagonism-related extrapyramidal adverse effects of deramciclane (Naukkarinen et al. 2005). The effects of deramciclane at single anxiolytic doses (3 and 10 mg/kg) on mesolimbic and nigrostriatal dopaminergic systems in our study resembled those of ritanserin (1 mg/kg). Both drugs had no significant effect on either extracellular levels of dopamine or dopamine metabolites. In the light of previous observations in ritanserin studies (Di Matteo et al. 1998; Gobert et al. 2000), these effects are evidently mediated via 5-HT2A/2C receptor antagonism. In contrast, deramciclane has induced behavioral effects which are indicative of antidopaminergic activity, but only at doses greatly exceeding anxiolytic doses (Gacsályi et al. 1997; Ingman et al. 2004). Various studies in rodents have clearly demonstrated the antidopaminergic activity of deramciclane at doses of 20 – 40 mg/kg or higher, which are much higher than those needed to achieve anxiolytic effects (Gacsályi et al. 1997; Ingman et al. 2004). These findings support our microdialysis results showing that 30 mg/kg of deramciclane has dopamine receptor blocking properties, particularly in nucleus accumbens. It seems that deramciclane fumarate has antidopaminergic activity similar to neuroleptics or buspirone on the nigrostriatal and particularly on the mesolimbic system at least after a high single dose of 30 mg/kg. However, the present results do not fully explain if the increase in accumbal dopamine levels by deramciclane 30 mg/kg occurred via antagonism at D2 receptors or 5-HT2A/2C receptors or via combined 5-HT2/D2 receptor action. There is, however, at least a 5-fold margin between the anxiolytic and neuroleptic doses of deramciclane in the rat.

6.4 Lack of protective effect of quercetin in two types of unilateral 6-OHDA- lesions (IV)

Unilateral 6-OHDA-lesioned rat is commonly used to model degeneration of the nigrostriatal system. Since the neurotoxicity of 6-OHDA is evidently mediated by non- enzymatic oxidative stress and excessive generation of free radicals (Zigmond et al. 1992; Kumar et al. 1995; Choi et al. 1999), it could be hypothesized that the oxidative damage should be prevented by antioxidants. In fact, compounds like melatonin and vitamin E, have been found to be effective against 6-OHDA-induced neurodegeneration in rats (Cadet et al. 1989; Aguiar et al. 2002; Sharma et al. 2006). Bioflavonoids, such as quercetin (3,5,7,3′,4′-pentahydroxyflavone), are known to possess oxygen radical 91

scavenging properties and some neuroprotective effects in rat mesencephalic cell cultures in vitro (Nobre Junior et al. 2003; Mercer et al. 2005). On the other hand, repeated administration of quercetin has shown some beneficial effects against neurotoxicity in vivo (Naidu et al. 2003; Singh et al. 2003). In the present study, the relation between the severity of dopaminergic and existence of serotonergic lesions in two types of rat 6-OHDA-lesion models, and the effect of quercetin in these two situations was estimated (IV). Interestingly, quercetin has been reported to exhibit effects on the serotonergic system, particularly for 5-HT3 receptor subtype, in vitro (Lee et al. 2005). 5-HT3 receptors are known to mediate rapid and transient membrane depolarizing effect of 5- HT in the CNS via transient inward current and subsequent opening of the cation channels (Hoyer et al. 2002). Quercetin seems to be able to dose-dependently modulate

5-HT3 receptor channel activity (at 10 – 100 µM concentration) by blocking the 5-HT- induced current in a reversible manner (Lee et al. 2005; 2008). Indeed, Lee et al. (2005) have suggested that quercetin can act as a natural 5-HT3 receptor antagonist, which might be beneficial in the same conditions where 5-HT3 blockers such as ondansetron are used. The herb Hypericum perforatum (St. John’s wort) contains numerous biological active constituents, including quercetin, hypericin and hyperforin (De Vry et al. 1999; see Di Carlo et al. 2001). Hypericum extracts have been shown to upregulate

5-HT1 and 5-HT2 receptors (Teufel-Mayer and Gleitz 1997) and to modify dopamine, 5- HT and 5-HIAA levels (Calapai et al. 1999) in the rat brain. Furthermore, it seems that the neurotransmitter modulating effects in the CNS are more widespread when the extract contains high concentration of flavonoids (Calapai et al. 1999). In this study, we explored the effect of various quercetin treatment schedules (pre- and post-treatment and their combination) on 6-OHDA-induced dopaminergic neurotoxicity in vivo using two types of unilateral 6-OHDA-lesion rat models of PD. In the full lesion model, a high dose of 6-OHDA was infused to the MFB leading to a severe dopaminergic damage. In the partial lesion model, small doses of 6-OHDA were administered into four sites of the striatum leaving part of the nigrostriatal dopaminergic innervation intact. In this model it was anticipated that there would be more scope for the putative neuroprotective effect. Furthermore, since we knew from our previous studies that 6-OHDA-infusion into the right MFB could evoke subsequent decrease in ipsilateral 5-HT levels, and that quercetin could exhibit effects on serotonergic system by possible 5-HT3 receptor antagonism, we studied if quercetin would have any effect on non-selective striatal serotonergic damage. However, repeated systemic administration of quercetin one week before or one week after 6-OHDA infusion to the MFB was not able to prevent the dopaminergic or serotonergic neuronal toxicity: no effects on striatal levels of dopamine, 5-HT or their metabolites were observed with any treatment schedule. While 5-HT and 5-HIAA levels were unaffected by striatal 6-OHDA-lesions, quercetin had no effect on the levels of 92

dopamine or its metabolites. Equally, in rotation tests, no significant effect of quercetin against MFB- or striatal lesions was observed. However, quercetin tended to decrease (n.s.) rotational behavior in some MFB-lesioned rats, and the same was observed in striatal lesioned rats at quercetin dose of 100 mg/kg. Furthermore, TH was practically undetectable in the lesioned side in MFB lesions, while the striatal lesion partly spared dopaminergic innervation. Quercetin treatment did not have any significant effect on TH-positive cells with either type of 6-OHDA-lesion. However, in parallel with the rotation results, in histochemical studies (TH-positive nigral cell numbers and striatal fiber density) we found that the quercetin pretreatment at 100 mg/kg had only a minor (n.s.) restorative effect against striatal 6-OHDA toxicity. At 200 mg/kg, no such a trend was observed. In our studies, quercetin has shown beneficial effects in catecholaminergic SH- SY5Y cells in vitro. The dual effect of quercetin was observed in cell viability experiments; small concentrations of quercetin (10 – 50 µM) provided some protection against 6-OHDA toxicity but at a high concentration (100 µM) this effect was reduced, or even enhanced toxicity occurred (IV). Furthermore, the dose range of the protective and toxic effects of quercetin seems to be quite narrow (Ossola et al. 2008). This biphasic effect has been shown previously also for two other bioflavonoids, epigallocatechin-3-gallate and genistein in vitro (Ramos 2007). In our studies, quercetin (10 – 100 µM) also significantly restored the elevated levels of ROS in 6-OHDA-treated neuronal SH-SY5Y cells (IV). Recently, Datla et al. (2007) described that plant extracts containing bioflavonoids administered orally in a 4-day pretreatment schedule possess protective properties on the nigrostriatal dopaminergic system in 6-OHDA lesioned rat. Our present in vivo results do not support these findings. Rather, they agree with the findings of Zbarsky et al. (2005) which demonstrated that quercetin (20 mg/kg, orally) for 4 days before the MFB lesion did not have any effect on the TH-positive cell loss in SN or the decrease in striatal dopamine, DOPAC and HVA levels in rats. Collectively, our findings suggest that quercetin does not reliably prevent the neurotoxic properties of 6-OHDA in vivo. Potential beneficial effects of bioactive compounds depend on their after administration. The antioxidant efficacy of quercetin has been directly related to the levels of quercetin in the plasma (Nakamura et al. 2000; Murota and Terao 2003). The elimination half-life of 17 h of quercetin in humans implies that repeated administration can cause an accumulation of significant quercetin concentration in plasma (Hollman et al. 1996; Egert et al. 2008). Quercetin also penetrates the blood-brain barrier, although less effectively than some other flavonoids (Youdim et al. 2004; de Boer et al. 2005). Consequently, the drug levels in the brain may well be variable, even during repeated administration at 100 mg/kg twice a day for two weeks. It is difficult to extrapolate in vitro concentrations to the in vivo situation. However, it seems evident that the levels of quercetin in the brain remain lower than the 93

concentrations used in in vitro studies. Theoretically, depending on the metabolism and the brain penetration properties of quercetin, one would predict low micromolar levels in the brain at the doses utilized in the present study. These are much lower than the quercetin concentrations used in our in vitro studies. Since the concentrations of 6- OHDA infused locally to the brain exceed 10 mM, it is probable that predicted quercetin concentrations are not high enough to scavenge the 6-OHDA produced ROS in vivo and thus to effectively protect dopaminergic cells from the damage. The site of 6-OHDA infusion may be very critical in determining how much serotonergic damage occurs. Serotonergic projections originating from the dorsal raphe nuclei innervate all parts of the basal ganglia circuitry (Lavoie and Parent 1990). Ascending 5-HT projections run in close proximity to the MFB (Jacobs and Azmitia 1992) and densely innervate several brain areas, including striatum (Dahlström and Fuxe 1964; Ungerstedt 1971). Thus, they may be readily susceptible to nonselective oxidative damage evoked by the 6-OHDA-infusion into MFB (Carta et al. 2006). The effects of 6- OHDA-lesions on striatal 5-HT and 5-HIAA content have been quite inconsistent in several previous studies in adult rats. Although the dopamine levels in lesioned striatum have been depleted, 5-HT levels have remained unchanged (Erinoff and Snodgrass 1986; Tanaka et al. 1999) or declined from 30 to 50% (Abrous et al. 1990; Karstaedt et al. 1994). Furthermore, regional differences in the patterns of 5-HT innervation have been reported after unilateral 6-OHDA-lesion in rats (Takeuchi et al. 1991; Zhou et al. 1991). Clinically, reduced striatal levels of all the key serotonergic markers (5-HT, 5- HIAA, transporter protein SERT, 5-HT synthesizing enzyme TrH; from 30 to 70% decreases have been observed) in PD have provided strong evidence for a serotonergic disturbance in this disease (Birkmayer and Birkmayer 1987; Jellinger 1991; Kerenyi et al. 2003; Kish et al. 2008). In our studies (I, II, IV), 6-OHDA-lesion of the MFB evoked a significant decrease in ipsilateral striatal 5-HT and 5-HIAA levels, while serotonergic innervation appeared to remain unaffected in the partial lesion after intrastriatal 6-OHDA-infusion. Obviously, there are anatomical reasons to account for these changes. 6-OHDA is taken up into dopaminergic nerve endings by DAT and subsequent damage of these cells is induced mainly by the formation of ROS (Choi et al. 1999; Blum et al. 2001). When 6- OHDA is infused intrastriatally in several sites, serotonergic neurons in the MFB remain unaffected. Surprisingly, the effect of 6-OHDA-lesions on the serotonergic system has been discussed only occasionally in studies with hemilesioned parkinsonian rats (Carta et al. 2006; 2007 and references therein). 6-OHDA-infusion into the MFB produces almost complete striatal dopamine depletion in the lesioned side, whereas striatal 6- OHDA-lesion decreases dopamine levels to a lesser extent, typically by about 50% (Yuan et al. 2005). The striatal lesion may be more sensitive at revealing the neuroprotective effect of compounds since it leaves more nigrostriatal dopaminergic neurons intact than is the case with MFB lesions (Kirik et al. 1998) and thus may 94

represent a more relevant model to study neuroprotective effects. One can hypothesize that the suggested neuroprotective effect of quercetin against dopaminergic cell damage in vivo, if it exists at all, should have been detected in the striatal lesion model. In the case of the serotonergic damage seen after MFB lesions (~50%), the unilateral 6- OHDA-lesion model apparently represents rather well the simultaneous serotonergic disturbance observed in PD. However, quercetin had no significant neuroprotective effects against either dopaminergic or serotonergic neuronal damage occurring 6- OHDA-lesioned rats. 95

7 CONCLUSIONS

In general, the present results point to an involvement of 5-HT neurons in the dopaminergic imbalance that is responsible for the rotational behavior of unilaterally 6- OHDA-lesioned rats and that 5-HT neurons play a role in the actions of exogenous intrastriatal L-dopa in the dopamine depleted striatum. This study provides new information about the serotonergic effects, such as putative 5-HT neuron sprouting, occurring after local exogenous L-dopa in dopamine depleted rat striatum, which may help to further clarify the role of the serotonergic system in the action of L-dopa under dopamine deficiency. In two types of unilateral 6-OHDA-lesion models (striatal and MFB lesion), the striatal 5-HT content was differently affected, highlighting the importance of site of administration in determining the effects of 6-OHDA-infusion. The clear unilateral 5-HT damage in this widely used rat model should be taken into account in further studies. This study also confirms earlier reports of dopamine modulating properties of 5-HT1A and 5-HT2A/2C ligands, particularly in the mesolimbic system, and provides further evidence that combined affinity of 5-HT/D2 receptors may be effective in modulating dopaminergic tone in the CNS. In these models, quercetin had no effect indicative of neuroprotection against nigrostriatal dopaminergic or serotonergic 6-OHDA-induced neuron damage in vivo. The lack of effect of quercetin in 6-OHDA-rats casts serious doubt on the neuroprotective effects of this bioflavonoid on experimental PD in vivo. Further studies are needed to evaluate the benefit of putative protective agents in neurodegeneration.

Based on the results of the experimental section (I – IV), the following specific conclusions can be drawn:

1. Acute intrastriatal L-dopa treatment decreases contralateral rotation of unilaterally 6-OHDA-lesioned rats apparently by desensitization of ipsilateral dopamine receptors. Attenuated rotational behavior and 6-OHDA-induced decrease in ipsilateral 5-HT levels are abolished by TrH enzyme inhibition. The lack of L-dopa-induced desensitization in rotation tests after acute intrastriatal L- dopa treatment appears to be associated with the lack of 5-HT imbalance between intact and lesioned striata. The results suggest a likely involvement of 5-HT neurons in the dopaminergic imbalance that is responsible for the rotational behavior of unilaterally 6-OHDA-lesioned rats.

2. Continuous 2-week intrastriatal L-dopa treatment in unilaterally 6-OHDA- lesioned rats decreases contralateral rotation and induces long-term alterations in serotonergic markers (5-HIAA, TrH, 5-HT uptake) in the ipsilateral striatum. 96

The present results indicate that 5-HT neurons play a role in the actions of exogenous intrastriatal L-dopa in the dopamine depleted striatum.

3. The 5-HT2A/2C antagonist deramciclane fumarate at single anxiolytic doses of 3 and 10 mg/kg has no significant effect on mesolimbic and nigrostriatal

dopaminergic systems, which resembles the effect of the 5-HT2A/2C antagonist ritanserin at low doses. A high single dose of 30 mg/kg of deramciclane

fumarate has antidopaminergic activity similar to the 5-HT1A partial agonist buspirone or to the neuroleptics particularly on the mesolimbic system.

Increased accumbal dopamine levels may occur by antagonism at D2 receptors

or 5-HT2A/2C receptors or via combined activity at 5-HT2/D2 receptors. There is at least a 5-fold margin between the anxiolytic and neuroleptic doses of deramciclane in the rat.

4. The site of the unilateral 6-OHDA infusion is critical in relation to ipsilateral serotonergic damage in rats. 6-OHDA-lesion of the MFB produces a significant decrease in ipsilateral striatal 5-HT and 5-HIAA, while serotonergic innervation seems to be unaffected in the partial lesion induced by intrastriatal 6-OHDA- infusion. In view of the fact that significant serotonergic damage was observed after MFB lesions (50% decrease in striatal 5-HT levels), this unilateral 6- OHDA-lesion rat model apparently represents rather well the serotonergic disturbance observed in PD. The striatal lesion may generally be more sensitive at revealing the neuroprotective effect of compounds since it leaves more nigrostriatal dopaminergic neurons intact than is the case with MFB lesions. Finally, the bioflavonoid, quercetin, had no significant effect which could be interpreted as being indicative of neuroprotection against either dopaminergic or serotonergic neuronal damage in 6-OHDA-lesioned rats. 97

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ORIGINAL PUBLICATIONS

This doctoral dissertation is based on the following publications, referred to in the text by Roman numerals I – IV.

I Tiina M. Kääriäinen, J. Arturo García-Horsman, Marjo Piltonen, Pekka T. Männistö: L-dopa induced desensitization depends on 5-HT imbalance in hemiparkinsonian rats. NeuroReport, in press.

II Tiina M. Kääriäinen, J. Arturo García-Horsman, Marjo Piltonen, Marko Huotari, Pekka T. Männistö: Serotonergic activation after 2-week intrastriatal infusion of L- dopa and slow recovery of circling in rats with unilateral nigral lesions. Basic and Clinical Pharmacology and Toxicology 102: 300-307, 2008.

III Tiina M. Kääriäinen, Marko Lehtonen, Markus M. Forsberg, Jouko Savolainen, Mikko Käenmäki, Pekka T. Männistö: Comparison of the effects of deramciclane, ritanserin and buspirone on extracellular dopamine and its metabolites in striatum and nucleus accumbens of freely moving rats. Basic and Clinical Pharmacology and Toxicology 102: 50-58, 2008.

IV Tiina M. Kääriäinen, Marjo Piltonen, Bernardino Ossola, Heli Kekki, Šárka Lehtonen, Terhi Nenonen, Anne Lecklin, Atso Raasmaja, Pekka T. Männistö: Lack of robust protective effect of quercetin in two types of 6-hydroxydopamine-induced parkinsonian models in rats and dopaminergic cell cultures. Brain Research 1203: 149-159, 2008.

Reprinted with permission from the publishers. Kuopio University Publications A. Pharmaceutical Sciences

A 96. Haapalinna, Antti. The Effects of on Brain Neurochemistry and Behaviour in Laboratory Rodents – Possible Implications for the Treatment of Neurodegenerative Diseases with an Alpha2-adrenoceptor Antagonist. 2006. 119 p. Acad. Diss.

A 97. Parkkari, Teija. Synthesis of Novel Cannabinoid CB1 Receptor Ligands. 2006. 148 p. Acad. Diss.

A 98. Jarho, Elina. Synthesis, Structure-Activity Relationships and Physico-Chemical Properties of Novel Prolyl Oligopeptidase Inhibitors. 2007. 121 p. Acad. Diss.

A 99. Turunen, Juha. Pain and Pain Management in Finnish General Population. 2007. 110 p. Acad. Diss.

A 100. Toropainen, Elisa. Corneal epithelial cell culture model for pharmaceutical studies. 2007. 81 p. Acad. Diss.

A 101. Mannila, Janne. Cyclodextrins in intraoral delivery of delta-9-tetrahydrocannabinol and . 2007. 90 p. Acad. Diss.

A 102. Männistö, Marjo. Polymeric carriers in non-viral gene delivery: a study of physicochemical properties and biological activity in human RPE cell line. 2007. 65 p. Acad. Diss.

A 103. Mauriala, Timo. Development of LC-MS methods for quantitative and qualitative analyses of endogenous compounds, drugs, and their metabolities to support drug discovery programs. 2007. 126 p. Acad. Diss.

A 104. Kumpulainen, Hanna. Novel prodrug structures for improved drug delivery. 2007. 129 p. Acad. Diss.

A 105. Korjamo, Timo. Improvement of the Caco-2 permeability model by genetic and hydrodynamic modifications. 2008. 134 p. Acad. Diss.

A 106. Pappinen, Sari. The organotypic culture of rat epidermal keratinocytes (ROC) in pharmaceutical and chemical testing. 2008. 83 p. Acad. Diss.

A 107. Mönkkönen, Kati. GĮɿͅ in ciliated tissues: physiological role in regulation of ependymal ciliary function and characteristics in human female reproductive tissues. 2008. 85 p. Acad. Diss.

A 108. Matilainen, Laura. Cyclodextrins in peptide deliverty: in vitro studies for injectable and inhaled formulations. 2008. 144 p. Acad. Diss.

A 109. Lääkepäivät : lääkkeitä myös terveille? 25.-26.4.2008, Kuopio. 2008. 120 p. Abstracts.

A 110. Myöhänen, Timo. Distribution of prolyl oligopeptidase and its colocalizations with neurotransmitters and substrates in mammalian tissues. 2008. 101 p. Acad. Diss.