5-HT2C SEROTONIN RECEPTORS: CELLULAR LOCALIZATION AND CONTROL OF DOPAMINERGIC PATHWAYS IN THE RAT BRAIN By Katherine Demetra Alex Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Thesis Adviser: Dr. Elizabeth A. Pehek Department of Neurosciences CASE WESTERN RESERVE UNIVERSITY January, 2007 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ______________________________________________________ candidate for the Ph.D. degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. 2 Table of Contents: List of Tables 4 List of Figures 5-6 Acknowledgements 7 Abstract 8-9 Chapter 1: Introduction 10-37 Introduction to Dopamine 10-13 Introduction to Serotonin 13-14 Evidence for Serotonergic Control of DA Activity 15-31 Roles of 5-HT2 Receptors in Modulating DA Activity 15-16 5-HT2A 16-21 Localization 16-18 Nigrostriatal Pathway 18 Mesolimbic Pathway 18-19 Mesocortical Pathway 19-21 Summary 21 5-HT2B 21 5-HT2C 21-30 Localization 21-22 Nigrostriatal Pathway 22-26 Mesolimbic Pathway 26-28 Mesocortical Pathway 28-29 Summary 29-30 Summary and Implications 30-31 The Unique Importance of the 5-HT2C Receptor 31-37 Chapter 2: Modulation of Dopamine Release by Striatal 5-HT2C Receptors 38-63 Summary 38 Introduction 39-41 Methods 41-47 Results 47-50 Discussion 50-56 Figures 57-63 Chapter 3: Colocalization of 5-HT2C Receptors and 5-HT2A Receptors in Rat Cortex and Hippocampus 64-92 Summary 64 Introduction 65-69 Methods 69-72 Results 72-74 Discussion 74-81 Figures 82-92 3 Chapter 4: Discussion 93-116 Using Microdialysis to Measure Changes in DA Release 93-95 Differential Regulation of the Nigrostriatal and Mesocortical Pathways 95-96 Tonic Inhibition of DA by 5-HT2C Receptors may be GABA-Mediated 96-98 The Heterogeneous Composition of the Striatum 98-100 Elucidating the Circuitry Involved in Control of DA by Striatal 5-HT2C Receptors 100-106 Cellular Localization of 5-HT2C Receptors in the Cortex and Hippocampus 106-111 Clinical Implications 111-113 Summary 113-114 Figures 115-116 Bibliography: 117-154 4 List of Tables: Table 1: Quantification of 5-HT2C Receptors with Parvalbumin and 5-HT2A Receptors in the Cortex and Hippocampus 92 5 List of Figures: Chapter 2: Figure 1: Intrastriatal Administration of a 5-HT2C Receptor Inverse Agonist Increases Dialysate Dopamine in the Striatum of the Rat 57-58 Figure 2: Coadministration of a 5-HT2C Receptor Agonist Attenuates the 5-HT2C Receptor Inverse Agonist-Induced Increase in Striatal Dopamine 59-60 Figure 3: Intracortical Administration of Low Concentrations of a 5-HT2C Receptor Inverse Agonist does not Affect Dialysate Dopamine in the Prefrontal Cortex 61 Figure 4: Intracortical Administration of Higher Concentrations of a 5-HT2C Receptor Inverse Agonist does not Affect Basal or High K+-Stimulated Dopamine Release in the Prefrontal Cortex 62-63 Chapter 3: Figure 1: Western Blot Illustrating the Specificity of the 5-HT2C Receptor Antibody 82 Figure 2: Immunofluorescence in Cultured Cells 83 Figure 3: 5-HT2C Receptors in Rat Choroid Plexus 84 Figure 4: Regions of Cortex Examined 85 Figure 5: Lack of Colocalization of 5-HT2C Receptors with Parvalbumin in the Cortex of the Rat 86 Figure 6: Lack of Colocalization of 5-HT2C Receptors with Parvalbumin in the Hippocampus of the Rat 87 Figure 7: Rostro-Caudal Level of Hippocampus Examined 88 Figure 8: 5-HT2C Receptors Colocalize with 5-HT2A Receptors in Rat Cortex 89 Figure 9: 5-HT2C Receptors Colocalize with 5-HT2A Receptors in Rat Hippocampus 90 6 Figure 10: 5-HT2C do not Colocalize with GAD65/67 in the Rat Striatum 91 Chapter 4: Figure 1: Putative circuitry of the regulation of nigrostriatal DA by 5-HT2C receptors under basal conditions 115 Figure 2: Putative circuitry of the regulation of nigrostriatal DA by 5-HT2C receptors in the presence of SB 206553 116 7 Acknowledgements: This work was supported by grant MH52220 from the National Institute of Health and a Merit Review Award from the Department of Veterans Affairs to E.A.P. I wish to thank SmithKline and Beecham for their generous donation of SB 206553. I would also like to acknowledge the data contributions of Gregory Yavanian, Hewlett McFarlane, Charlie Pluto, and Atheir Abbas. I would like to acknowledge the support that I have received from my advisor, Dr. Elizabeth Pehek throughout the past 6 years. I would also like to thank the members of my thesis committee for providing constructive criticism and support. In addition, I would like to thank Dr. Bryan Roth for his mentoring and technical help during the collection of my immunofluorescence data. I am grateful to the colleagues that I have worked with in both the Pehek and the Roth laboratories. Lastly, I would like to thank my friends and family for their encouragement and support during this time. 8 5-HT2C Serotonin Receptors: Cellular Localization and Control of Dopaminergic Pathways in the Rat Brain Abstract By Katherine Demetra Alex Dopamine (DA) is known to play a role in the pathology and/or treatment of schizophrenia, drug abuse and Parkinson’s disease. Serotonin (5-HT) is capable of modulating dopamine release through actions at 5-HT receptors. In particular, 5-HT2 receptor binding, and subsequent effects on DA release, may be involved in the efficacy of atypical antipsychotic drugs and recent work suggests that they may be promising targets for the treatment of depression, anxiety, obesity, and drug abuse as well. 5-HT2C receptors have been shown to tonically inhibit DA release in the striatum and the prefrontal cortex (PFC). The localization of the receptors that mediate these effects has not been studied. These data show that 5-HT2C receptors in the terminal region of the nigrostriatal pathway are, at least in part, responsible for the tonic inhibition of DA release in the striatum. In addition, data is presented that suggests that the mesocortical pathway is not modulated by 5-HT2C receptors in the terminal region. The cellular localization of 5-HT2C receptors has not been extensively studied, in part due to a lack of specific antibodies. Here, a selective 5-HT2C receptor antibody was used in immunofluorescence studies to examine the cellular localization of the 5-HT2C receptors that mediate the tonic inhibition of DA release in the brain. These studies show that 5- HT2C receptors do not colocalize with markers for parvalbumin-containing GABAergic cells in the cortex and hippocampus. Importantly 5-HT2C receptors show a high degree of 9 colocalization with 5-HT2A receptors in these regions. 5-HT2A and 5-HT2C receptors are similar in structure and couple to the same intracellular signaling pathways upon activation. Differences in their levels of constitutive activity and desensitization in response to chronic ligand exposure have, however, been shown. Thus, by these mechanisms 5-HT2A and 5-HT2C receptors expressed in the same cortical and hippocampal pyramidal neurons may finely tune cortical efferents. The results presented here have implications for the development of new therapeutics for the treatment of diseases and disorders in which a 5-HT2 receptor-mediated manipulation of DA is beneficial. 10 CHAPTER 1: Introduction to Dopamine: The first known function of dopamine (DA) was its role in many species as a precursor to norepinephrine (NE) and epinephrine – the other neurotransmitters in the catecholamine family. It wasn’t until the late 1950’s that evidence began to accumulate favoring an independent role for DA in the brain. At this time it was discovered that DA and NE were manufactured in different brain regions, in essence the regions with the highest levels of DA produced low levels of NE and vice versa (Bertler and Rosengren, 1958; Carlsson, 1959) and that in mammals the vast majority of the body’s DA is found in the brain (Carlsson, 1959). Further research led to the discovery of dopaminergic pathways in which cell bodies in one brain region send projections to another brain region where DA is released from axon terminals. The first to be characterized was the nigrostriatal pathway in which cell bodies in the substantia nigra pars compacta (SNpc) project their axons to the caudate and putamen, collectively called the striatum (Anden et al., 1964). Soon after, two additional pathways had been discovered: the mesolimbic pathway in which cell bodies in the ventral tegmental area (VTA) project axons to the nucleus accumbens (NA), and the tuberoinfundibular pathway in which cell bodies in the arcuate nucleus of the hypothalamus project axons to the median eminence (for review see McClure, 1973; Glowinski, 1975). Later, evidence was found for a fourth group of neurons, dubbed the mesocortical pathway, that send dopaminergic projections from the VTA to the cerebral cortex, specifically the prefrontal cortex (PFC) (Glowinski, 1975). DA has binding affinity for and adrenergic receptors and it was not until it was recognized that DA has a distinct pattern of expression from NE that it became apparent that there are also distinct DA receptors (see Woodruff, 1971 for review). It is 11 now known that there are 5 distinct DA receptor subtypes which can be classified as D1- like (D1 and D5) and D2-like (D2, D3, and D4) based on their coupling to G-protein signal cascades. D1-like receptors couple positively to adenylyl cyclase while D2-like receptors couple negatively to adenylyl cyclase and thus cAMP production (for review, see Missale et al., 1998). DA receptors are found in both the cell body and terminal regions of the dopaminergic pathways. Both D1-like and D2-like receptor subtypes are found both pre and postsynaptically but only D2 receptors are known to serve as autoreceptors (see Missale et al., 1998, for review and Paspalas and Goldman-Rakic, 2005).